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Nanomechanics of Barnacle Proteins and Multicomponent LipidBilayers Studied by Atomic Force Microscopy
by
Ruby May Arana Sullan
A thesis submitted in conformity with the requirementsfor the degree of Philosophy
Graduate Department of ChemistryUniversity of Toronto
© Copyright by Ruby May Arana Sullan 2010
ii
Nanomechanics of Barnacle Proteins and Multicomponent LipidBilayers Studied by Atomic Force Microscopy
Ruby May Arana Sullan
Doctor of Philosophy
Graduate Department of ChemistryUniversity of Toronto
2010
Abstract
Owing to atomic force microscopy�’s (AFM) high resolution in both imaging and force
spectroscopy, it is very successful in probing not only structures, but also nanomechanics of
biological samples in solution. In this thesis, the nanomechanical properties of lipid bilayers of
biological relevance and proteins of the barnacle adhesive were examined using AFM
indentation, AFM based force mapping, and single molecule pulling experiments. Through high
resolution AFM based force mapping, the self organized structures exhibited in phase
segregated supported lipid bilayers consisting of dioleoylphosphatidylcholine / egg
sphingomyelin / cholesterol (DEC) in the absence and presence of ceramide (DEC Ceramide)
were directly correlated with their breakthrough forces, elastic moduli, adhesion, and bilayer
thickness. Results were presented as two dimensional visual maps. The highly stable ceramide
enriched domains in DEC Ceramide bilayers and the effect of different levels of cholesterol as
iii
well as of diblock copolymers, on the nanomechanical stability of the model systems studied
were further examined. For the proteins of the barnacle adhesive, scanning electron
microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and chemical staining with
amyloid selective dyes, in addition to AFM imaging, indentation, and pulling experiments were
performed to study the structure and nanomechanics of the polymerized barnacle glue.
Nanoscale structures exhibiting rod shaped, globular, and irregularly shaped morphologies
were observed in the bulk barnacle cement by AFM. SEM coupled with energy dispersive x ray
(EDX) makes evident the organic nature of the rod shaped nanoscale structures while FTIR
spectroscopy on the bulk cement gave signatures of sheet and random coil conformations.
Indentation data yielded higher elastic moduli for the rod shaped structures as compared to the
other structures in the bulk cement. Single molecule AFM force extension curves on the matrix
of the bulk cement often exhibited a periodic sawtooth like profile, observed in both extend
and retract portions of the force curve. Rod shaped structures stained with amyloid protein
selective dyes (Congo Red and Thioflavin T) revealed that about 5% of the bulk cement are
amyloids.
iv
Acknowledgments
I owe my deepest gratitude to my supervisor Gilbert C. Walker who is very instrumental in my
career as a novice researcher. His breadth of knowledge, quantitative approach, and way in
addressing key scientific questions using the simplest methods possible taught me creativity in
research. Being in the Walker�’s group under Gilbert�’s direction has catalyzed my growth in the
field and as a person. Also, Gilbert�’s deep concern to the welfare of his students is a living
example that great scientists are also excellent mentors! Indeed, it is a great fortune to be in
the Walker�’s Lab!
The year I spent in Ottawa has been truly amazing and I have Shan Zou, my local supervisor at
NRC SIMS to thank for. Her exemplary passion for Science has somehow been contagious and
heightened my love for it. I have acquired important scientific skills from working closely with
Shan. Our long discussions on Science and philosophy have always been intellectually
stimulating, thought provoking, and insightful.
James K. Li, my lab mate and a co author in a number of my papers, for providing the extremely
useful script I used in my analysis. James�’ help in everything is just invaluable. His presence
reduced the typical load associated with being a grad student by a significant much. And
everyone agrees that James is the superhero in the lab!
Nikhil Gunari �“Niki�”, for being patient when I was first learning how to �“fish�” for molecules. The
hours we spent pulling on the barnacle proteins has taught me a lot about force spectroscopy.
Niki also has unlimited patience answering my questions, half of which I asked more than once.
To the members of my supervisory committee: Prof. Chris Yip, for his invaluable and excellent
suggestions in every meeting. Also, his combinatorial approach to biological problems has been
inspiring and has always provided me with useful insights to my own research. Prof. Mark Nitz,
for his relevant feedbacks to my work and for agreeing to review my thesis even while on
Sabbatical.
v
I am particularly indebted to Dr. Linda Johnston, the group leader in the Biomembrane and
Imaging lab at NRC SIMS, for sharing her expertise on membranes, which has been
instrumental to my graduate work. I owe a thank you to Daniel Carter for meticulously showing
me how to prepare lipid bilayers. My warm thanks to Lucie for making my stay in NRC and
Ottawa delightful! To the rest of the Biomembrane Group: Dusan, Zhengfang, Zygmunt, Ryan,
Rory, and Kirk, for being friendly and generous with their time and guidance. Special thank you
to Maohui Chen who helped me a lot in almost anything and for great discussions.
To the great bunch of individuals in the Walker�’s lab who made graduate school truly happy,
full of vigor, meaningful, and memorable: Melissa, Adrienne, Weiqing, Christina, Claudia, Jane,
Zahra, Niki, Sudipta, Srini, Shell, Isaac, James. Adrienne, for wonderfully organizing events in the
Walker�’s Lab and for greatly helping me with SEM and FTIR soooooo gooood! Weiqing, for
patiently answering my anything under the sun questions. Shell, whose creativity, wit, and
complicated character (like Sherlock Holmes driving a Ferrari), have resulted to a number of
improvised yet fully functional equipment in the lab useful to my experiments. Christina, for the
always relaxing chats in random places in the lab and in the building. Melissa and Isaac, BPS
conference and Jollibee buddies, for all the great and stimulating discussions and the awe
inspiring road trip along the pretty coast from SFO to LA! People in the Walker�’s labs is
simply amazing!!!
Machine shop in the Department of Chemistry, U of T, in particular Johnny Lo and John Ford
who always attend to my machining needs with utmost care and accuracy.
Helane Chan, a summer student, whom I had a pleasure of working with. Her presence made
this final year packed with thesis writing and experiments more manageable. Her inquisitive
character also brought me new avenues to think of.
Cynthia Goh, for her support and advise from day 1 of my life at the University of Toronto to
present and am sure till eternity. Jane Goh and Richard Loo, of the Goh�’s lab, for their advice of
all sorts from Schlenk line to fit adapter to surface modification and functionalization
Chemistry.
vi
To my friends in the department: Jordan �“Manong Jordan�”, who�’s a living oracle and helped me
in so many ways from my initial settling years in Toronto to my final year contemplating on
the next steps to take. Yes Jordan, may bayad na yan! Mayrose �“Manay Mayrose�”, for making
my transition to grad school smooth and easy. Emina, for the great chats which are always a
source of comfort. Zaldy, for teaching me how to kayak and canoe. Yoshi, for serving the Filipino
community since 2001. Hannah, for being a great chef, while I was writing this thesis and for
the profound conversations!
Nesha May Andoy, a fellow graduate student in Cornell, for numerous exciting and fruitful
discussions. It is a pleasure to have a friend going through the same things as you do.
During this work I have collaborated with many colleagues for whom I have great regard, and I
wish to extend my warmest thanks to Hong Zhang, Gary Dickinson, Chris Kavanagh, Eugenia
Kumacheva, Dan Ritschoff, Bea Orihuela, Maja Weigemann, and Changchun Hao.
To my friends outside the department: Gena, Rheea, Evelyn, Anna, and David, I have enjoyed
our all embracing conversations and your unwavering friendship!
To papang, mamang, kuya Anjo, te Fel, Vj, my nieces and nephews for their support, well
wishes, prayers, and ready smiles throughout. Despite the distance, all through these years,
their love kept me company. This thesis is dedicated to them.
Any task of this enormity would have been impossible if not for the hand that guided me. A big
THANK YOU to the Lord God Almighty, for being with me in this journey and for bringing all the
right people at the right time. All glory, honor, and praise to You!!!
vii
Table of Contents
ACKNOWLEDGMENTS ........................................................................................................................................ IV
TABLE OF CONTENTS ........................................................................................................................................ VII
LIST OF TABLES ................................................................................................................................................. XI
LIST OF ACRONYMS .......................................................................................................................................... XII
LIST OF FIGURES.............................................................................................................................................. XIII
LIST OF EQUATIONS ......................................................................................................................................... XVI
LIST OF APPENDICES .......................................................................................................................................XVIII
1 NANOMECHANICS OFMODEL AND BIOLOGICAL SYSTEMS STUDIED THROUGH ATOMIC FORCEMICROSCOPY (AFM): AN INTRODUCTION ............................................................................................ 1
1.1 PRINCIPLES OF ATOMIC FORCEMICROSCOPY.............................................................................................2
1.2 STUDY OF NANOMECHANICAL PROPERTIES THROUGH AFM.........................................................................5
1.2.1 FORCE CURVE..........................................................................................................................6
1.2.2 AFM INDENTATION AND SINGLE MOLECULE PULLING EXPERIMENT.................................................8
1.3 MULTICOMPONENT LIPID BILAYERS........................................................................................................12
1.3.1 AFM IMAGING AND FORCEMAPPING ON LIPID BILAYERS.............................................................14
1.4 THE BIOFOULING PROBLEM: BARNACLE AS A CULPRIT...............................................................................15
1.5 OVERVIEW OF THE THESIS ....................................................................................................................16
1.6 REFERENCES.......................................................................................................................................18
2 MULTICOMPONENT LIPID BILAYERS: EXPERIMENTALMETHODS ............................................................. 23
2.1 MATERIALS ANDMETHODS ..................................................................................................................23
2.1.1 MATERIALS ...........................................................................................................................23
2.1.2 PREPARATION OF SMALL UNILAMELLAR VESICLES........................................................................24
2.1.3 PREPARATION OF THE BILAYER .................................................................................................24
2.1.4 AFM IMAGING AND FORCEMAPPING.......................................................................................25
2.1.5 FLUORESCENCE IMAGING ........................................................................................................26
viii
2.1.6 BATCH ANALYSIS OF THE FORCE CURVES....................................................................................26
2.2 REFERENCES.......................................................................................................................................30
3 DIRECT CORRELATION OF STRUCTURES AND NANOMECHANICAL PROPERTIES OFMULTICOMPONENT LIPIDBILAYERS ........................................................................................................................................ 31
3.1 OVERVIEW.........................................................................................................................................31
3.2 SETPOINT DEPENDENT AFM IMAGING OFMULTICOMPONENT LIPIDMIXTURE..............................................32
3.3 FORCEMAPPING ON DEC BILAYERS.......................................................................................................34
3.4 FORCEMAPPING ON DEC CERAMIDE ....................................................................................................38
3.5 COMPARISON OF BREAKTHROUGH FORCES: DEC VS DEC CERAMIDE ..........................................................39
3.6 COMPARISON OF YOUNG�’S MODULUS: DEC VS DEC CERAMIDE ................................................................40
3.7 CERAMIDE INCREASE THEMECHANICAL STABILITY IN DEC CERAMIDE BILAYER .............................................41
3.8 HETEROGENEITY IN DISTINCT PHASES OBSERVED AT THE NANOSCALE LEVEL THROUGH FORCEMAPPING ..........43
3.9 CONCLUSION......................................................................................................................................45
3.10 REFERENCES.......................................................................................................................................46
4 QUANTIFICATION OF THE NANOMECHANICAL STABILITY OF CERAMIDE ENRICHED DOMAINS ...................... 47
4.1 OVERVIEW.........................................................................................................................................47
4.2 CERAMIDE NOT A TYPICAL GEL PHASE ....................................................................................................48
4.3 INCUBATION WITH METHYL CYCLODEXTRIN (M CD) AND CHLOROFORM ..................................................49
4.4 FORCEMAPPING ONM CD AND CHLOROFORM TREATED DEC CERAMIDE .................................................55
4.5 CONCLUSION......................................................................................................................................58
4.6 REFERENCES.......................................................................................................................................59
5 CHOLESTEROL DEPENDENT NANOMECHANICAL STABILITY OF PHASE SEGREGATEDMULTICOMPONENT
LIPID BILAYERS ................................................................................................................................ 60
5.1 OVERVIEW.........................................................................................................................................60
5.2 CHOLESTEROL.....................................................................................................................................61
5.3 MODELS FOR RUPTURE OFMOLECULARLY THIN FILMS..............................................................................62
5.4 RUPTURE ACTIVATION ENERGY CALCULATION..........................................................................................63
ix
5.5 BILAYER MORPHOLOGY AS A FUNCTION OF CHOLESTEROL CONCENTRATION ..................................................64
5.6 LOADING RATE DEPENDENCE OF THE BREAKTHROUGH FORCE......................................................................70
5.7 CHOLESTEROL DEPENDENCE OF BREAKTHROUGH FORCES...........................................................................73
5.8 RUPTURE ACTIVATION ENERGY OF DOPC/SM/CHOL AT VARYING CHOL CONCENTRATIONS.............................75
5.9 DISCUSSION .......................................................................................................................................79
5.10 CONCLUSION......................................................................................................................................82
5.11 REFERENCES.......................................................................................................................................83
6 NANOMECHANICS OF LIPID POLYMER/PEPTIDE INTERACTIONS .............................................................. 86
6.1 OVERVIEW.........................................................................................................................................86
6.2 INTRODUCTION...................................................................................................................................86
6.3 PREPARATION OF LIPID POLYMER/PEPTIDE SYSTEMS................................................................................88
6.4 RESULTS AND DISCUSSION....................................................................................................................89
6.4.1 LIPID POLYMERMORPHOLOGY ................................................................................................89
6.4.2 NANOMECHANICAL STABILITY OF THE LIPID POLYMER SYSTEMS ....................................................91
6.4.3 PROPOSEDMECHANISM FOR THE OBSERVED ENHANCEDMECHANICAL STABILITY ............................92
6.4.4 LIPID PEPTIDE SYSTEM............................................................................................................94
6.4.5 CONTROL PS(3.6) PEO(25) ...................................................................................................96
6.4.6 IMPLICATIONS .......................................................................................................................96
6.4.7 CURRENT AND FUTUREWORK .................................................................................................97
6.5 REFERENCES.......................................................................................................................................98
7 NANOSCALE STRUCTURES ANDMECHANICS OF BARNACLE CEMENT .......................................................100
7.1 OVERVIEW.......................................................................................................................................100
7.2 INTRODUCTION.................................................................................................................................101
7.2.1 BIOFOULING........................................................................................................................101
7.2.2 BARNACLE REMOVAL............................................................................................................101
7.2.3 BARNACLE CEMENT ..............................................................................................................102
x
7.3 MATERIALS AND METHODS .................................................................................................................103
7.3.1 BARNACLE REARING..............................................................................................................103
7.3.2 AFM IMAGING, FORCE SPECTROSCOPY, AND INDENTATION.........................................................104
7.3.3 SCANNING ELECTRON MICROSCOPY ENERGY DISPERSIVE X RAY (SEM EDX) ..................................105
7.3.4 FTIR SPECTROSCOPY ............................................................................................................105
7.3.5 CHEMICAL STAINING WITH AMYLOID SELECTIVE DYES.................................................................106
7.4 RESULTS ..........................................................................................................................................107
7.4.1 AFM IMAGING AND FORCE SPECTROSCOPY..............................................................................107
7.4.2 ELASTIC MODULI OF THE CEMENT............................................................................................111
7.4.3 SEMWITH EDX ..................................................................................................................113
7.4.4 FOURIER TRANSFORM INFRARED SPECTROSCOPY .......................................................................114
7.4.5 CHEMICAL STAINING WITH AMYLOID SELECTIVE DYES.................................................................116
7.5 DISCUSSION .....................................................................................................................................117
7.5.1 ADHESIVITY OF THE BARNACLE CEMENT ...................................................................................117
7.6 CONCLUSION....................................................................................................................................123
7.7 REFERENCES.....................................................................................................................................124
8 CONCLUSIONS AND RECOMMENDATIONS ...........................................................................................127
8.1 MULTICOMPONENT LIPID BILAYERS......................................................................................................127
8.2 BARNACLE PROTEINS .........................................................................................................................130
8.3 REFERENCES.....................................................................................................................................131
9 APPENDIX......................................................................................................................................132
9.1 RUPTURE ACTIVATION ENERGY CALCULATION........................................................................................132
9.2 REFERENCES.....................................................................................................................................134
xi
List of Tables
TABLE 5.1 RUPTURE ACTIVATION ENERGIES OF THE COEXISTING PHASES IN DOPC/SM (1:1) WITH 10 40% CHOLESTEROL
BILAYERS. 78
TABLE 7.1 IR PEAKS AND THE CORRESPONDING FRACTION OF THE OBSERVED SECONDARY STRUCTURES FOUND IN A GUMMY
BARNACLE CEMENT SAMPLE. 115
xii
List of Acronyms
2D TWO DIMENSIONAL
AFM ATOMIC FORCE MICROSCOPE
CHOL CHOLESTEROL
DEC DIOLEOYLPHOSPHATIDYLCHOLINE/EGG SPHINGOMYELIN/CHOLESTEROL
DEC CER DEC CERAMIDE
DPPC 1,2 DIPALMITOYL SN GLYCERO 3 PHOSPHOCHOLINE
DOPC 1,2 DIOLEOYL SN GLYCERO 3 PHOSPHOCHOLINE
DOPS DIOLEOYLPHOSPHATIDYLSERINE
DOTAP DIOLEOYLOXYPROPYL TRIMETHYLAMMONIUM CHLORIDE
EDX ENERGY DISPERSIVE X RAY
ESM EGG SPHINGOMYELIN
FTIR FOURIER TRANSFORM INFRARED
M CD METHYL BETA CYCLODEXTRIN
PC PHOSPHATIDYLCHOLINE
SEM SCANNING ELECTRON MICROSCOPY
SM SPHINGOMYELIN
TR DHPE TEXAS RED® 1,2 DIHEXADECANOYL SN GLYCERO 3 PHOSPHOETHANOLAMINE
TRIETHYLAMMONIUM SALT
xiii
List of Figures
FIGURE 1.1 PRINCIPLE OF ATOMIC FORCEMICROSCOPY (AFM)................................................................................2
FIGURE 1.2 SEM IMAGES OF MICROFABRICATED AFM CANTILEVERS AND TIPS. ...........................................................5
FIGURE 1.3 TYPICAL FORCE CURVES AND SCHEMATIC ILLUSTRATION OF A BREAKTHROUGH EVENT OF A SUPPORTED LIPID
BILAYER INDENTED BY AN AFM TIP. ..............................................................................................................7
FIGURE 1.4 AFM INDENTATION AND PULLING EXPERIMENTS....................................................................................8
FIGURE 1.5 FORCE EXTENSION CURVE UPON STRETCHING OF A SINGLE IMMUNOGLOBULIN (IG8) TITIN FRAGMENT
SHOWING A CHARACTERISTIC SAWTOOTH PATTERN. ......................................................................................10
FIGURE 1.6 SCHEMATIC ILLUSTRATION OF SUPPORTED PHASE SEGREGATED DOPC/SPHINGOMYELIN/CHOLESTEROL (DEC)
LIPID BILAYERS AND CONSTITUENT LIPID COMPONENTS. .................................................................................13
FIGURE 2.1 FORCE CURVES ILLUSTRATING THE BREAKTHROUGH FORCE, INDENTATION, AND ADHESION FORCE.................28
FIGURE 3.1 AFM IMAGES AND HEIGHT PROFILES OF A DEC CERAMIDE BILAYER AT DIFFERENT IMAGING SETPOINTS.........33
FIGURE 3.2 AFM HEIGHT IMAGE AND ADHESION FORCE MAP OF A DEC BILAYER........................................................35
FIGURE 3.3 BREAKTHROUGH FORCE, BREAKTHROUGH CONTOUR REPRESENTATION, AND THE YOUNG�’S MODULUS MAP....37
FIGURE 3.4 AFM HEIGHT IMAGES OF A DEC BILAYER BEFORE AND AFTER FORCE MAPPING AND THE CORRESPONDING
BREAKTHROUGH FORCE AND ADHESION MAPS. .............................................................................................37
FIGURE 3.5 AFM HEIGHT IMAGE AND FORCE MAPS OF A DEC CERAMIDE BILAYER. ....................................................38
FIGURE 3.6HISTOGRAMS OF THE BREAKTHROUGH FORCES OF A DEC AND OF A DEC CERAMIDE BILAYER. .....................39
FIGURE 3.7HISTOGRAMS OF THE ELASTIC MODULUS OF THE INDIVIDUAL PHASES IN A DEC AND A DEC CERAMIDE BILAYER.
............................................................................................................................................................40
FIGURE 3.8HISTOGRAM OF THE BREAKTHROUGH FORCES OF A DEC 111 LIPID BILAYER. .............................................42
FIGURE 3.9 INDENTATION VERSUS BREAKTHROUGH FORCE OF A DEC BILAYER............................................................44
FIGURE 4.1 AFM HEIGHT IMAGES BEFORE AND AFTER FORCE MAPPING, AND HISTOGRAM OF THE BREAKTHROUGH FORCES
OF A DPPC GEL PHASE IN A DOPC/DPPC LIPID BILAYER................................................................................48
FIGURE 4.2 AFM HEIGHT IMAGES OF A DEC CERAMIDE AND CHLOROFORM TREATED DEC CERAMIDE BILAYER AND THE
CORRESPONDING BREAKTHROUGH FORCE MAP. ............................................................................................50
FIGURE 4.3 AFM HEIGHT IMAGE OF DEC CERAMIDE BILAYER AND CORRESPONDING FORCE MAPS. ..............................51
FIGURE 4.4 AFM HEIGHT IMAGE AND CORRESPONDING MAPS OF DEC CERAMIDE BILAYER AFTERM CD TREATMENT. ..52
FIGURE 4.5HISTOGRAM OF THE BREAKTHROUGH FORCES AND ADHESION OFM CD TREATED DEC CERAMIDE BILAYERS.
............................................................................................................................................................53
FIGURE 4.6 AFM HEIGHT IMAGES OF DEC CERAMIDE BILAYERS WITHOUT ANDWITH 1 MMM CD TREATMENT. ..........54
xiv
FIGURE 4.7OPTICAL IMAGE OF DEC BILAYER AND AFM HEIGHT IMAGES WITHOUT ANDWITH 10 MMM CD...............54
FIGURE 4.8 AFM HEIGHT IMAGE AND CORRESPONDING FORCE MAPS OF DEC CERAMIDE BILAYER AFTER TREATMENTS WITH
1MMM CD AND CHLOROFORM VAPOR. ..................................................................................................56
FIGURE 4.9 TYPICAL FORCE CURVES FROM OUTSIDE AND WITHIN THE CERAMIDE ENRICHED DOMAINS. INSETS SHOW THE
HISTOGRAM OF BREAKTHROUGH FORCES AND PENETRATION DEPTHS................................................................57
FIGURE 5.1 AFM HEIGHT IMAGES OF SUPPORTED LIPID BILAYERS. ...........................................................................66
FIGURE 5.2 OPTICAL IMAGES OF DOPC/SM/CHOL BILAYERS AT 5 40 MOL% CHOL. .................................................67
FIGURE 5.3 AFM HEIGHT IMAGES OF DOPC/SM/CHOL BILAYER WITH 5 MOL% CHOLESTEROL...................................68
FIGURE 5.4 AFM HEIGHT IMAGES OF OF DOPC/SM BILAYERS AFTER FORCE MAPPING. ..............................................70
FIGURE 5.5 REPRESENTATIVE BREAKTHROUGH FORCE MAPS AND CORRESPONDING HISTOGRAMS OF DOPC/SM/CHOL
BILAYERS AT A SERIES OF LOADING RATES. ...................................................................................................72
FIGURE 5.6 CORRELATED AFM HEIGHT IMAGES AND BREAKTHROUGH FORCE MAPS OF DOPC/SM/CHOL BILAYERS WITH
20% CHOL AT 2000 NM/S, 800 NM/S, AND 200 NM/S LOADING RATE. .........................................................73
FIGURE 5.7 BREAKTHROUGH FORCES OF DOPC/SM/CHOL BILAYERS WITH 10 40% CHOL AT 200 NM/S......................74
FIGURE 5.8 LOADING RATE DEPENDENCE OF THE BREAKTHROUGH FORCES AT DIFFERENT CHOLESTEROL CONCENTRATIONS.
............................................................................................................................................................76
FIGURE 5.9 RUPTURE ACTIVATION ENERGIES OF THE COEXISTING PHASES (LO AND LD) IN DOPC/SM/CHOL BILAYERS WITH
10 40% CHOLESTEROL. ...........................................................................................................................77
FIGURE 6.1 AFM HEIGHT IMAGES OF PURE DEC, AND DEC WITH 0.05 AND 2 MOL% OF PS(3.6) B PEO(25). .............90
FIGURE 6.2 BREAKTHROUGH FORCEMAP AND CORRESPONDING HISTOGRAM OF A DOPC/ESM/CHOL BILAYER. ...........91
FIGURE 6.3 BREAKTHROUGH FORCEMAP AND CORRESPONDING HISTOGRAM OF A DOPC/ESM/CHOL BILAYER WITH 0.05
MOL% PS(3.6) B PEO(25)......................................................................................................................92
FIGURE 6.4 BREAKTHROUGH FORCEMAP AND CORRESPONDING HISTOGRAM OF A DOPC/ESM/CHOL BILAYER WITH 0.05
MOL% PS(19) B PEO(6.4)......................................................................................................................92
FIGURE 6.5 BREAKTHROUGH FORCEMAP AND CORRESPONDING HISTOGRAM OF A DOPC/ESM/CHOL BILAYER WITH 2
MOL% 26 AA ZN BINDING PEPTIDE. ...........................................................................................................95
FIGURE 6.6 AFM DEFLECTION IMAGE OF CONTROL PS(3.6) B PEO(25). .................................................................96
FIGURE 7.1 AFM TOPOGRAPHIC IMAGES OF THE BARNACLE CEMENT IN 35 PPT SEA WATER AND IN AIR........................107
FIGURE 7.2 DIFFERENT MORPHOLOGIES COMPRISING THE BARNACLE CEMENT. .......................................................108
FIGURE 7.3 FORCE EXTENSION PROFILES OBTAINED WHEN PULLING ON THE BULK CEMENT OF AMPHIBALANUS AMPHITRITE
(=BALANUS AMPHITRITE)........................................................................................................................108
FIGURE 7.4 SINGLE MOLECULE PULLING ON THE CEMENT SAMPLE. ........................................................................110
FIGURE 7.5 IDENTIFICATION OF SELF ASSEMBLY PROPERTY OF THE BARNACLE CEMENT. .............................................111
xv
FIGURE 7.6 IN SITU DETERMINATION OF THE ELASTIC MODULUS OF THE BARNACLE CEMENT. .....................................112
FIGURE 7.7 SEM IMAGES AND EDX SPECTRA OF THE BARNACLE CEMENT RESETTLED ON ALUMINUM FOIL. ..................114
FIGURE 7.8 FTIR SPECTRA OF THE BULK CEMENT FROM AMPHIBALANUS AMPHITRITE. ..............................................115
FIGURE 7.9 CHEMICAL STAINING IMAGES OF THE BARNACLE CEMENT WITH AMYLOID SELECTIVE DYES..........................116
FIGURE. 7.10NOVEL MECHANISM FOR PROVIDING A STRONG, ADAPTABLE GLUE. ....................................................119
List of Equations
F kx EQUATION 1.1 ..............................................................................................................................8
x(F) L cothFlK
kBTkBTFlK
EQUATION 1.2 ..........................................................................................11
F (x)kBTlP
14
1xL
2 xL
14
EQUATION 1.3 .................................................................................11
F 2E tan(1 2)
2EQUATION 2.1 ..............................................................................................................29
F 4E R3(1 2)
3 / 2EQUATION 2.2............................................................................................................29
Ea (F0) kBT ln0.693k
AdvdF0
EQUATION 5.1 ...................................................................................63
F0 a b ln v EQUATION 5.2 .................................................................................................................64
Abk
bFaTkFE Ba
60.1ln30.2)( 00 EQUATION 5.3....................................................................64
Gh giAii
EQUATION 5.4..................................................................................................................78
RF aN 3 / 5 EQUATION 6.1.......................................................................................................................93
2/32 )1(3
4vREF EQUATION 7.1 ..........................................................................................................111
dN k r Ndt EQUATION 9.1 ................................................................................................................132
dP kr(t)Pdt EQUATION 9.2..............................................................................................................132
lnP(t) kr(t )dt 0
tEQUATION 9.3 ....................................................................................................132
TkFE
rB
a
Aetk)(
)( EQUATION 9.4...........................................................................................................133
F=KVT EQUATION 9.5 ...............................................................................................................................133
lnP(F)Akv
eEa (F )kBT dF
0
FEQUATION 9.6 .....................................................................................133
vA
0.693ke
Ea (F )kBT dF
0
F0EQUATION 9.7 ..........................................................................................133
xvi
Ea (F0) kBT ln0.693k
AdvdF0
EQUATION 9.8 .................................................................................133
F0 a b ln v EQUATION 9.9 ................................................................................................................134
Abk
bFaTkFE Ba
60.1ln30.2)( 00 EQUATION 9.10 ................................................................134
xvii
xviii
List of Appendices
9.1 RUPTURE ACTIVATION ENERGY CALCULATION ..................................................................................132
1
1 Nanomechanics of Model and Biological Systems Studiedthrough Atomic Force Microscopy (AFM): An Introduction
Hailed as a versatile tool in biophysics, atomic force microscopy or AFM has been widely and
effectively used to shed light to a broad range of biophysical problems and systems.1 Since
AFM�’s invention in 1986, it has evolved from primarily an imaging tool to a technique capable
of examining mechanical, chemical, and functional properties of molecules and
macromolecules in great detail.2 5 Furthermore, the development of tip functionalization,
surface modification, and molecular manipulation procedures,6,7 as well as the emergence of
multimodal imaging methods such as AFM coupled with confocal, Total Internal Reflection
Fluorescence (TIRF), or Attenuated Total Reflectance (ATR) FTIR among others have led to
breakthroughs utilizing AFM in addressing a wide range of biological problems and continues to
bring great advances in the field of life science, materials science, and nanotechnology.8 14
AFM�’s high resolution in both imaging (~0.1 nm vertical, several nm�’s lateral) and force
spectroscopy (pN range) in liquid environment makes it successful in probing not only
structures, but also nanomechanics of biological samples in solution. Owing to its refined
technology and relative ease of usage,15 it has become a common tool to study DNAs,16,17
proteins, DNA protein interactions,18 20 lipid bilayers,21 23 and cells24,25. This thesis utilizes the
atomic force microscope to study structures and nanomechanics of multicomponent lipid
bilayers26 28 and proteins of the barnacle adhesive.29 Two experiments with AFM, namely
indentation and single molecule pulling, were primarily used.
2
1.1 Principles of Atomic Force Microscopy
Figure 1.1 Principle of Atomic Force Microscopy (AFM).30 A laser source is shone onto the back of acantilever with a nanoscale tip attached to a sensitive piezo scanner. Depending on the interactions withthe sample surface and the tip, the cantilever deflects whose signal is processed by an optical leverdetection system. The vertical movement of the scanner, together with its x, y position, then constitutesthe sample�’s topography. Figure 1.1 is adapted from reference 30.
Figure 1.1 is a schematic of how an AFM operates.30 An atomically sharp tip attached to the
cantilever is scanned in a regular pattern over a sample surface. Local attractive or repulsive
forces existing between the tip and the sample cause the cantilever to bend. The cantilever
bending or deflection monitored by a position sensitive photodetector, is converted to an
electrical signal. This is then translated into a topographic image. The forces acting between the
sample surface and cantilever tip can be due to steric repulsion, van der Waals, electrostatic,
magnetic, as well as specific interactions (e.g. ligand receptor binding such as antigen antibody
recognition events, base pairing of DNA duplex, and enzymatic reactions).31 These interactions
are reflected in the deflection of the cantilever, whose signal is processed by an optical lever
detection system.32,33 The optical lever operates by reflecting a laser beam off the cantilever to
a four segment photodetector. The difference between four photodiode signals indicates the
3
position of the laser spot on the detector. Because the cantilever to detector distance is several
times longer than the length of the cantilever, the optical lever greatly magnifies (~2000 fold)
the motions of the tip.32 34
To regulate the force on the sample, AFM uses a feedback mechanism. The feedback loop
consists of the scanner, the cantilever, optical lever detection scheme, and a feedback circuit
that keeps the cantilever deflection constant by adjusting the voltage applied to the scanner.34
The tube scanner, as depicted in Fig. 1.1 is usually made up of piezoelectric ceramics that
expand or contract in proportion to the applied voltage. Piezoelectric materials as such, are
used to precisely control sample tip movement to obtain sub Angstrom accuracy while
scanning a sample. The piezoelectric scanner together with optical detection system give AFM
Angström resolution in the z direction and subnanometer resolution in the x, y direction.6,35 The
feedback loop is what renders AFM more sophisticated than other stylus based instruments; it
does not only measure the force on the sample but also regulates it, making it capable of
acquiring images at very low forces. The feedback mechanism depends on the imaging mode
used.
The feedback mechanism, which could either be a constant force, constant height, or constant
oscillation amplitude, is determined by the AFM imaging mode used. Different imaging modes,
choice of which normally depends on the sample being imaged, are employed to monitor the
force between the AFM tip and the sample. Relevant to the experiments that made up this
body of work are the contact mode and intermittent contact or tapping mode. In contact mode,
the applied force measured by the cantilever deflection, is kept constant by the feedback
system as the probe scans the sample. Thus, depending on the surface topography, the piezo
height is adjusted accordingly to maintain such constant deflection or setpoint. The sample
however, is subjected to more drag in the lateral direction, which renders the contact mode not
suitable for soft biological samples and samples loosely bound to the surface. On the other
hand, in intermittent contact, the cantilever is oscillated at near its resonant frequency and the
4
oscillation amplitude is monitored and used as the feedback signal. Features on the sample
surface leads to damping of the oscillation amplitude. Since there is minimal contact between
the tip and the sample in this imaging mode, the dragging forces are greatly reduced.
AFM utilizes a flexible cantilever that deflects as a consequence of the interaction between the
sample surface and the tip. Since the imaging process greatly depends on these interactions,
cantilevers or the probe tip is considered to be a crucial part of an AFM. Different types of
cantilevers should be chosen for different imaging modes, silicon nitride and silicon for contact
and intermittent contact (tapping) mode, respectively. Figure 1.2 shows SEM images of
micromachined silicon nitride (A) and silicon (C) tips attached on the cantilever and the
corresponding magnified image, (B) and (D), respectively. Generally, soft cantilevers with high
resonant frequency to withstand vibrational instabilities are suited for contact mode imaging of
soft samples. For intermittent contact, stiffer probes with larger spring constants and resonant
frequencies should be used.
5
Figure 1.2 SEM images of microfabricated AFM cantilevers and tips. Silicon nitride (A) and Silicon (C)cantilevers and the corresponding high magnification images for A (B) and C (D), respectively. Figure isadapted from reference 1.
1.2 Study of Nanomechanical Properties through AFM
AFM is also very sensitive to minute forces. Thus, in addition to providing the sample
topography, the ability of AFM to measure intra and inter molecular interactions contained in
force distance or force curves, enables one to determine the sample elasticity, hardness,
adhesion, surface charge densities, and other local material properties.15,31,36,37 Before one can
fully appreciate these local mechanical properties that can be obtained from AFM
measurements, it is necessary to understand the nature of a force curve.
This figure was reproduced with permission from Alessandrini, A. and Facci, P.Measurement Science andTechnology 2005, 16, R65 R92. (© 2005 IOS Publishing).
6
1.2.1 Force Curve
Force curves consist of two parts�— the approach, extend, or trace portion (solid line in Fig. 1.3)
and the retract portion (dashed line in Fig. 1.3). Different information can be obtained from
each region of a force curve. Fig. 1.3 illustrates the physical events associated with different
regions of typical force curves when the tip is indenting into a lipid bilayer. Though this force
curve is specific to a lipid bilayer, it nevertheless illustrates different aspects of a force curve
and what information can be obtained from such a plot. The non contact region (Fig. 1.3 O A)
before the contact point (Fig. 1.3 A) indicates very little to no interaction between the tip and
the bilayer, which results in a relatively constant force on the plot. As the AFM tip continues to
approach and indent into the bilayer, a repulsive region (Fig. 1.3 A B) then follows. This region
of increasing force is attributed to a combination of steric and hydration forces as well as
effective surface charges arising from the bilayer water interface.38 At a certain force threshold,
when the bilayer can no longer withstand the applied load, a sudden jump to contact or
breakthrough occurs (Fig. 1.3 B C). The magnitude of these breakthrough events is used as a
measure of the bilayer�’s mechanical stability.28,39 41
7
Figure 1.3 Typical force curves and schematic illustration of a breakthrough event of a supported lipidbilayer indented by an AFM tip. In the non contact region (O A), the tip is far from the surface and theforce remains constant. At the contact point (A), the tip starts to contact the lipid surface and continuesto indent into it (A B), followed by a sudden breakthrough (B C) of the bilayer with sufficient load. Thetip reaches the supporting substrate (C) after which the cantilever itself begins to deflect (C D).
8
1.2.2 AFM Indentation and Single Molecule Pulling Experiment
The two types of experiments with AFM mainly reported in this thesis are indentation and
single molecule pulling, as shown in Fig. 1.4.
Figure 1.4 AFM indentation (A) and single molecule pulling (B) experiments.42 In AFM indentation (A), ananoscale AFM tip applies a compressive load into the sample while in AFM single molecule pulling (B),the AFM tip stretches and relaxes a molecule chain or a bundle of it. The black dot in the insetrepresents the position of the tip as a function of force. From the force�–distance curves, we can extracta number of mechanical properties of the sample as well as details of the substructures comprising it.Figure 1.4 is adapted from reference 42.
In an indentation experiment (Fig. 1.4 A), a compressive load is applied to a nanoscale contact
area of the sample. Since the cantilever acts like a spring, the cantilever deflection is converted
to force using Hook�’s law given by this equation,
F kzF Equation 1.1
where zF is the deflection of the cantilever and k is the cantilever�’s spring constant. To obtain an
accurate measurement of force, tip calibration must be initially performed. To do this, the AFM
tip is usually pressed against a hard reference substrate (e.g. glass, mica, silicon) to determine
the cantilever sensitivity. This is then followed by a procedure to obtain the cantilever�’s spring
constant. In the experiments reported here, the thermal noise method of Hutter and
9
Bechhoefer was used to obtain the spring constant.43 Data in the form of force distance or
force curves are then analyzed for their mechanical properties such as stiffness, hardness,
adhesion, and viscoelasticity.31
AFM indentation experiments have been extensively used to study different systems ranging
from interface to materials to colloids to life sciences. Among the earlier work utilizing AFM
indentation to determine the elasticity of biological molecules include that of Radmacher et al.
(aggregates of lysozyme, single molecules of DNA, different organelles of human platelet),44 46
Weisenhorn et al. (cartilage and living cells),47 Vinckier et al. (microtubules),48 Noy et al. (DNA
duplexes),49 and several others succeeding these pioneering work. Furthermore, force curves
obtained from an AFM indentation experiment shed light on the understanding of van der
Waals, double layer and solvation forces in liquids. These forces in particular proved to be
useful in the understanding of the different interaction forces present in single and binary
mixtures of phospholipids.21,22,50 53
In AFM based single molecule pulling experiment (Fig. 1.4 B), the molecule is being stretched
and relaxed using the AFM tip. With this method, it is possible to study and control
conformations of molecules and to obtain information on the details of the substructures
comprising the sample, e.g. protein and polymer.54 59 Among the first experiments utilizing AFM
single molecule manipulation technique is that of the giant protein titin which contains a large
number of immunoglobulin (Ig) domains.58 When this protein is stretched using the AFM tip, a
sawtooth like force profile, as shown in Fig. 1.5, is observed which is attributed to the unfolding
of the individual Ig domains.
10
Figure 1.5 Force extension curve upon stretching of a single immunoglobulin (Ig8) titin fragmentshowing a characteristic sawtooth pattern. The seven peaks correspond to the unfolding of individualtitin Ig domains. Figure 1.5 is adapted from reference 58.
Great advances in the mechanical unfolding of proteins field have been made when protein
engineering is combined with AFM based single molecule pulling experiments. The strength of
interaction between a ligand and a corresponding receptor can also be measured with this
method.5,59 63 Lee et al. were first to show the strength of interaction between biotin and
streptavidin molecular pairs.5 In the most recent study utilizing AFM based single molecule
force spectroscopy, folding and unfolding of single calmodulin proteins in the presence of
calcium ions and target peptides were observed at equilibrium conditions, using a home built
low drift AFM.64 Results of the study show that calcium ions affect the folding kinetics of the
protein while the target peptides stabilize its folded structure.
In a single molecule pulling experiment, the two ways usually implemented to optimize the
grabbing of a single molecule are: 1) fly fishing mode, where the tip, after approaching the
surface without indenting, is retracted step by step until a binding occurrence is observed; and
This figure was reproduced with permission from Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M., Gaub, H.E.Science 1997, 276 (5315), 1109 1112. (© 1997 AAS Science).
11
2) manual disentanglement of individual chains by slowly pulling back the tip while observing
rupture of multiple bonds until only one filament is left.59 To ensure that a single molecule is
attached to the AFM tip, the force extension profiles are fitted with polymer models from
statistical mechanics. The two most commonly used models that describe polymer behavior are
the worm like chain (WLC) and the freely jointed chain (FJC), Eqn. 1.2 and Eqn. 1.3, respectively.
F (x)kBTlP
14
1xL
2 xL
14
Equation 1.2
x(F) L cothFlK
kBTkBTFlK
Equation 1.3
where x is the extension length, F is the pulling force, L is the contour length, lK is the Kuhn
length, lP is the persistence length, kB the Boltzmann constant, and T the temperature. The
contour length, L is given by L=nl
B
K where n is the number of polymer segments. In the FJC
model, monomers are treated as rigid rods of a fixed length lK, and their orientation is
completely independent of the orientations and positions of neighboring monomers while in
WLC, it is treated as a continuous curve, thus a worm like chain. From the fit, the parameters lK
and lP for FJC and WLC models, respectively, are obtained.37
The aforementioned examples demonstrate the power of AFM force spectroscopy in studying
the nanomechanics of biological samples. In this work, single molecule pulling and indentation
experiments were used to probe the mechanical properties of the barnacle proteins and of
multicomponent lipid bilayers.
12
1.3 Multicomponent Lipid Bilayers
Cell membrane defines the limiting boundary of eukaryotic cells from their external
environment, and plays a vital role in regulation of nutrients and metabolites as well as in signal
transduction.65 Lipids and proteins are the primary constituents of the plasma membrane. The
fluid mosaic model of Singer and Nicholson represents an early effort to depict the two
dimensional organization of proteins and lipids in the plasma membrane. It renders the plasma
membrane as a multicomponent environment of functional proteins distributed in a
homogeneous lipid bilayer.66 This model does not take into account any functional significance
to physical heterogeneities in the lipid organization as a consequence of thermal fluctuations
and non ideal mixing. However, in recent years, numerous studies have provided a much more
sophisticated picture of the organization of lipids and proteins in the plasma membrane.67 72 In
particular, the �“lipid raft�” hypothesis70 72 has elicited interest in the field of membrane structure
and assembly as well as in membrane biophysics.
Lipid rafts are lateral structural components of the plasma membrane enriched in cholesterol
and glycosphingolipids (sphingomyelin in particular), onto which specific proteins attach within
the bilayer.70 These functional platforms have been implicated in a number of important cell
functions such as protein sorting, signal transduction, transcytosis, potocytosis, as well as HIV 1
assembly and release.67,73,74 In exploring this hypothesis, lipid mixtures consisting of cholesterol
(Chol), phosphatidylcholine (PC), and sphingomyelin (SM) (Fig. 1.6 C E), have often been used to
mimic the rafts in cells. It is well established that this ternary mixture gives rise to coexisting
liquid ordered (Lo) domains (SM/Chol enriched) and fluid disordered (Ld) phase (PC enriched) in
the bilayer (Fig. 1.6 A).69,75 77 In this thesis, DOPC/SM/Chol bilayers will be referred to as DEC.
Another sphingolipid ceramide (Fig. 1.6 F), an important signaling molecule involved in a wide
range of cellular processes such as apoptosis, cell developmental processes, and bacterial
13
pathogenesis,78 80 is also investigated. Several recent studies have determined the effects of
ceramide incorporation on coexisting fluid phase and ordered domains in phase separated
binary and ternary lipid mixtures.81 87 Ceramide has been shown to increase membrane
heterogeneity, displace cholesterol from rafts, and gives rise to ceramide enriched domains, in
both model and cellular membranes.81,88 92 These ceramide enriched domains seems to
promote the assembly of smaller inactive rafts to larger signaling platforms.78,80 The generation
of ceramide enriched domains from the enzymatic hydrolysis of the SM phosphocholine
headgroups has also been reported.81,84 When ceramide is directly incorporated into DEC
bilayers (DEC Ceramide), a third phase (ceramide enriched domains) is generated, in addition
to the coexisting Lo and Ld phases in pure DEC bilayers (Fig. 1.6 B).
Figure 1.6 Schematic illustration of supported phase segregated DOPC/ sphingomyelin/ cholesterol(DEC) lipid bilayers (A), DEC with ceramide (DEC Ceramide) (B), and skeletal structures of cholesterol (C),DOPC (D), sphingomyelin (E), and ceramide (F).
14
1.3.1 AFM Imaging and Force Mapping on Lipid Bilayers
Knowledge of the structures as well as of the mechanical stability of physiologically relevant
lipid bilayers is important in understanding the functions of biological membranes. In the
literature, a number of studies have exploited the physical properties of two component lipid
bilayers but few data are available on multicomponent lipid mixtures of biological
relevance.21,22,93,94 Dufrene et al. for instance, have observed phase separation in mixed
distearoylphosphatidylethanolamine (DSPE) and dioleoylphosphatidylethanolamine (DOPE)
bilayers. Their AFM studies revelaed the different height, adhesion, friction, and information on
short range repulsive hydration/steric force observed in both phases.21 The same group went
further and used functionalized AFM tips to probe the interaction forces and topography of
mixed phospholipid and glycolipid bilayers.22 This particular work provided evidence for the
influence of the structure and mechanical properties of lipid bilayers on their interaction with
another lipid or biomolecule with a particular functionality or charge. The effects of ionic
strength on the adhesion between chemically modified AFM tips and lipid bilayers have also
been reported.95,96 The force required to puncture the dimyristoylphosphatidylcholine (DMPC)
bilayer has been shown to increase with increasing concentration of monovalent sodium ions
and a much higher force is needed when divalent magnesium ions are added to the
solution.96,97 The effect of temperature on the stability of bilayers have also been
investigated.38,95 For dipalmitoylphosphatidylcholine (DPPC) membrane, the force needed to
break through the bilayer decreases with increasing temperature and that DPPC exhibits similar
instability as fluid phase DOPC and DOTAP bilayers at elevated temperatures.38 By combining
force spectroscopy with temperature controlled AFM imaging, the temperature dependent
property of the force needed to puncture the lipid bilayer has also been demonstrated.95
Though the aforementioned studies have provided excellent insights on the biophysics of
membrane, an important component, cholesterol, is lacking in the bilayer systems studied to
date. Thus, the mechanical stability of the coexisting phases in a multicomponent lipid mixture
of biological relevance, and correlation of composition and structure with membrane
mechanical properties, have not yet been adequately addressed.
15
AFM based force mapping, where you simultaneously measure the morphology and interaction
forces has been utilized in this thesis. Force maps have an added advantage of directly
correlating the morphology to mechanical properties, making it a valuable AFM technique in
membrane research. Force mapping has been available for some time with applications in early
experiments to synaptic vesicles and polymer surfaces.98 101 In relation to membrane research,
previous efforts involved reconstruction of a sequence of force curves to create two
dimensional (2D) adhesion maps for bilayer domains.21,22,93 Although these studies have
provided valuable information on the physical properties of a binary lipid mixture,
nanomechanical properties of multicomponent lipid mixtures with biological relevance such as
PC/SM/Chol and PC/SM/Chol with ceramide is scarce. This paucity of data arises from the
lengthy analysis of an enormous number of force separation curves collected in high resolution
force mapping.15,94 Because of this, relevant issues such as the mechanical stability of different
phases in phase segregated multicomponent bilayers have yet to be resolved.
1.4 The Biofouling Problem: Barnacle as a Culprit
Barnacles are common marine fouling organisms that has been shown to comprise 60% of the
surface area of a heavily fouled ship.102,103 Removal of these barnacles remains a practical
challenge in marine ship hull husbandry.104,105 To date, the use of non toxic, non fouling
elastomeric silicone material coatings with low surface energy serves as good alternatives to
the now prohibited traditional paints and poisons that were previously used to remove the
biofoulants.106 However, this only provides temporary solution, as these alternative coatings
are not as robust and effective as the conventional paints. A greater understanding of the
adhesion process is therefore important in the search for more effective solutions to biofouling.
Our approach is to study the mechanical properties of the nanoscale structures comprising the
barnacle adhesive to better understand the mechanics of cement adhesion. In particular, AFM
single molecule manipulation and indentation experiments were performed to gain
fundamental insights on the toughness of the barnacle adhesive.
16
1.5 Overview of the Thesis
This thesis primarily used AFM indentation and single molecule pulling experiments to study
the nanomechanical properties of the proteins in the barnacle adhesive as well as the
coexisting phases in multicomponent lipid bilayers of biological importance. The chosen
systems stem from two different motivations: (1) In the case of lipid bilayers, the goal is to
provide a more robust platform to characterize the coexisting phases in multicomponent lipid
systems. The succeeding work, investigating the effect of ceramide, of cholesterol, and of
added polymer and peptide, were mostly built from this framework; (2) For the barnacle
proteins, studying the nanomechanics of the barnacle adhesive itself might shed light to the
adhesion mechanism (i.e. how the macro and nano scale assembly of the protein constituents
resulted to a highly adhesive material). The study might also provide insights to the
development of alternative antifouling coatings.
Chapter 2 of this thesis lays down the experimental techniques in the study of the planar
supported lipid bilayers. A detailed description of the methodology is presented: lipid film to
bilayer preparation, AFM imaging and force mapping, and analysis of the data. Chapter 3
demonstrates the effectiveness of AFM based force mapping in directly correlating the self
organized structures exhibited in phase segregated supported lipid bilayers consisting of
dioleoylphosphatidylcholine/egg sphingomyelin/cholesterol (DEC) in the absence and presence
of ceramide (DEC Cer) with their breakthrough force, elastic modulus, adhesion, and
penetration depths. AFM based force mapping is further applied to quantify the mechanical
stability of the highly rigid and extremely stable ceramide enriched domains,81,107 as presented
in chapter 4. Here, an experiment was designed that enabled probing the ceramide enriched
regions in DEC Cer bilayers. Chapter 5 is a systematic study on the influence of different levels
of cholesterol in the lateral organization and mechanical stability of DEC bilayers. In this
chapter, force mapping at a series of tip velocities was performed. In addition, breakthrough
activation energies were calculated using the model for rupture of molecular thin films.50,108,109
17
Chapter 6 continues the exploration of multicomponent lipid bilayers. Polymers and peptides
are incorporated to the bilayer system. This chapter presents how the presence of the
polystyrene b polyethylene oxide (PS b PEO) diblock copolymer of varying lengths and
concentrations as well as of a helical zinc binding peptide affects the mechanical stability of
DEC bilayers. Based on the results, two possible mechanisms that could explain the observed
enhanced stability in the presence of the polymer and peptide were proposed.
Chapter 7 embodies the work on the natural biological system studied: proteins of the barnacle
adhesive. The first section of this chapter gives an overview of the biofouling problem, the
challenge with barnacle removal, and what has been known to date with the barnacle cement.
The succeeding section presents the different techniques used: AFM indentation and single
molecule pulling experiments, scanning electron microscopy (SEM) with energy dispersive x ray
(EDX), fourier transform infrared (FTIR) microscopy, and chemical staining with amyloid
selective dyes. This host of complementary techniques was used to better understand the
mechanics of cement adhesion and to study the nanomechanical properties of the barnacle
glue. The polymerized glue of the barnacle adhesive of Balanus amphitrite gave nanoscale
structures with robust mechanical properties (stiff nature), which could be a possible source of
the cement�’s strong adhesion on the molecular scale. The study also identified the presence of
a small fraction of amyloid like rod shaped structures that may provide specific insights on the
toughness of the barnacle cement. Results from the molecular pulling experiment revealed a
modular nature of the cement�’s matrix that contributes to an increase in the cement�’s
resistance to fracture.
Chapter 8 summarizes the body of work comprising this thesis: multicomponent lipid bilayers,
lipid bilayers with added polymer, and proteins of the barnacle adhesive. Recommendations for
future work are also mentioned.
18
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23
2 Multicomponent Lipid Bilayers: Experimental Methods
2.1 Materials and Methods
2.1.1 Materials
All lipids: 1,2 dioleoyl sn glycero 3 phosphocholine (DOPC, 18:1), N stearoyl D erythro
sphingosylphosphorylcholine or sphingomyelin (SM, 18:0), egg sphingomyelin (ESM), ovine
wool cholesterol (Chol), N palmitoyl D erythro sphingosine (ceramide) and 1,2 dipalmitoyl sn
glycero 3 phosphocholine (DPPC) were purchased from Avanti Polar Lipids (Alabaster, AL) and
used as received. Texas Red® 1,2 dihexadecanoyl sn glycero 3 phosphoethanolamine
triethylammonium salt (TR DHPE) was from Invitrogen Canada (Burlington, ON). HPLC grade
chloroform (ACP Chemicals Inc., Montreal, QC), ACS grade methanol from Fisher Scientific
(Ottawa, ON), and Milli Q water, deionized to a resistivity of 18 M cm were used in all of the
experiments.
24
2.1.2 Preparation of Small Unilamellar Vesicles
Lipid mixtures were obtained by combining the appropriate molar ratios of the different lipid
components using chloroform and methanol as solvents: DOPC/ESM/Chol in a 2:2:1 molar ratio,
referred to as DEC, DOPC/ESM/Ceramide/Chol in a 4:3:1:2 molar ratio (25% of the ESM in DEC
was replaced with ceramide resulting to 10 mol% ceramide of the total lipid mixture) as DEC
Ceramide, and DOPC/SM (1:1) in a series of cholesterol concentrations (5 40 mol%). To bilayer
samples for fluorescence microscopy, 0.3 mol% TR DHPE was added to the lipid mixture. The
resulting solution was then exposed to a gentle stream of nitrogen and placed under vacuum
overnight to further remove the solvents. The lipid film was hydrated with 18 M cm Milli Q
H2O to a final lipid concentration of 0.5 mg/mL for the DEC and 1 mg/mL for the DEC Ceramide
prior to use. Small unilamellar vesicles were obtained by sonicating the lipid solution to clarity
(~ 20 30 min) using a bath sonicator (Cole Parmer, Montreal, QC). Vesicle solutions are used
within a week after sonication. In some instances, vesicle solutions are resonicated for 5 10
mins before use.
2.1.3 Preparation of the Bilayer
Vesicle fusion protocols for both DEC and DEC Ceramide bilayer preparation were followed.1,2
Vesicle solutions of DEC containing 25 g lipids and a final concentration of 10 mM CaCl2 were
deposited on freshly cleaved mica substrates (20�–30 m thick) glued (Norland Optical Adhesive
88, Norland Products, Inc., Cranbury, NJ, 08512) on glass cover slips affixed to a liquid cell. The
sample was incubated at 45 C for an hour, and slowly cooled to room temperature. Extensive
washing using ~150 mL with 18 M cm Milli Q H2O followed the incubation. For the DEC
Ceramide, vesicle solutions of 50 g lipids and a final concentration of 10 mM CaCl2 were
incubated for 30 minutes at room temperature. Similarly, washing with excess 18 M cm Milli
Q H2O followed the incubation. About 15 DEC and 10 DEC Ceramide samples were prepared
and examined in the experiments.
25
2.1.4 AFM Imaging and Force Mapping
All AFM images were obtained using the Nanowizard ® II BioAFM (JPK Instruments, Berlin,
Germany) mounted on an Olympus 1X81 inverted confocal microscope. Both contact and
intermittent contact modes were used. Silicon nitride cantilevers (DNP S, Veeco, CA) were used
in contact mode imaging and force mapping measurements unless stated otherwise. The
spring constant, typically in the range of 0.06 0.28 N/m, was determined by the thermal noise
method3 after the determination of the cantilever deflection sensitivity. Deflection sensitivity is
obtained by pressing the AFM tip against a hard reference glass substrate. Silicon nitride
cantilevers (TR08 35, Veeco, CA) were used in the intermittent contact mode experiments with
nominal spring constant of 0.57 N/m. All AFM imaging measurements were carried out on
mica on glass substrates fixed to a liquid cell, and the samples were kept hydrated at all times.
All AFM images were plane fit (1st order) using the JPK SPM Image Processing Software (JPK
Instruments, Berlin, Germany).
In force mapping, arrays of force distance curves were collected on bilayer samples with
selected grid sizes (e.g., 128 x 64). At the center of every pixel, the scanner performed a single
force spectroscopy experiment and acquired approaching and retracting force distance curves.
For DOPC/SM bilayers with 10 40% Chol, force mapping at a series of tip velocities was
performed. Two dimensional (2D) visual maps of the breakthrough forces were reconstructed
from 64 × 64 grids of 3 m × 3 m scan size. An applied load within the range of 8 20 nN was
used, unless stated otherwise. With the unmodified Si3N4 tips, one can reliably distinguish
between the DOPC rich phase and the (ESM/Chol) rich phase in topography, adhesion, and
other force based 2D maps and plots.
26
2.1.5 Fluorescence Imaging
Epifluorescence images of DOPC/SM/Chol bilayers with various Chol concentrations with 0.3%
Texas Red dye and of M CD treated DEC bilayers were taken using an Olympus 1X81 inverted
optical microscope equipped with a high resolution CCD camera (CoolSNAP, Photometrics, US),
a 100x/1.40 N.A. oil immersion objective (UPlanSApo, Olympus), and a TRITC WF filter set
(Chroma Technology). All measurements were carried out on mica on glass substrates fixed to
a liquid cell. The samples were kept hydrated at all times.
2.1.6 Batch Analysis of the Force Curves
In the analysis of the data, a batch analysis algorithm to rapidly process the large number of
force curves captured in high resolution force mapping measurements, i.e. 128 x 64 pixels
versus a previously achieved maximum of 32 x 32 pixels was developed. In addition to
conventional adhesion data, mechanical properties such as breakthrough force and elastic
modulus of different phases in the supported lipid bilayers consisting of
dioleoylphosphatidylcholine / egg sphingomyelin / cholesterol (DEC) in the absence and
presence of ceramide (DEC Ceramide) bilayers were extracted automatically. Two dimensional
visual maps reconstructed using this analysis code enable the correlation of the structures of
different phases in the bilayer to their nanomechanical properties. The mechanical response of
the different phases in DEC and DEC Ceramide bilayers was systematically determined using
AFM topography imaging and force mapping.
27
The collection of force curves (approximately 4000 to 8000 curves per set) comprising the force
map were batch analyzed using the self developed algorithm implemented in IGOR Pro 6
(Wavemetrics, Portland, OR). Individual force curves were read in as deflection force F (in N),
and piezo distance z (in m), along with (x,y) position. For each curve breakthrough force,
indentation, elastic moduli, adhesion, penetration depth (bilayer thickness) were calculated or
extracted. And, using the (x,y) positions, force maps, contour plots, and pair wise scatter plots
of these quantities were generated to ascertain any correlation of clusters of data with the
spatial distribution of the phases in the lipid bilayer. Following is a brief description of the
analysis algorithm.
The piezo distance z is calculated as: s=(z z0) �– (d d0)/c, where the location (z0,d0) defines the
contact point of the tip with the sample, and c is a minor correction factor that is ideally close
to unity, but can fluctuate for each force curve due to the slight change of deflection sensitivity
during force map collection.
Breakthrough Force. The breakthrough force is defined as the difference in force between the
contact point (A), and the entrance point (B) in the extension curve. To calculate breakthrough
force, indentation, and elastic moduli, it was necessary to locate the contact point (A), the
indentation region (AB), and the substrate (C) of the extension curve (Fig. 2.1).
The analysis script is written by James K. Li of the Walker�’s Lab.
28
Figure 2.1 Force curves indicating quantities extracted: breakthrough force, indentation, and adhesionforce.
An algorithm was utilized to automatically search for the breakthrough points. Briefly, the F(d)
curve was box smoothed and its first derivative was found. A segment of the non contact
region was identified as the baseline and its associated standard deviation was calculated. To
locate the breakthrough region, the first derivative graph was incrementally searched for a
sharp peak (arising from the abruptness of the B C breakthrough) that exceeded a pre defined
multiple of the standard deviation from the non contact baseline. The minimum of this peak
corresponds to point C, the breakthrough exit point. To find point B, the local maximum prior
to point C is searched for. The threshold multiplier can be adjusted, depending on the quality
of the entire batch of force curves, to be more strict or relaxed in searching out the
breakthrough region. If a certain force curve does not meet the threshold criteria, a
breakthrough region is not identified and the force curve is dropped from the analysis. This is
useful for dropping curves not exhibiting a breakthrough event, or those with multiple ruptures.
A contact point finding algorithm was also implemented to facilitate the batch analysis process.
The algorithm assumes that the breakthrough entrance has already been found, and searches
points only prior to point B. To speed up the analysis, only a portion of the curve prior to point
B is searched. Each point in the selected portion is evaluated as a candidate contact point with
29
the following algorithm: the candidate point is the junction of a piece wise function. Any points
before the candidate contact point is considered the non contact region, and is fit to a straight
line. Points beyond this point up to (B) belong to the indentation region and are also fit to a
line. Although a 3/2 power fit in this region may have been more accurate, use of a second
linear fit is much faster, and given the evaluation scheme to be described, was not deemed
necessary. The average mean square error of the proposed piecewise function and the
experimental data is calculated and the candidate point that provides the lowest mean square
error becomes the contact point to be used for subsequent calculations. To further prevent
false identification, an added criterion to ensure the candidate point is sufficiently close to the
y value of the non contact region was implemented.
Indentation. Indentation is the total distance between the contact point (A) and the onset of
breakthrough (B).
Elastic Modulus. The elastic modulus is a mechanical property of the lipid layer that can be
obtained by fitting the indentation region of the extension curve to the Sneddon model for a
semi infinite sample contacted by a conical or paraboloidal shaped tip. (Equations 2.1 and 2.2
respectively):
F 2E tan(1 2)
2
Equation 2.1
F 4E R3(1 2)
3 / 2
Equation 2.2
where E is Young�’s modulus, is Poisson�’s ratio, R is the tip radius, is half the semi vertical
angle of a conical tip, F is the load, and is indentation. In the calculations, a tip radius of 25
30
nm and a Poisson ratio of 0.5 were used. SEM images of the DNPS AFM tips (k ~ 0.25 N/m) were
obtained to affirm the tip radius used in the calculation.
Adhesion Force. Adhesion is the minimum value of the retract portion of the force curve
relative to the non contact region, that is, when the tip is lifted sufficiently far away from the
sample and experiences no tip sample force.
2.2 References
(1) Chiantia, S.; Kahya, N.; Ries, J.; Schwille, P. Biophys. J. 2006, 90, 4500.
(2) Ira; Johnston, L. J. Langmuir 2006, 22, 11284.
(3) Hutter, J. L.; Bechhoefer, J. Rev. Sci. Instrum. 1993, 64, 1868.
31
3 Direct Correlation of Structures and NanomechanicalProperties of Multicomponent Lipid Bilayers*
3.1 Overview
Exploring the fine structures and physicochemical properties of physiologically relevant
membranes is crucial in understanding biological membrane functions including membrane
mechanical stability. In this chapter, AFM imaging and high resolution force mapping is used to
directly correlate the self organized structures in phase segregated supported lipid bilayers
consisting of dioleoylphosphatidylcholine / egg sphingomyelin / cholesterol (DEC) in the
absence and presence of ceramide (DEC Ceramide) with their nanomechanical properties.
Direct incorporation of ceramide into phase segregated supported lipid bilayers formed
ceramide enriched domains, where the height topography was found to be imaging setpoint
dependent. In contrast, liquid ordered domains in both DEC and DEC Ceramide presented
similar heights regardless of AFM imaging settings. The intrinsic breakthrough forces, regarded
as fingerprints of bilayer stability, along with elastic moduli, adhesion forces, and indentation of
the different phases in the bilayers were systematically determined at the nanometer scale,
and the results were presented as two dimensional visual maps using a self developed code for
force curves batch analysis, as outlined in the previous chapter. The mechanical stability and
*This chapter was reproduced in part with permission from Sullan, R.M.A., Li, J.K., Zou, S. Langmuir, 25 (13), 7471
7477. © 2009 American Chemical Society.
32
compactness were increased in both liquid ordered domains and fluid disordered phases of
DEC Ceramide, attributed to the influence of ceramide in the organization of the bilayer, as well
as the displacement of cholesterol as a result of the generation of ceramide enriched domains.
The use of AFM force mapping in studying phase segregation of multicomponent lipid
membrane systems is a valuable complement to other biophysical techniques such as imaging
and spectroscopy, as it provides unprecedented insight into lipid membrane mechanical
properties and functions.
3.2 Setpoint Dependent AFM Imaging of Multicomponent LipidMixture
While high spatial resolution images can be obtained from the AFM, caution must be exercised
in their interpretation, as the topography generated can be a strong function of imaging
parameters. Figures 3.1 A and 3.1 B show AFM height images of the phase segregated DEC
Ceramide bilayer using contact mode at a low setpoint of F ~ 0.5 nN. Three distinct phases were
observed: (1) features with the shortest height are ascribed to the fluid disordered (DOPC rich)
phase; (2) the tallest height to the ceramide enriched domains or subdomains and; (3) the
intermediate height to the liquid ordered (ESM/Chol rich) domains or Lo phase, consistent with
previous studies.1,2 The subdomains that are 0.3 ± 0.1 nm higher than the Lo phase were
obtained when a low setpoint was applied to the DEC Ceramide lipid bilayer (Fig. 3.1 E).
However, with a larger imaging setpoint of F ~ 5.0 nN in contact mode, the difference in height
between the subdomains and the Lo phase increased to 0.6 ± 0.1nm (Fig. 3.1 F), and the Lo
phase are 0.8 ± 0.1 nm taller than the fluid disordered phase, similar to that of the 0.7 ± 0.1 nm
observed in the lower setpoint image (Fig. 3.1 A and 3.1 E). Applying an even smaller force in
the AFM topography imaging, the same area was re imaged using intermittent contact mode
(Fig. 3.1 D). The height profiles show the liquid ordered domains are 1.2 ± 0.1 nm above the
fluid disordered phase, and the subdomains 0.2 ± 0.1 nm higher than the Lo phase (Fig. 3.1 G).
33
Figure 3.1 AFM images and height profiles of a DEC Ceramide bilayer on mica in contact mode using alow setpoint of F ~ 0.5 nN (A, E) with corresponding lateral deflection image (B), and using a highsetpoint of F ~ 5.0 nN (C, F), and in the intermittent contact mode (D, G). Height profiles show that thehighest features (subdomains) are 0.3 ± 0.1, 0.6 ± 0.1, and 0.2 ± 0.1 nm higher than the intermediateliquid ordered domains, which are 0.7 ± 0.1, 0.8 ± 0.1, and 1.2 ± 0.1 nm above the lowest fluiddisordered phase, respectively. Regions outlined in circles in the height profiles indicate thesubdomains.
Comparison of the height profiles obtained in different AFM imaging modes shows that
different heights of ceramide enriched domains can be obtained as a consequence of varying
setpoints, suggesting a sensitive mechanical response of the subdomains in DEC Ceramide
bilayers. In contrast, the liquid ordered domains in DEC Ceramide exhibited similar heights
regardless of the imaging settings. These results indicate that in DEC Ceramide bilayers, one
cannot only rely on the AFM height image in understanding the behavior of the different
phases in the lipid mixture.3,4 In addition, the topography itself cannot sufficiently provide
accurate composition and structural information of the bilayer. Force mapping coupled with
AFM imaging is thus needed to systematically characterize the different coexisting phases in
multicomponent lipid bilayers, on the basis of their nanomechanical properties.
34
3.3 Force Mapping on DEC Bilayers
The major driving force that holds the amphiphilic molecules together in bilayers is not due to
strong covalent or ionic bonds but rather, arises from weaker Van der Waals, hydrophobic,
hydrogen bonding, and screened electrostatic interactions, and is greatly influenced by external
conditions.5,6 It is therefore necessary to first consider how an applied force and the presence
of certain molecules, i.e., cholesterol and ceramide, will influence the organization of the lipid
bilayer, and consequently its properties and functions.
In contrast to the significant height differences of ceramide enriched domains upon imaging
settings, the Lo phase in DEC Ceramide were consistently ~0.8 nm taller than the matrix of the
fluid disordered phases (Fig. 3.1 A, C), which is in good agreement with reported values in pure
DEC ternary mixtures.1,7,8 As an initial step in understanding how the addition of one more
component (i.e. ceramide) to the ternary lipid mixture alters the mechanical response of the
bilayer, force mapping was first performed on DEC bilayer without ceramide. In Fig. 2.2, a
typical force extension curve illustrating each characteristic feature of the force profile
corresponding to its measured quantities is shown.
35
Figure 3.2 AFM height image (A), corresponding height profile (B), typical force curves for liquid ordereddomains and fluid disordered phase, showing the expected height difference of ~ 0.8 nm (C), adhesionforce map (D) and corresponding histogram (E) of a DEC bilayer. Solid bars correspond to the liquidordered domains while the hollow bars to the fluid disordered phase.
The AFM height image of a DEC bilayer showing the coexistence of liquid ordered domains
(brighter regions) and fluid disordered phase (darker matrix) is shown in Fig. 3.2 A. The Lo phase
is 0.8 nm above the DOPC rich phase consistent with literature data (Fig. 3.2 B).1,7,8
Superimposing more than 200 curves recorded in both phases shows two distinct groups of
breakthrough events (Fig. 3.2 C). Measuring the distance at a given force (~ 1 nN) within the
indentation region yielded the expected height difference of ~ 0.8 nm, consistent with what has
been observed in the AFM height image (Fig. 3.2 A, B). The adhesion map and the
corresponding histogram of the same area are shown in Fig. 3.2 D and E, respectively. The
lighter regions represent areas with high adhesion and are ascribed to the liquid ordered
domains, while the darker regions are areas with low adhesion and correspond to fluid
disordered phase. The slight difference in the domain sizes is due to the different pixel densities
between an AFM topographical image (512 x 512) and the map constructed from force
separation curves (128 x 64) as well as to the significant difference in the applied force between
imaging and force mapping (i.e. AFM images are taken with less than a nN force while greater
36
than 8 nN is used when force maps are obtained). In general, adhesion force is related to the
contact area of the tip lipid interaction and depends on external forces and other
environmental conditions such as temperature, humidity, surface roughness, contaminant, and
total interfacial contact time.9 Hence, it is not an intrinsic property of a bilayer and should not
be used to discriminate mechanical properties of the different phases.
Breakthrough forces and Young�’s moduli, in contrast to adhesion, are two mechanical
quantities that can be used to reliably characterize the properties of each phase in a natural
multicomponent lipid mixture. A breakthrough force is the maximum force that the bilayer is
able to withstand before rupture.10,11 It has been shown to be an intrinsic property of a bilayer,
and can be regarded as a fingerprint of bilayer stability. The Young�’s modulus, on the other
hand, is a measure of the stiffness of a material,12,13 and provides information on the strength
of cohesive forces among the lipid components within a bilayer. Values of the Young�’s modulus
in this work were obtained by fitting the indentation region of the force curve (see Fig. 3.2 C) to
the Sneddon model for a semi infinite sample contacted by a paraboloidal shaped tip14 (Eqn.
2.2).
In order to quantitatively compare the mechanical properties of different phases in the DEC
bilayer, high resolution 2D visual maps of breakthrough forces and elastic moduli,
reconstructed from the analysis of 8192 force separation curves (128 x 64 array of force curves)
using the self developed code, are shown in Figs. 3.3 A and C, respectively.
37
Figure 3.3 Breakthrough force map (A), contour map representation of the breakthrough force (B), andthe Young�’s modulus map (C).
From these visual maps, the liquid ordered domains exhibit higher breakthrough forces, higher
Young�’s moduli and higher adhesion than the fluid disordered phase. In both phases,
heterogeneity can be resolved at the nanometer scale and is more apparent in the contour map
representation (Fig. 3.3 B). Here, the boundary between the coexisting phases is clearly
defined, and the interface has breakthrough forces intermediate to that of the two phases.
Comparison of the AFM height images of a DEC bilayer before (Fig. 3.4 A) and after (Fig. 3.4 D)
force mapping shows that the liquid ordered domains retained their general shapes and
relative positions from each other, indicating that no significant restructuring of the bilayer is
being induced by force mapping. The breakthrough force map (Fig. 3.4 B) and adhesion map
(Fig. 3.4 C) are included to show the state of the bilayer while force mapping is being
performed.
Figure 3.4 AFM height images of a DEC bilayer before (A) and after (D) force mapping and thecorresponding breakthrough force (B) and adhesion (C) maps.
38
3.4 Force Mapping on DEC Ceramide
Similar to DEC, high resolution force mapping measurements were carried out on the DEC
Ceramide bilayers. Fig. 3.5 A shows the adhesion force map of a DEC bilayer with ceramide. The
low adhesion regions (black areas) in this map correspond to locations of ceramide enriched
domains, as can be inferred from both AFM height (Fig. 3.5 B) and lateral deflection (Fig. 3.5 C)
images collected right after force mapping.
Figure 3.5 Adhesion force map (A), AFM height image after force mapping (B), the corresponding lateraldeflection image (C), breakthrough force map (D), contour representation of the breakthrough forces(E), and Young�’s modulus map (F) of a DEC Ceramide bilayer.
A typical breakthrough force map, its contour plot, and the Young�’s modulus map, constructed
from a total of 4096 force separation curves (64 x 64 array of force curves) were presented in
Figs. 3.5 D, E, and F, respectively. High breakthrough forces at the boundary and within the
vicinity of the subdomains were observed. Similar to DEC, heterogeneities in the phases are
visible in the force maps and contours. Force curves without breakthrough and adhesion events
(termed hereafter as a no profile curve) were consistently observed within the subdomains. As
will be discussed in greater detail in a subsequent section, the lack of breakthrough and
39
adhesion events does not mean zero values for these quantities but indicates a packing
behavior different from typical Lo phase. Hence, the AFM tip could not penetrate into these
regions.
3.5 Comparison of Breakthrough Forces: DEC vs DEC Ceramide
The histograms of breakthrough forces of the DEC and DEC Ceramide bilayers were constructed
and are respectively shown in Figs. 3.6 A and B. For the DEC bilayer, a bimodal distribution with
peaks at F ~1.4 nN, ascribed to the fluid disordered phase, and at F ~ 3.2 nN, corresponding to
the liquid ordered domains was obtained. Shifts to much higher breakthrough forces were
observed in DEC Ceramide bilayer. The histogram of the breakthrough forces in the rafts (solid
bars in Fig. 3.6 B) in DEC Ceramide shows a peak at F ~ 5 nN while the fluid disordered phase
(hollow bars in Fig. 3.6 B) at F ~ 4.1 nN. Breakthrough forces greater than 5 nN are from the
regions within the vicinity of the ceramide enriched domains (see Fig. 3.5 D). It should be noted
that the histogram of breakthrough forces for DEC Ceramide only accounts for the liquid
ordered domains and fluid disordered phases, as the subdomains exhibited no breakthrough
events. High resolution visual maps together with the histogram distribution of the intrinsic
breakthrough forces could directly correlate the fine structures of multicomponent lipid
mixtures with their mechanical stability.
Figure 3.6 Histograms of the breakthrough forces of a DEC (A) and of a DEC Ceramide (B) bilayer. Solidbars correspond to the liquid ordered domains while hollow bars to the fluid disordered phase.
40
3.6 Comparison of Young�’s Modulus: DEC vs DEC Ceramide
Fitting the indentation region in each of the extension curve (see Fig. 3.2 C and Fig. 2.2) with the
Sneddon model yielded the Young�’s modulus values, and the distribution of these values of the
individual phases for DEC bilayers were grouped and plotted in Fig. 3.7 A. The peak at ~ 80 MPa
corresponds to the fluid disordered phase (hollow bars), and the peak at ~ 140 MPa to the
liquid ordered domains (solid bars), indicating less elastic deformation or higher degree of
compactness in the latter. These values are in close agreement with what has been obtained
using electrocompression experiments.15,16 The two phases have overlapping elastic moduli in
the 50 130 MPa range, and the distribution is broader in liquid ordered domains. Both
observations point to the heterogeneity within the liquid ordered domains, which is not evident
in the height topography images (Fig. 3.2 A, 3.5 B). The histogram of Young�’s modulus values
measured on a DEC Ceramide (Fig. 3.7 B) shows peaks at ~ 185 MPa for the liquid ordered
domains and at ~ 175 MPa for the fluid disordered phase, with greater overlap between the
two phases. This indicates that heterogeneity and compactness are increased in both phases.
This result provides direct evidence that it is the high nanomechanical stability and
compactness of the rafts that accommodates proteins and other signaling molecules.
Figure 3.7 Histograms of the elastic modulus of the individual phases in a DEC bilayer (A) and a DECCeramide bilayer (B). The hollow bars correspond to the fluid disordered phase while the solid bars tothe liquid ordered domains.
41
3.7 Ceramide Increase the Mechanical Stability in DEC CeramideBilayer
Breakthrough forces increased from 1.4 nN (in Ld phase, DEC) to 4.1 nN (in Ld phase, DEC
Ceramide), and from 3.2 nN (in Lo phase, DEC) to 5.0 nN (in Lo phase, DEC Ceramide). The
increase of the breakthrough forces on the Lo phase can be explained by the presence of
ceramide in the domains�— ceramide has a strong affinity for H bonding (between the �–OH
groups and the amine moieties) with sphingomyelin and favored hydrophobic interactions
among the saturated alkyl chains of both lipids. In the case of the fluid disordered phase, the
increase in breakthrough forces can be attributed to two possible scenarios: (1) the saturation
of ceramide in the Ld phase and (2) the effect of displaced cholesterol as a result of ceramide
enriched domains generation. To assess the latter, force mapping was carried out on DEC111
(dioleoylphosphatidylcholine / egg sphingomyelin / cholesterol in a 1:1:1 molar ratio), a lipid
bilayer with 13% more cholesterol in composition than a DEC or DEC Ceramide. The effect of
13% more cholesterol in DEC 111 on the changes of breakthrough forces (if there are any) is
comparable to the effect of the 10 mol% cholesterol at the maximum (10 mol % is the total
ceramide content) that could be expelled by the formation of ceramide subdomains in DEC
Ceramide.
The histogram of the breakthrough forces in DEC111 (Fig. 3.8) has a bimodal distribution with
peaks at F ~ 1.4 nN and at F ~ 3.4 nN. It is not surprising that the breakthrough force peak of
the fluid disordered phase remains unchanged even at higher cholesterol concentration
because cholesterol preferentially packs in the ESM domains than in the fluid DOPC.17 19 The
increase of about 200 pN in the breakthrough force of the liquid ordered domains is within
margin of error so is not suggestive of cholesterol increasing the mechanical stability. It is more
likely therefore that the increase in mechanical stability in DEC Ceramide bilayers is due to the
presence of ceramide.
42
Figure 3.8 Histogram of the breakthrough forces of a DEC 111 lipid bilayer. Solid bars correspond to theliquid ordered domains while the hollow bars to the fluid disordered phase.
The 13% more cholesterol which led to the slightly higher breakthrough forces of the liquid
ordered domains in DEC111, however, could not account for the significant breakthrough force
increase in the liquid ordered domains (i.e., from 3.2 nN to 5.0 nN in DEC and DEC Ceramide,
respectively). This significant increase of breakthrough forces suggests that it cannot be
attributed to the displacement of cholesterol by the generation of ceramide enriched domains,
but primarily to the effects of ceramide itself on the bilayer.
It should be noted that the high forces (8 12 nN) observed at the boundary and within the
vicinity of the subdomains (Fig. 3.5 D), which is a strong indication that there is a high
localization of ceramide in those regions. The increase in breakthrough force values by 2.7 nN in
the fluid disordered phase, and by 1.8 nN in the liquid ordered domains suggested that the
enhanced stability of the bilayer is due to the presence of ceramide in both phases.
It is worth noting that in the ceramide enriched regions, no profile (no breakthrough no
adhesion) curves were observed. To ensure that this observation is not a consequence of
having �“no molecular contact between the probe and the lipid layer surface�”,3 or due to some
strong repulsive forces between the tip and the lipid surface, stiffer silicon cantilevers (k ~ 7
43
N/m) were used to probe those ceramide enriched regions. Consistent with the results using
the softer cantilevers (k < 0.25 N/m), no profile force curves were obtained even at loading
forces as high as 70 nN. This may provide an evidence to an earlier study which suggested that
the ceramide enriched region is a highly ordered and tightly packed gel phase, composed of
both ceramide and ESM.1
3.8 Heterogeneity in Distinct Phases Observed at the Nanoscale Levelthrough Force Mapping
In addition to how cholesterol and ceramide affect the mechanical properties of a bilayer, it is
worthwhile to examine the heterogeneity at the nanoscale level observed in the different
coexisting phases. The histograms of breakthrough forces and elasticity reflect this wide
distribution. This heterogeneity is more apparent in the visual maps and contour maps
presented. In particular, the contour map representation of the breakthrough forces clearly
defines the boundary between the liquid ordered domains and fluid disordered phases in the
DEC system (Fig. 3.3 B), and for the subdomains in the case of DEC Ceramide (Fig. 3.5 E). It is
known that the major force that governs the self assembly of amphiphiles into well defined
structures, such as micelles and bilayers is derived from the hydrophobic attraction at the
hydrocarbon water interface, which induces the molecule to associate. The hydrophilic,
electrostatic, or steric repulsion of the headgroups thus impose the opposite requirement and
remain in contact with water. Measuring distinct phase boundaries is an important practical
consideration, because reliable data interpretation of multicomponent lipids with different
compositions is needed in understanding the properties of a bilayer.20 AFM force mapping
employed in this work strongly provides such lateral heterogeneity and distinct phase boundary
information.
44
Figure 3.9 Scatter plot of indentation versus breakthrough force of a DEC bilayer.
In addition to the 2D visual maps, pair wise scatter plots of the measured mechanical
quantities, allocate the lipid organizations to those physicochemical properties. The scatter plot
in Fig. 3.9 shows two distinct clusters: higher indentation lower breakthrough force and lower
indentation higher breakthrough force regions. This scatter plot further confirms that the
higher indentation lower breakthrough force region can be correlated to the fluid disordered
phase while the lower indentation higher breakthrough force region to the liquid ordered
domains. The high resolution force mapping and pair wise scatter plots of the measured
nanomechanical quantities allow the direct determination of the breakthrough forces,
adhesion, and indentation of a lipid bilayer, as well as a straightforward correlation of those
properties to the bilayer structures.
45
3.9 Conclusion
High resolution force mapping coupled with AFM imaging was demonstrated to provide direct
correlation of the organization of multicomponent lipid mixtures to their nanomechanical
properties that were not previously achieved. The 2D visual maps of the intrinsic breakthrough
forces, elastic moduli, and adhesion generated from the self developed batch analysis code can
directly correlate the fine structures of multicomponent lipid mixtures with their mechanical
stability. In the ceramide incorporated bilayer system being studied, an increase in membrane
mechanical stability, which was attributed to the influence of ceramide in the lipid organization,
as well as the displacement of cholesterol as a result of the generation of ceramide enriched
domains, was observed. It was shown that ceramide enriched domains exhibit a different
packing behavior from well known gel phases, indicating a high mechanical rigidity. The results
in this chapter suggests that mechanical stability and compactness is the basis of the ceramide
induced formation of signaling platforms in cell membranes and hence, AFM force mapping is a
valuable complement to other biophysical techniques currently used in studying
multicomponent lipid bilayer mixtures. The results on the effects of direct incorporation of
ceramide into a ternary lipid mixture on the properties of the bilayer are closely related to
several recent studies that examined the influence of ceramide on membranes treated with
enzyme Sphingomyelinase.1,7,21 23 Sphingomyelin in cells undergoes hydrolysis through the
action of Sphingomyelinase which consequently generates ceramide. Studying the
nenomechanical stability of the model membrane with this enzymatic generation of ceramide is
a good comparison to the direct incorporation (of ceramide) study described in this chapter.
46
3.10 References
(1) Chiantia, S.; Kahya, N.; Ries, J.; Schwille, P. Biophys. J. 2006, 90, 4500.
(2) Ira; Johnston, L. J. Langmuir 2006, 22, 11284.
(3) Dufrene, Y. F.; Boland, T.; Schneider, J. W.; Barger, W. R.; Lee, G. U. FaradayDiscuss. 1998, 111, 79.
(4) Schneider, J.; Dufrene, Y. F.; Barger, W. R.; Lee, G. U. Biophys. J. 2000, 79, 1107.
(5) Garcia Manyes, S.; Oncins, G.; Sanz, F. Electrochim. Acta 2006, 51, 5029.
(6) Oncins, G.; Picas, L.; Hernandez Borrell, J.; Garcia Manyes, S.; Sanz, F. Biophys. J.2007, 93, 2713.
(7) Ira; Johnston, L. Biochim. Biophys. Acta, Biomembr. 2008, 1778, 185.
(8) Rinia, H. A.; de Kruijff, B. FEBS Lett. 2001, 504, 194.
(9) Israelachvili, J. Electrostatic Forces Between Surfaces in Liquid. Intermolecularand Surface Forces; Academic Press, 2002.
(10) Kunneke, S.; Kruger, D.; Janshoff, A. Biophys. J. 2004, 86, 1545.
(11) Schneider, J.; Barger, W.; Lee, G. U. Langmuir 2003, 19, 1899.
(12) Guo, S.; Akhremitchev, B. B. Langmuir 2008, 24, 880.
(13) Guo, S. L.; Akhremitchev, B. B. Biomacromolecules 2006, 7, 1630.
(14) Sneddon, I. Int. J. Eng. Sci. 1965, 3, 47.
(15) Cevc, G.; Marsh, D. Phospholipid Bilayers: Physical Principles and Models; JohnWiley and Sons: New York, 1987.
(16) Marsh, D. Handbook of Lipid Bilayers; CRC Press: Boca Raton, FL, 1987.
(17) Sankaram, M. B.; Thompson, T. E. Biochemistry 1990, 29, 10670.
(18) Simons, K.; Ikonen, E. Science 2000, 290, 1721.
(19) Slotte, J. P. Chem. Phys. Lipids 1999, 102, 13.
(20) Feigenson, G. W. Annu. Rev. Biophys. Biomol. Struct. 2007, 36, 63.
(21) Ira; Zou, S.; Carter, D.; Vanderlip, S.; Johnston, L. J. Struct. Biol. 2009, 168, 78.
(22) Lopez Montero, I.; Velez, M.; Devaux, P. F. Biochim. Biophys. Acta, Biomembr.2007, 1768, 553.
(23) Silva, L. C.; de Almeida, R. F. M.; Castro, B. M.; Fedorov, A.; Prieto, M. Biophys. J.2007, 92, 502.
B
47
4 Quantification of the Nanomechanical Stability ofCeramide enriched Domains�†
4.1 Overview
Quantification of the mechanical stability of lipid bilayers is important in establishing the
composition structure property relations, and shed light on understanding functions of
biological membranes. In this chapter, an experiment was designed to directly probe and
quantify the nanomechanical stability and rigidity of the extremely stable, tightly packed, and
somewhat impenetrable ceramide enriched domains,1 5 known to play an essential role as
signaling molecules in programmed cell death, as well as in a wide range of cellular processes.6
AFM based force mapping5,7 on the methyl cyclodextrin (M CD) and chloroform treated
ceramide enriched regions revealed an organization not typical of gel states, and supported the
long standing hypothesis that ceramide displaces cholesterol from sphingolipid/cholesterol
enriched domains of model membranes.8 The work reported in this chapter provides
quantitative information on the nanomechanical stability and rigidity of coexisting phase
segregated lipid bilayers with the presence of ceramide enriched platforms, indicating that
generation of ceramide in cells drastically alters the structural organization and the mechanical
property of biological membranes.
�† This chapter was reproduced in part with permission from Sullan, R.M.A., Li, J.K., Zou, S. Langmuir, 25 (22),12874 12877. © 2009 American Chemical Society.
48
4.2 Ceramide NOT a typical gel phase
As presented in the previous chapter, breakthrough and adhesion events were not observed in
the ceramide enriched domains of DEC Cer bilayers, which could signify the inability of the AFM
tip to penetrate into these regions. To compare the physical state of the ceramide enriched
domains with a well known gel phase, force mapping was applied to another phase separated
supported lipid bilayer consisting of DOPC and 1,2 dipalmitoyl sn glycero 3 phosphocholine
(DPPC) in a 1:1 molar ratio. DPPC is known to form an ordered gel phase in lipid bilayers.9,10 The
histogram of the breakthrough forces of the DPPC gel phase (Fig. 4.1 C) shows higher values of
forces, F ~ 6 12 nN, consistent with its more ordered packing. However, contrary to DEC
Ceramide, breakthrough and adhesion events were observed in the DPPC gel phases. This
indicates that the lipids in the ceramide enriched domains may have an even more ordered
organization than a typical gel phase such as DPPC. AFM height images of the DOPC/DPPC
bilayer before (Fig. 4.1 A) and after (Fig. 4.1 B) force mapping showed holes (~ 4 nm deep) in the
DPPC domains, highlighting the less fluid nature of the DPPC gel phase. This comparison further
indicates that the ceramide enriched regions exhibit an organization different from a typical gel
phase.
Figure 4.1 AFM height images before (A), after (B) force mapping, and histogram of the breakthroughforces (C) of a DPPC gel phase in a DOPC/DPPC (1:1 molar ratio) lipid bilayer.
49
4.3 Incubation with methyl cyclodextrin (M CD) and Chloroform
To be able to probe and quantify the stability of the ceramide enriched regions, an experiment
that would allow the AFM tip to access the seemingly impenetrable ceramide enriched domains
in the bilayer was designed. Here, the DEC Ceramide bilayers were first treated with methyl
beta cyclodextrin or M CD, which is known to extract cholesterol. It was then further incubated
with chloroform to weaken the acyl chain packing. In the chloroform alone treatment, pre
formed DEC Ceramide bilayers were placed in a sealed desiccator with saturated chloroform
vapor for 13 hours. AFM images were taken in less than 15 mins after removal from chloroform
vapor without further rinsing. For the M CD alone treatment, an appropriate volume of M CD
solution was added to the sub phase of the pre formed DEC Ceramide and DEC bilayers to a
final concentration of 1 mM and 10 mM M CD. AFM images were taken at least 5 mins after
M CD was added and optical images were taken in situ. For M CD and chloroform treatment,
the pre formed DEC Ceramide bilayers were first treated with M CD of 1 mM final
concentration. AFM images were then obtained in a chosen area. When no further changes in
the bilayer morphology were observed, the samples were rinsed extensively with Milli Q water
and placed in a desiccator saturated with chloroform vapor for 13 hours. AFM images were
taken less than 15 mins after removal from chloroform vapor without further rinsing.
50
Figure 4.2 AFM height images of a DEC Ceramide bilayer (A), chloroform treated DEC Ceramide bilayer(B) with line profiles below each image, zoomed in area of chloroform treated DEC Ceramide bilayershowing smaller liquid ordered domains (C), and the corresponding breakthrough force map (D).
Consistent with the reported ability of ceramide to form microdomains,1,5,6,11 three distinct
phases (schematic shown in Fig. 2.1 B): the shortest height (1) ascribed to DOPC rich fluid
disordered phase, intermediate height (2) to sphingomyelin/cholesterol (ESM/Chol) rich liquid
ordered domains, and the tallest (3) to ceramide enriched domains, were observed in AFM
height images (Fig. 4.2 A) of DEC Ceramide supported bilayers. This phase separation arises
from the differential packing of the various lipid components (Fig. 2.1, B E) in the bilayer.
Ceramide (Fig. 2.1 F) for instance, has a strong affinity for H bonding (between the �–OH groups
and the amine moieties) with sphingomyelin (Fig. 2.1 E), and, favored hydrophobic interactions
among the saturated acyl chains of both lipids give rise to the tallest height phase (�“3�” in Fig.
4.2 A).6 Cholesterol (Fig. 2.1 C) on the other hand, is a small molecule that readily inserts itself
51
in between acyl chains of ESM. The presence of an �–OH moiety forms H bonds with ESM
headgroups, resulting to its more favored interaction with sphingomyelin in comparison to
DOPC.12,13 Different heights of ceramide enriched domains were also obtained with varying
imaging setpoints (Fig. 3.2), suggesting a sensitive mechanical response of the ceramide
enriched regions in DEC Ceramide bilayers not observed for a pure DEC mixture (Fig. 3.2 A).
Figure 4.3 AFM height image of DEC Ceramide bilayer with line profile below the image (A),corresponding maps of breakthrough force (B) and adhesion (C), and histogram of breakthrough forces(D). Images are 3 × 3 m2. Outlines in (A) are to guide the eye.
It is worth noting that although force mapping on the DEC Ceramide bilayers gave
breakthrough forces at ~ 6.0 nN for the liquid ordered domains and ~ 5.0 nN for the fluid
disordered phase (Fig. 4.3), ceramide enriched regions were impenetrable (dark regions in Fig.
4.3 B) even at applied forces that have otherwise led to breakthrough events in well known gel
phases such as DPPC and DMPC supported lipid bilayers.5,7 It is noted that the absence of
breakthroughs in the ceramide enriched regions does not imply zero breakthrough forces but
indicates the inability of the AFM tip to rupture these domains. As has been demonstrated in
52
chapter 3, this illustrates the very tight lipid packing in the ceramide enriched domains that is
not typical of gel states.
As mentioned earlier, DEC Ceramide bilayers were incubated in chloroform vapor to loosen the
acyl chain packing of the ceramide enriched regions. Chloroform is known to have a strong
fluidizing action on membranes.14 However, even in this case, ceramide enriched domains still
persisted and remained impenetrable (Fig. 4.2 B). In addition, smaller liquid ordered domains
were observed to disperse over the fluid disordered phase (Fig. 4.2 C) and lower breakthrough
forces (Fig. 4.2 D), ~ 1.2 nN and ~ 0.5 nN, for Lo and Ld phase, respectively, were obtained. This
indicates that while chloroform weakened the packing in both liquid ordered domains and fluid
disordered phase, it was not able to soften the ceramide enriched regions sufficiently to obtain
breakthrough events.
Figure 4.4 AFM height image (A), corresponding maps of breakthrough force (B), and adhesion (C) ofDEC Ceramide bilayers after 1mMM CD treatment. Asterisk indicates a defect in the bilayer.
Previous studies have suggested that generation of ceramide in both biological and model
membranes lead to expulsion of cholesterol from sphingolipid/cholesterol enriched
domains.1,8,15 17 To test this hypothesis, 1 mM M CD was added to the pre formed DEC
Ceramide bilayers to extract cholesterol.18 It was observed that the ceramide enriched domains
fuse within the liquid ordered domains and expand towards the boundary (Fig. 4.4 A). Force
53
mapping on these regions still gave no breakthrough and no adhesion events (Fig. 4.4 B, C). In
contrast, the area indicated by an asterisk is a bilayer defect, and gives adhesion values. The
absence of breakthrough forces and adhesion in the ceramide enriched domains indicates
strong interaction between ESM and ceramide causing it to be impenetrable by the AFM tip.
Compared to untreated DEC Ceramide samples (Fig. 4.3), decreases in breakthrough forces for
both fluid disordered phase and liquid ordered domains were observed after M CD treatment
(Fig. 4.5 A), suggesting a less ordered lateral organization in these phases.
Figure 4.5 Histogram of the breakthrough (A), and adhesion (B) forces of 1 mM M CD treated DECCeramide bilayers.
Consistent results were observed when higher M CD concentration (10 mM) was used (Fig. 4.6
B). We found that the ceramide enriched regions remained intact (e.g. dashed outline area in
Fig. 4.6 C) even after prolonged incubation with M CD while significant restructuring (i.e.
holes/dissolution, solid outline area in Fig. 4.6 C) was observed in the liquid ordered domains.
This could be a direct evidence that supports the ceramide induced displacement of cholesterol
from ESM/Chol enriched domains, reinforcing the literature proposed model that ceramide and
cholesterol compete for association with liquid ordered domains.10 The increase in size of the
ceramide enriched domains may reveal the increasing interaction probabilities among acyl
chains of ceramide and ESM, causing the ceramide enriched domains to spread to regions
where ESM exists. Similar M CD treatment on DEC bilayers without ceramide (Fig. 4.7) led to
54
the disappearance of liquid ordered domains,19 further confirming the highly ordered packing
of the bilayer in the presence of ceramide.
Figure 4.6 AFM height images of DEC Ceramide bilayers without M CD (A), 40 min (B) and 265 min (C)after 10 mM M CD treatment. All images are 10 × 10 m2, with Z range (height): 0 4 nm, from dark tobright.
Figure 4.7 Optical image of DEC bilayer with 0.3% Texas Red DHPE (A), AFM height images of DECbilayers without M CD (B), and 15 min (C), 25 min (D), 75 min (E), 135 min (F) after treatment with 10mMM CD. Images are 25 × 25 m2 (A) and 30 × 30 m2 (B F).
55
4.4 Force Mapping on M CD and Chloroform treated DEC Ceramide
To quantify the mechanical strength and rigidity of the ceramide enriched domains, the M CD
treated DEC Ceramide bilayers were further incubated in chloroform vapor. Incubation for
approximately 13 hours yielded inhomogeneous ceramide enriched domains (inside outlined
areas, 3�’ in Fig. 4.8 A) that are 0.4 0.6 nm greater in height than the remaining liquid ordered
domains (2�’). In this case, breakthrough forces (~ 4 7 nN) and adhesion values (~ 5 nN) (Fig. 4.8
B, C) were eventually obtained in the ceramide enriched regions.20 Thus, it is inferred that for
untreated ceramide enriched domains, the breakthrough forces are larger than 7 nN, which
implies a greater degree of stability and rigidity. Interestingly, the penetration depths within
the ceramide enriched domains are approximately half of that obtained from the other
coexisting phases in the bilayer (Fig. 4.8 D, Fig. 4.9 and inset). This reflects the possibility of
interdigitation (interpenetration of the acyl chains across the width of the bilayer) in these
regions,21 consistent with a study on the very long chain asymmetric nervonoylceramide.22
Interdigitation may also explain the greater variations in the breakthrough forces (Fig. 4.9)
within the ceramide enriched regions due to the inhomogeneous lateral organization after
M CD and chloroform treatments. This is contrary to the excellent overlap of force curves from
both liquid ordered domains and fluid disordered phase (Fig. 4.9), which may be explained by
the leveling effect of chloroform in membranes.14 It is also clear from the force curves in Fig. 4.9
that the ceramide enriched regions exhibit less elastic deformation than either phase in the
bilayer (Fig. 4.8 E).
56
Figure 4.8 AFM height image (A), corresponding maps of breakthrough force (B), adhesion (C),penetration depths (D), and Young�’s modulus (E) after treatments with 1mM M CD and chloroformvapor. Outlines in (A) are to guide the eye.
57
Figure 4.9 Typical force curves from outside (blue) and within (red) the ceramide enriched domains.Insets show the histogram of breakthrough forces and penetration depths.
The high mechanical stability exhibited by the ceramide enriched regions is due to the ability of
ceramides to form extensive hydrogen bonds23 and their strong hydrophobic interactions with
sphingomyelin. In addition to cholesterol, the ESM may also be extracted by M CD,19 which
leads to a restructuring of the liquid ordered domains, thus weakening the stability of the
bilayer. With further incubation in chloroform, the chain packing between ESM and ceramide is
loosened to a greater extent due to the insertion of chloroform molecules,14 allowing
penetration of the bilayer and subsequently obtaining breakthrough forces and adhesion.
58
4.5 Conclusion
The results reported in this chapter indicate that ceramide enriched domains require both
M CD and chloroform treatments to weaken their highly rigid organization. It also suggests
lipid packing different from typical gel states. Force mapping on phase segregated regions
enabled to quantify the nanomechanical stability of the supported multicomponent bilayers.
The force mapping results demonstrate the expulsion of cholesterol from
sphingolipid/cholesterol enriched domains as a result of ceramide incorporation. This work
gives quantitative information on the mechanical stability and rigidity of the ceramide enriched
platforms. Furthermore, quantification of the nanomechanical stability provides further insights
on how the presence of ceramide in membranes could dramatically alter their properties.
59
4.6 References
(1) Chiantia, S.; Kahya, N.; Ries, J.; Schwille, P. Biophys. J. 2006, 90, 4500.
(2) Goni, F. M.; Alonso, A. Biochim. Biophys. Acta Biomembr. 2009, 1788, 169.
(3) Kolesnick, R. N.; Goni, F. M.; Alonso, A. J. Cell. Physiol. 2000, 184, 285.
(4) Sot, J.; Bagatolli, L. A.; Goni, F. M.; Alonso, A. Biophys. J. 2006, 90, 903.
(5) Sullan, R. M. A.; Li, J. K.; Zou, S. Langmuir 2009, 25, 7471.
(6) Bollinger, C. R.; Teichgraber, V.; Gulbins, E. Biochim. Biophys. Acta Mol. Cell Res.2005, 1746, 284.
(7) Nussio, M. R.; Oncins, G.; Ridelis, I.; Szili, E.; Shapter, J. G.; Sanz, F.; Voelcker, N.H. J. Phys. Chem. B 2009, 113, 10339.
(8) Megha; London, E. J. Biol. Chem. 2004, 279, 9997.
(9) Choucair, A.; Chakrapani, M.; Chakravarthy, B.; Katsaras, J.; Johnston, L. J.Biochim. Biophys. Acta, Biomembr. 2007, 1768, 146.
(10) Giocondi, M. C.; Vie, V.; Lesniewska, E.; Milhiet, P. E.; Zinke Allmang, M.; LeGrimellec, C. Langmuir 2001, 17, 1653.
(11) Ira; Johnston, L. J. Langmuir 2006, 22, 11284.
(12) Sankaram, M. B.; Thompson, T. E. Biochemistry 1990, 29, 10670.
(13) Slotte, J. P. Chem. Phys. Lipids 1999, 102, 13.
(14) Turkyilmaz, S.; Chen, W. H.; Mitomo, H.; Regen, S. L. J. Am. Chem. Soc. 2009, 131,5068.
(15) Ali, M. R.; Cheng, K. H.; Huang, J. Biochemistry 2006, 45, 12629.
(16) Ira; Zou, S.; Ramirez, D. M. C.; Vanderlip, S.; Ogilvie, W.; Jakubek, Z. J.; Johnston,L. J. J. Struct. Biol. 2009, 168, 78 89.
(17) Ito, J.; Nagayasu, Y.; Yokoyama, S. J. Lipid Res. 2000, 41, 894.
(18) Lawrence, J. C.; Saslowsky, D. E.; Edwardson, J. M.; Henderson, R. M. Biophys. J.2003, 84, 1827.
(19) Giocondi, M. C.; Milhiet, P. E.; Dosset, P.; Le Grimellec, C. Biophys. J. 2004, 86,861.
(20) only trace portions of the force curves are shown for clarity.
(21) Slater, J. L.; Huang, C. Prog. Lipid Res. 1988, 27, 325.
(22) Pinto, S. N.; Silva, L. C.; De Almeida, R. F. M.; Prieto, M. Biophys. J. 2008, 95,2867.
(23) Pasher, I. Biochim. Biophys. Acta 1976, 455, 433.
60
5 Cholesterol Dependent Nanomechanical Stability of PhaseSegregated Multicomponent Lipid Bilayers�‡
5.1 Overview
Cholesterol is involved in endocytosis, exocytosis, and the assembly of sphingolipid/cholesterol
enriched domains, as has been demonstrated both in model membranes and in living cells. In
this chapter, the influence of different cholesterol levels (5 40 mol%) on the morphology and
nanomechanical stability of lipid bilayers consisting of dioleoylphosphatidylcholine /
sphingomyelin / cholesterol (DOPC/SM/Chol) was explored by means of atomic force
microscopy (AFM) imaging and force mapping. Breakthrough forces were consistently higher in
the SM/Chol enriched liquid ordered domains (Lo) than in the DOPC enriched fluid disordered
phase (Ld) at a series of loading rates. The activation energies ( Ea) for the formation of an AFM
tip induced fracture, calculated by a model for the rupture of molecular thin films, were also
reported. The obtained Ea values agree remarkably well with reported values for fusion
related processes using other techniques. Furthermore, within the Chol range studied, the
lateral organization of bilayers can be categorized into three distinct groups. The results are
rationalized by fracture nanomechanics of a ternary phospholipid/ sphingolipid/ cholesterol
mixtures using correlated AFM based imaging and force mapping, which demonstrates the
�‡ This chapter was reproduced in part with permission from Sullan, R.M.A., Li, J.K., Hao, C.C., Walker, G.C., Zou, S.Biophysical Journal, 99 (2), 507 516. © 2010 Elsevier.
61
influence of a wide range of cholesterol content on the morphology and nanomechanical
stability of model bilayers. This provides fundamental insights on the role of cholesterol in the
formation and stability of sphingolipid/cholesterol enriched domains, as well as in membrane
fusion.
5.2 Cholesterol
Abundant studies on cholesterol demonstrate its importance in understanding various
membrane structures and processes in cells. Cholesterol�’s diverse roles range from building
block to key regulatory molecule in highly specialized membrane fusion processes such as
endocytosis and exocytosis.1 5 Large variations in a number of structural and dynamical
parameters of the lipid bilayer are also observed in the presence of cholesterol, illustrating its
influence on the phase properties of membranes.2,3,5 10 In addition, cholesterol is closely
associated with phase segregated micro or nano domains in cells termed as rafts, which have
been proposed as platforms for the preferential sorting of proteins.5,11 13 While a recent study
has demonstrated the presence of ~ 20 nm sized cholesterol mediated rafts in the plasma
membrane of living cells, nevertheless there is still ongoing debate about their existence.11,14 19
The size of the rafts, its physical state, molecular composition, longevity/existence in the cell
membrane, and functional roles are among the subjects in question.12
Several studies have investigated the effects of varying levels of cholesterol in both model and
biological membranes using different techniques.3,7 9,20,21 In the work reported in chapters 3
and 4 of this thesis,22,23 atomic force microscopy (AFM) based force mapping was used to
directly correlate the self organized structures exhibited in phase segregated supported lipid
bilayers consisting of dioleoylphosphatidylcholine / sphingomyelin / cholesterol
(DOPC/SM/Chol), known to mimic rafts in cells15,24,25, with their nanomechanical properties. It
was quantitatively shown by means of the breakthrough force and elastic modulus, the greater
62
stability and degree of compactness exhibited in the Chol/SM enriched liquid ordered domains
(Lo phase) over the DOPC enriched fluid disordered phase (Ld phase).
In this chapter, AFM based force mapping is further used to investigate the influence of
different levels of cholesterol on the morphology, lateral organization, and mechanical stability
of coexisting phases in supported DOPC/SM/Chol bilayers. It is noted that compared to the
studies reported in chapters 3 and 4, the egg sphingomyelin (ESM) has been replaced with pure
sphingomyelin (SM). This was done to eliminate complications that could arise from the
presence of other constituent lipids in egg sphingomyelin which is composed of ~85%
sphingomyelin and other lipids.
5.3 Models for Rupture of Molecularly Thin Films
In addition to the breakthrough forces in Lo and Ld phases that were measured at different Chol
concentrations at a series of loading rates reported in this chapter, activation energies ( Ea) of
bilayer breakthrough were also calculated, using the AFM tip induced film rupture theory
introduced by Butt et al.26,27 This formalism assumes that an energy barrier must be overcome
by the tip before the film ruptures; dependence of the activation energy on the force and
loading rate was then obtained following this premise. This theory is analogous to earlier
theories of rupture of single molecular bonds under the influence of loading rate, relating
microscopic parameters of the film with measurable quantities obtained from force
measurements.28 31 Here, the activation energy is the energy required to create a hole
(fracture) in the film/bilayer large enough to initiate bilayer rupture. Furthermore, two specific
models for Ea calculations were presented: a continuum nucleation model and a discrete
molecular model.26 The former considers a thin, molecular and homogenous film confined
between the solid substrate and the solid surface of the AFM tip. The prerequisite is that the
film exhibits fluidity; it is laterally mobile with a well defined vertical structure. In the discrete
molecular model, the film is treated as having energetically favorable binding sites, formed by
the substrate or surrounding molecules. When molecules move from one binding site to an
adjacent binding site, a potential energy barrier has to be overcome. Once a critical number of
63
molecules moves out from or reorganizes within the contact area, the large pressure on the
remaining molecules triggers film rupture and tip breakthrough. In our study, we use the
general formalism to calculate the rupture activation energies of DOPC/SM bilayers with 10
40% Chol. It is noted that the general theory used in the present study does not refer to neither
the continuum nucleation model nor to the discrete molecular model as described above, but
only to this general assumption�— that an energy barrier must be overcome by the tip before
the film ruptures. The results demonstrate the influence of a wide range of cholesterol
concentrations on the morphology and nanomechanical stability of model lipid bilayers that
simulate the rafts in cells. Also, possible implications of membrane rupture events in the
presence of cholesterol in providing fundamental nanomechanical insights on other cellular
processes are discussed.
5.4 Rupture Activation Energy Calculation
The rupture of a lipid bilayer is considered as an activated process wherein an energy barrier
needs to be overcome to form a hole or fracture in the bilayer by the AFM tip, and is observed
as a jump or kink in a force curve (Fig. 1.3 B C).26,27 The theory introduced by Butt et al. to
calculate the activation energy of rupture of lipid membranes was used.27 Details of the rupture
kinetics are presented in the Appendix.
Ea (F0) kBT ln0.693k
AdvdF0
Equation 5.1
Equation 5.1 describes the dependence of the rupture activation energy, Ea on the mean
breakthrough force, F0. kB is Boltzmann�’s constant, T is the Kelvin temperature, v is the loading
rate (loading rate is calculated using the approaching velocity of the base of the cantilever), k is
the cantilever spring constant, and A is assumed to be the frequency at which the tip attempts
to penetrate the film. In the case where the distribution of mean breakthrough forces is
relatively narrow ( F/F
B
27
0 << 1, where F is the half width of the yield force distribution), F0 can
64
be obtained from the histogram of breakthrough forces; the loading rate demonstrates a
logarithmic dependence on the breakthrough force, namely,
F0 a b lnv Equation 5.2
where a and b are parameters obtained from fits to the experimental data. The dependence of
activation energy on loading rate can be explicitly expressed as:
Abk
bFaTkFE Ba
60.1ln30.2)( 00 Equation 5.3
Ea can then be written as a function of force that is proportional to kBT. When this relation is
extrapolated to zero mean yield force (F
B
27
0 = 0), it provides the rupture activation energy of lipid
membranes in the absence of an applied force.
In the experiments, the mean breakthrough force, F0 was extracted from histograms compiled
for each phase (Ld and Lo), at each loading rate. In the calculations, k of ~0.25 N/m,
temperature of 296.2 K, and A equal to the cantilever�’s resonant frequency under water (~15 ×
103 Hz) were used.
5.5 Bilayer morphology as a function of cholesterol concentration
AFM imaging was employed to visualize how different cholesterol compositions influence the
lateral organization of phase separated supported lipid bilayers prepared from
dioleoylphosphatidylcholine / sphingomyelin/ cholesterol (DOPC/SM/Chol). The AFM height
images in Fig. 5.1 show the coexistence of the SM/Chol enriched liquid ordered domains, Lo
phase (lighter regions) and DOPC enriched fluid disordered, Ld phase (darker matrix) in
DOPC/SM/Chol with 10 40 mol% Chol. This result is in good agreement with previous studies
reporting phase separation in model membranes with similar lipid mixtures. 15,25,32 Specifically,
extended phase separation starts to occur at 10 mol% Chol and the round shape of the domains
65
suggests the coexistence of both Lo and Ld phases. The height difference between the Lo and Ld
phases are also consistent with previous studies of similar lipid mixtures: ~0.8 1.2 nm in 10 35%
and ~0.6 nm in 40% Chol.18,19,23,33 39 The decrease in height difference at 40% Chol was
attributed to the saturation of Chol in the Lo phase and the subsequent fluidizing effect. As a
result of more Chol in the domains, SM packing is loosened, which then leads to a decrease in
height of the Lo phase, and hence a smaller height difference between the coexisting phases.
This decrease at 40% Chol could also be due to the ability of Chol to thicken fluid bilayers.18,40,41
Owing to Chol saturation in the domains, some Chol may now tend to interact with the DOPC
enriched fluid phase. As a result, the thickness of the Ld phase is increased, thus decreasing the
height difference between the coexisting Lo and Ld phases. This smaller height difference
observed in DOPC/SM at 40% Chol closely agrees with a separate study on same lipid mixtures
in which phase separation was observed for 50% Chol bilayers with the Lo phase about 0.4 nm
taller than the Ld phase.18
66
Figure 5.1 AFM height images of supported lipid bilayers. Visualization of phase separation for ternarylipid mixtures of DOPC/SM/Chol bilayers with 5 40% cholesterol (A H). Bilayers with 5% Chol exhibitphase separation of smaller domains (A). More distinctive phase separation between the coexistingliquid ordered domains (Lo) and fluid disordered phase (Ld) were observed with 10 35% Chol (B G).Bilayers with 40% Chol also showed distinct phase separation but with reversed contrast to thoseobserved in 10 35% (Ld phase seemed to be dispersed in the Lo matrix) (H). Cross sectional line profilesare shown below each image. The color scale bars on the left side of 1 A and 1 H provide heightreference for Fig. 5.2 A G and Fig. 2 H, respectively. All images are 10 × 10 m2.
Fluorescence images of larger areas of the bilayers also show the coexistence of the Lo phase
(darker domains) and Ld phase (lighter matrix) (Fig. 5.2). The Texas Red dye displays strong
partitioning into the fluid disordered phase, consequently causing the liquid ordered domains
to appear as micron sized dark spots.36 Large dye excluded regions (dark patch with asterisk)
are visible in DOPC/SM/Chol bilayer sample with 5% Chol. This dark patch arises from the
coalescence of small individual domains and bilayer defects (Fig. 5.3 A). It is evident from both
AFM and fluorescence images that the role of cholesterol is to promote phase separation into
Lo and Ld phases in DOPC/SM/Chol bilayers.
67
Figure 5.2 Optical images of DOPC/SM/Chol bilayers with 0.3% Texas Red DHPE at 5% (A), 10% (B), 15%(C), 20% (D), 25% (E), 30% (F), 35% (G), and 40% (H) Chol. Images are 88 × 66 m2.
68
Figure 5.3 AFM height images of DOPC/SM/Chol bilayer with 5% Cholesterol. 30 × 30 m2 area showingthe dark patch also visible in Fig. 5.2 A (A); 10 × 10 m2 of the features within the dashed circle in Fig. 5.3A (B). The line profile in Fig. 5.2 B indicates that the dark patch is a combination of bilayer defects (~ 6nm) and individual domains that coalesced. Color scale bar at the left side provides z range.
Figures 5.1 and 5.2 also demonstrate how the morphology of the DOPC/SM/Chol bilayers
evolves with increasing Chol concentration. Lipid bilayers with 5 10% Chol (Fig. 5.1 A B) showed
small domains that appear to be taller and tend to form close networks with other domains.
Distinctive phase separated, round, and well separated domains are observed with 15 30%
Chol (Fig. 5.1 C F). Elongated domains start to appear in 30% Chol bilayer (Fig. 5.1 F), become
more dominant in 35% Chol (Fig. 5.1 G), and continues to coalesce with 40% Chol (Fig. 5.1 H).
This gives rise to what appears to be a phase inversion: the Lo phase now constitutes the matrix
and the Ld phase assumes a more separated and rounded shape which is in contrast to that of
the liquid ordered domains observed in 15 30% Chol bilayers (Fig. 5.1 B F). In a previous study
of DOPC/SM/Chol bilayers, this phase inversion or change in percolation phase, has been
observed with 30% Chol.18 Interestingly, a change in percolating and non percolating phases
has been proposed as trigger mechanism in the control of membrane physiology.42 The
observed bilayer morphologies also suggest that three different morphological classifications
69
exist within the range of Chol concentration studied. In the first group (5 10% Chol), it was
inferred that the domains of bilayers with 5% Chol are predominantly in the gel phase (Fig. 5.3)
while the domains with 10% Chol appears to be a mixture of gel and Lo phases. This inference is
supported by AFM images after force mapping; the dips and holes in the line profile of the
bilayer with 10% Chol indicate the existence of a gel phase (Fig. 5.4 A) in contrast to
DOPC/SM/Chol bilayer with 25% Chol where no holes were seen, confirming the presence of
the more fluid (laterally mobile) liquid ordered state (Fig. 5.4 C). These images after force
mapping therefore indicate that the domains in 10% Chol are not solely in the Lo phase but are
a mixture of both gel and Lo phases. In the second group (15 30% Chol), the domains are more
rounded and separated, indicating that liquid ordered and fluid disordered phases coexist in
the bilayer (Fig. 5.4 C). Also, with increasing Chol content in the second group, a subtle increase
of the domain size is observed (Fig. 5.1, 5.2 C F), confirming the known affinity of cholesterol
towards sphingomyelin in the formation of sphingolipid/cholesterol enriched domains.14,20,38,43
The AFM image after force mapping for the bilayer with 15% Chol suggests the existence of a
minimal gel phase character in the domains (Fig. 5.4 B). In the third group (35 40% Chol), the Lo
phase occupies a relatively larger area in the bilayer, further indicating the increasing
interaction between the added Chol and SM. At Chol concentrations above 40%, conflicting
observations by different groups regarding the presence/absence of separated phases for the
same lipid system presently renders any hypothesis inconclusive.14,15,25,32 Phase separation was
not observed on DOPC/SM/Chol giant unilamellar vesicles (GUVs) with less than 10% Chol.42
The proposed classification of bilayer morphologies into three groups within the studied 5 40%
Chol range is in good agreement with the fluorescence microscopy study on Egg PC/brain
SM/Chol, in which different regions in the phase diagrams of the coexisting liquids were
observed.21,32,44
70
Figure 5.4 AFM height images of of DOPC/SM bilayers with 10% (A), 15% (B), and 25% (C) Chol, afterforce mapping. The dips/holes (inside the dashed circle) in the line profiles of the bilayers with 10% and15% Chol indicate the existence of a gel phase. On the contrary, the absence of holes in DOPC/SM with25% Chol confirms the presence of a liquid ordered state. Color scale bar at the left side provides the zrange.
5.6 Loading rate dependence of the breakthrough force
While the AFM height images in Fig. 5.1 do not provide quantitative nanomechanical
information on these bilayers, they do reveal some insight to how DOPC/SM/Chol bilayers
evolve with increasing Chol concentration. To investigate how cholesterol influences the
mechanical stability and lateral organization of the bilayers, AFM based force mapping on
DOPC/SM/Chol bilayers with 10 40 mol % Chol while keeping DOPC and SM at a 1:1 ratio, was
performed. Consistent with previous results reported in chapters 3 and 4 of this thesis,
breakthrough forces were obtained in the coexisting phases�— liquid ordered domains and fluid
disordered phase�— at different loading rates. 26,27,45 Results are reported as breakthrough force
maps (Fig. 5.5). As noted in the Materials and Methods section (chapter 2 of this thesis), the
breakthrough force is the maximum force that a bilayer can withstand before it ruptures and is
used as a measure of the bilayer�’s mechanical stability.
71
Representative breakthrough force maps of DOPC/SM/Chol bilayers with 15, 20, and 25% Chol
at 200, 800 and 2000 nm/s loading rates, respectively, are displayed in Fig. 5.5 A I. The
correlated AFM height images and breakthrough force maps for 20% Chol in Fig. 5.6 clearly
show that lighter regions and darker matrix in the breakthrough force maps respectively
correspond to the taller Lo and shorter Ld phase in AFM height images. Also, the Lo and Ld
phases correspond to the higher and lower mean breakthrough forces in the correlated
histograms. The mean breakthrough force peaks were obtained from Gaussian fits of the force
distribution histograms. The higher breakthrough force exhibited in the SM/Chol enriched Lo
phase reflects the more efficient packing between Chol and SM in the Lo phase in comparison to
DOPC enriched Ld phase. Figures 5.5 and 5.6 also reveal that mean breakthrough forces depend
on the loading rate, i.e., the breakthrough force increases with increasing loading rate.26,27,45 In
addition, at a fixed loading rate, the breakthrough forces tend to decrease with increased Chol
concentration. Furthermore, the difference between the breakthrough forces in the two phases
in the bilayer with 15% Chol is larger than between those with 20% and 25% Chol, confirming
the preferential affinity of Chol to SM.14,20,38,43 At a lower Chol concentration, Chol tends to fully
incorporate into the domains, resulting in more disparate breakthrough force values in the
coexisting phases.
72
Figure 5.5 Representative breakthrough force maps and corresponding histograms of DOPC/SM/Cholbilayers at a series of loading rates. Breakthrough force maps were reconstructed from 64 × 64 pixelforce mapping experiments and the corresponding histograms on bilayers with 15% (A C), 20% (D F),and 25% (G I) Chol at loading rates of 200 nm/s (C, F, I), 800 nm/s (B, E, H), and 2000 nm/s (A, D, G),respectively. All histograms consist of 4096 force curves and the numbers in the histogram represent themean breakthrough force values with standard deviations from Gaussian fits. The darker region in theupper left corner of Fig. 5.5 A is a defect in the bilayer. All breakthrough force maps are 3 × 3 m2.
73
Figure 5.6 Correlated AFM height images and breakthrough force maps of DOPC/SM/Chol bilayers with20% Chol at 2000 nm/s (A, B), 800 nm/s (C, D), and 200 nm/s (E, F) loading rate. It is apparent fromthese correlated images the lighter regions and the darker matrix in the breakthrough force mapsrespectively correspond to the Lo and Ld phases.
5.7 Cholesterol dependence of breakthrough forces
To clearly illustrate how the breakthrough force varies with Chol concentration, Fig. 5.7
presents the plot of mean breakthrough forces versus mol % Chol at 200 nm/s. The solid and
open circles correspond to the Lo and Ld phases, respectively. Four main results were observed
in this plot. First, in comparison to the Ld phase, higher mean breakthrough forces were
consistently detected in the Lo phase. This higher nanomechanical stability in the Lo phase is
due to the enhanced SM Chol interaction that results in a more ordered lipid organization
compared to interactions between Chol and unsaturated phospholipids.14,20,32,38,43 The second
74
significant result is bilayers with 10% Chol exhibit the largest difference between the domains
and Ld phases. This is indicative of domains that maintain a partial gel like nature (see Fig. 5.4
A). Another main finding is that for bilayers with 10 30% Chol, the breakthrough forces in both
phases tend to decrease as Chol concentration increases, consistent with the fluidizing effect of
Chol and its role in the formation of a liquid ordered phase with a more relaxed lipid packing
than the pure SM gel phase. This is consistent with a previous fluorescence correlation
spectroscopy (FCS) study of DOPC/SM/Chol GUVs, in which an increased Chol concentration
resulted in an increased lipid mobility in SM enriched domains.32 More Chol in the
DOPC/SM/Chol relieves bilayer stress; as a result, a lower force is necessary to induce rupture,
and hence a lower breakthrough force. The fourth result of interest is the sudden increase of
the breakthrough force in both Lo and Ld phases at 35% Chol; this transition is consistent with
the observed change in the bilayer morphology in Fig. 5.1 F G (a shift from rounded to more
elongated domains). This sudden increase could be due to the coalescence effect, i.e.,
individual domains start to form a network with neighboring domains, as revealed by the more
elongated features in bilayers with 35% (Fig. 5.1 G). At this Chol concentration, the interaction
of the constituent lipids in the coexisting phases may have led to an increase in the line tension
wherein the decrease in boundary energy dominates the unfavorable entropy of merging, and
consequently resulting in coalescence of the domains.46 Force mapping was not performed on
DOPC/SM with 5% Chol due to domains being relatively small and not well separated.
Figure 5.7 Breakthrough forces of DOPC/SM/Chol bilayers with 10 40% Chol at 200 nm/s. The error barsare the mean standard deviation from a set of 2 10 force maps, each containing 4096 force curves.
75
The breakthrough force versus Chol content plot in Fig. 5.7 supports the three group
classification observed in Fig. 5.1: (1) smaller domains in 5 10% Chol that could be
predominantly gel or a mixture of gel and Lo phases, (2) more rounded and separated domains
in 15 30% Chol indicating coexistence of liquid ordered and fluid disordered phases, (3) Lo
phase occupying a relatively larger area in bilayers with 35 40% Chol suggesting enhanced
interaction between the added Chol and SM. It can then be presumed that DOPC/SM/Chol
bilayers with 10% Chol falls in the first group, the 15 30% Chol in the second group, and 35%
40% Chol in the third group. These correlated results by AFM imaging and force mapping
demonstrate the effective use of force mapping in studying the influence of a wide range of
Chol concentrations on the nanomechanical stability and the lateral organization of coexisting
phases in model bilayers with compositions that simulate biological membranes.
5.8 Rupture activation energy of DOPC/SM/Chol at varying Cholconcentrations
As illustrated in Fig. 5.8, the same loading rate dependence of the breakthrough force obtained
in Fig. 5.5 (i.e. breakthrough forces in both Lo and Ld phases increase with increasing loading
rate) was observed for other cholesterol concentrations. The slope (b) and the y intercept (a)
obtained from linear fits to Equation 5.2 were used in the calculation of the rupture activation
energy. As noted earlier, Ea can be explicitly expressed as a function of force proportional to
kBT, when the mean breakthrough forces increase linearly with the logarithm of loading rates.
The relation presented in Equation 5.3, when extrapolated to zero mean breakthrough force,
provides the activation energy of the bilayer rupture in the absence of an applied force.
B
27
76
Figure 5.8 Loading rate dependence of the breakthrough forces with different cholesterolconcentrations. Breakthrough forces of the coexisting phases (Lo and Ld) of DOPC/SM/Chol (10 40%Chol) bilayers at different loading rates (200, 400, 800, 2000, and 2400 nm/s) (A G).
The values of the calculated rupture activation energy using Equation 5.3 while Fo=0, are
summarized in Table 5.1, and plotted as a function of Chol content in Fig. 5.9. The solid and
open squares correspond to Lo and Ld phases, respectively. The results yield a range of
activation energies that vary for Ld and Lo phases, depending on the Chol concentration.
77
DOPC/SM/Chol bilayers with 10, 25, 30, and 40% Chol provided Ea values in the range of 75
100 kJ/ mol, while higher Ea in the range of 100 125 kJ/mol were obtained for bilayers with 15,
20, and 35% Chol. The difference in Ea values between the coexisting phases with different
mol % Chol is within 10 kJ/mol, except for 15 mol% Chol where the Ld phase is 30 kJ/mol greater
than the Lo phase. The data did not show dependence in the activation energy for the different
lipid phases. The absence of a clear trend or a linear correlation between Ea and Chol content
underlines the complex nature of the rupture process in bilayers of this ternary mixture.47
Nevertheless, when compared to the reported activation energies of membrane fusion and
other cellular processes using different techniques, the Ea values (Table 5.1) obtained by force
measurements are remarkably in good agreement. For instance, activation energies of a lipid
flip flop in highly curved vesicles48 were found to be 100 kJ/mol while for lipid desorption from
a membrane49 they were 92 kJ/mol. In addition, pore formation during secretory granule
release50, has a Ea of 96 kJ/mol, along with 88 kJ/mol and 113 kJ/mol for PEG mediated fusion
of protein free model lipid bilayers51. As a further comparison, activation energies of 20 50
kJ/mol are obtained for the diffusion of lipid molecules in phosholipid bilayers without rafts
using proton NMR measurements and fluorescence excimer techniques.52,53
Figure 5.9 Rupture activation energies of the coexisting phases (Lo and Ld) in DOPC/SM/Chol bilayerswith 10 40% Chol.
78
Rupture Activation Energy (kJ/mol)CholesterolConcentration
Ld phase Lo phase
10% 73 ± 50 78 ± 123
15% 123 ± 12 93 ± 8
20% 114 ± 23 105 ± 83
25% 73 ± 36 75 ± 28
30% 76 ± 18 85 ± 21
35% 108 ± 15 109 ± 45
40% 85 ± 25 98 ± 74
Table 5.1 Rupture activation energies of the coexisting phases (liquid ordered domains, Lo and fluiddisordered phase, Ld) in DOPC/SM (1:1) with 10 40% cholesterol bilayers.
The number of lipid molecules involved in the rupture process was also approximated using
Equation 5.4 and the activation energy values in Table 5.1. Equation 5.4 represents the total
free energy of hydration of a solute molecule, given byGh
G h g i A ii
Equation 5.4
where gi is the proportionality constant pertaining to a functional group i, which in our case,
was taken as an aliphatic group to represent the aliphatic tails of the lipids; and Ai is the
conformation dependent accessible surface area.54 Comparison of the Ai obtained using
Equation 5.4 with the cross sectional area of a cylinder (assuming that exposed surface area of
the lipid bilayer has this conformation, 3 6 lipid molecules were obtained. This compares well
with the 4 7 lipid molecules obtained for dioleoylphosphatidylserine (DOPS) and
dioleoyloxypropyl trimethylammonium chloride (DOTAP) using the discrete molecular model.26
This approximation implies that the structural change at the breakthrough transition state
involves this number of lipid molecules which effectively corresponds to binding sites that are
in energetically favorable positions, as described in the discrete molecular model by Butt et al.26
79
5.9 Discussion
The work presented in this chapter draws attention to a number of important cell membrane
processes involving cholesterol. The AFM tip induced rupture of the DOPC/SM/Chol bilayers at
various Chol concentrations can serve as a model platform for studying relevant cellular
processes, such as membrane fusion and poration,55 58 and can represent proteins, viruses, or
other trigger particles that mediate pore formation and bilayer rupture. An example of this is
the well known fusion inducing agent, lysolecithin, which may operate by creating small defects
in the bilayer.56 The process of fusion requires a breaking of the membrane, at least during the
coalescence of the fusion vesicles.56 Since cholesterol is reported to be involved in a wide range
of significant cellular events, it is important to know how it influences the lateral organization
and mechanical stability of lipid bilayers. Using AFM based force mapping we revealed that
Chol content affects the morphology and the mechanical stability of the DOPC/SM/Chol model
membrane. Quantifying the breakthrough forces enabled the direct correlation of the
nanomechanical stability with the bilayer�’s morphology in a wide range of Chol concentrations.
This direct correlation is not apparent in conventional force measurements (e.g. non force
mapping experiments). Moreover, there is a significant improvement in statistics with the
massive number of force curves obtained from a force map. Additionally, force mapping
provides a dye label free means in the biophysical study of phase segregated lipid bilayers,
avoiding the occasionally inconsistent partitioning behavior of dye molecules, which affects
lipid organization.14,39,59,60
As for the possibility that the force mapping technique mechanically alters the organization of
the bilayer, AFM images obtained at the same location before and after force mapping in our
previous work indicate no significant restructuring of the bilayer by virtue of the unchanged
domain sizes and relative positions.23,61 Additionally, tip contamination as a result of force
mapping measurements, has already been addressed by Leonenko et al. in the study of
dipalmitoylphosphatidylcholine (DPPC) bilayers measured with loading rates in the range of 0.5
80
2500 nm/s.61 In that study, no evidence of lipids permanently coating the AFM tip was
observed. Continuous imaging of the bilayers did not show resolution degradation or changes in
force curves that could arise from uncontrollable lipid coating of the AFM tip. In the study
reported in this thesis, for 50% of all the tips (25 out of 50) used to collect 20 images and 10
force maps, permanent contamination was not observed; in other occasions contaminated
effects that show dull breakthroughs was apparent. These gradual breakthroughs did not affect
the statistics, as the force curves were still recognized and processed by the analysis algorithm.
The distribution of the breakthrough forces in Fig. 5.5 demonstrates that the observed AFM tip
induced rupture in the lipid bilayer is a statistical process that depends on the probability that
the AFM tip ruptures the bilayer at a certain breakthrough force.26 This probability increases
with increasing breakthrough force. Given this rupture probability and breakthrough force
relation, Equation 5.3 predicts that the activation energy decreases with increasing mean
breakthrough force, hence, with increasing loading rate. Since lower breakthrough forces in the
Ld phase in all Chol concentrations (Fig. 5.5, 5.7) were obtained, it is expected that Ld phase will
have higher activation energies than the Lo phase. This holds true with 15 20% Chol, but is not
the case for other Chol concentrations. Furthermore, the tendency of the breakthrough force to
decrease with increasing Chol content (Fig. 5.7) should also lead to higher Ea values with
increasing Chol content. However, there is no clear dependence of Ea and mol % Chol
observed in our study. This result implies the presence of a richer microscopic behavior of the
bilayers in the presence of Chol, where incremental addition of Chol do not always lead to the
same thermodynamic effect.9 Further evidence of the non conforming trend of the effect of
cholesterol levels can be found in a recent study using X ray scattering methods; it has been
demonstrated that the effect of cholesterol is not universal for lipids with varying numbers of
saturated chains.8
When a wider range of loading rates is used (e.g. different by orders of magnitude),
breakthrough force versus loading rate plots (Fig. 5.8) can be used to reveal the energy
81
landscape of the rupture process. This has been well demonstrated in unfolding of proteins62,63,
receptor ligand dissociation62,63 and fusion of floating lipid bilayers64. In the latter, a
pronounced dependence of the fusion force on the compression rate was observed for DMPC
and Egg PC, and has been interpreted as evidence for the presence of a single energy barrier in
the fusion of the lipid bilayers.
Lastly, the breakthrough force vs. Chol content plot in Fig. 5.7 can be compared to typical x y
composition plots that can be used to estimate the lipid composition of a given phase
segregated bilayer when the breakthrough force is known, which can easily be extracted from
breakthrough force maps. As demonstrated in the results presented in chapters 3 and 4 of this
thesis and by others, the breakthrough force is an intrinsic property of a bilayer that is strongly
dependent on the bilayer�’s chemical composition. 23,65 67 As such, it is regarded as a fingerprint
of bilayer stability analogous to the force needed in unfolding a single protein68, the transition
force of single DNA strand formation from double stranded DNA69, and the force needed to
indent single crystals70.
82
5.10 Conclusion
In this chapter, the influence of different cholesterol content on the morphology, lateral
organization, and nanomechanical stability of phase segregated lipid bilayers with compositions
that simulates cell membranes was examined. The AFM height images of DOPC/SM/Chol
bilayers with 5 40% Chol demonstrate the involvement of Chol in the formation of liquid
ordered domains in phase segregated bilayers. Force mapping on these bilayers reveal that the
nanomechanical stability of the coexisting Lo and Ld phases decrease with increasing Chol
content, confirming the fluidizing effect of Chol. The observed loading rate dependence of the
breakthrough forces in both phases allowed us to calculate the activation energies of bilayer
rupture at zero applied force. The obtained Ea values agree well with reported values for
biomembrane and model membrane fusion processes. This study demonstrates the influence
of cholesterol on the mechanical stability and lateral organization of model membranes,
providing fundamental nanomechanical insights on the role of cholesterol in the formation and
stability of sphingolipid/cholesterol enriched domains as well as in membrane fusion.
83
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86
6 Nanomechanics of Lipid Polymer/Peptide Interactions
6.1 Overview
Physiological functioning of membrane bound proteins, receptors, and enzymes is controlled to
a large extent by the structure and dynamics of the lipid bilayer matrix with which they are
associated. Membrane thickness and receptor function for instance, are correlated.1 In this
chapter, AFM based force mapping was further applied to study the mechanical stability of
phase separated lipid bilayers in the presence of a diblock copolymer consisting of stretches of
polystyrene and polyethylene oxide of varying molecular weight and concentrations as well as
in the presence of a helical zinc binding peptide. The presence of the polymer or peptide led to
an increase in the breakthrough forces in the bilayer. Two mechanisms that could have led to
the enhanced stability were proposed.
6.2 Introduction
Lipid polymer/peptide systems have been used as platforms to understand various cellular
components, functions, and processes. For instance, a number of studies have incorporated a
polymer or a peptide to model the cell glycocalyx,2 to understand hydrophobic mismatch (the
effect of mismatch between the hydrophobic regions of integral membrane proteins and the
hydrophobic core of the lipid bilayer),3 elucidate the mechanism of antimicrobial peptides,4 and
study the interaction of specific peptides with lipid bilayers.5 Several different techniques such
87
as fluorescence microscopy, foerster resonance energy transfer (FRET), differential scanning
calorimetry (DSC), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy,
neutron and X ray diffraction, and atomic force microscopy (AFM) were employed and have
addressed key questions regarding lipid protein interactions.1 3,5 7 Though these tools have
provided useful information on different aspects of both model and biological lipid protein
systems, none have yet reported quantitative information on the mechanical stability of phase
segregated multicomponent lipid bilayers of biological relevance in the presence of an added
polymer or peptide. To this end, the work presented in this chapter applies AFM based force
mapping to quantify the mechanical stability of lipid polymer systems. As demonstrated in
chapters 3 5 of this thesis, force mapping has an added advantage of direct correlation of
structures with its mechanical properties.8 10
Poloxamers or Pluronics (trade name) have attracted considerable attention because of its
potential as transport agents in diagnostics and therapy. Pluronics are nonionic amphiphilic
polymeric nanomaterials consisting of a hydrophilic poly(ethylene oxide) PEO and hydrophobic
poly(propylene oxide) PPO arranged in an alternating triblock fashion. Numerous studies in the
literature have tackled several aspects of Pluronics: the various molecular architecture it forms,
interactions with phospholipid vesicles and liposomes, all with an emphasis on its potential as
steric stabilizers for targeted drug delivery.6,11 18 A recent review has shown that Pluronic block
copolymers, in addition to being nanocarriers for drug delivery, also act as biological response
modifiers that cause alterations in cells.19 In earlier reports (and the references therein), the
presence of Pluronics in cell suspensions has been shown to protect both plants20 and
mammalian cells21,22 against mechanical damage. The aforementioned reasons prodded the
investigation of the effect of Pluronic mimics on the mechanical stability of model lipid bilayers
of biological relevance.
In this chapter, we used AFM based force mapping to study the mechanical stability of
phase separated DEC bilayers in the presence of hydrophobic polystyrene(PS) and hydrophilic
88
poly(ethylene oxide) arranged in a diblock fashion, which were taken as Pluronic mimics. Self
assembled PS PEO copolymer surfaces have been demonstrated to resist protein and cell
adhesion and were found to be biocompatible.18,23 Various concentrations and lengths of PS b
PEO chains were studied to simulate peptides with varying degree of hydrophobicity and
hydrophilicity. Breakthrough force was primarily used to quantify the mechanical stability,
which were reported as 2D visual maps. The effect of a short helical zinc binding peptide on
the stability of the bilayers was also examined.
6.3 Preparation of Lipid Polymer/Peptide Systems
The procedure previously described in sections 2.1.2 and 2.1.3 in the preparation of lipid films
was slightly modified to accommodate added polymer and peptide. Lipid mixtures were
obtained by combining the appropriate molar ratios of the different lipid components and
diblock polymers polystyrene b poly(ethylene oxide), (PS b PEO). For the diblock polymer,
PS(3.6) b PEO(25) and PS(19) b PEO(6.4) from Polymer Source Inc., Canada, were added to
form the lipid polymer system using chloroform and methanol as solvents: DOPC/ESM was kept
in a 1:1 molar ratio, while the Chol concentration is varied to accommodate the polymer which
were prepared in 0.05, 0.1, 0.5, and 2 mol% of the total lipid mixture. One mg mL 1 stock
solutions of the PS PEO were initially prepared, kept overnight, and were then diluted to 1 µg
mL 1 solution. Appropriate volume of this more diluted polymer solution was added to the DEC
lipid mixture. In the case of the lipid peptide, 2 mol% (of the total mixture) of a 26 amino acid
zinc binding peptide was added to the DEC lipid mixture. The resulting solution was then
exposed to a gentle stream of nitrogen and placed under vacuum overnight to further remove
the solvents. The lipid film was hydrated with 18 M cm Milli Q H2O to a final lipid
concentration of 0.5 mg mL 1 prior to use. Small unilamellar vesicles were obtained by
sonicating the lipid polymer solution to clarity (40 90 min) using a bath sonicator (Cole Parmer,
Montreal, QC). For bilayer preparation, 25 g of the lipid polymer mixture and a final
concentration of 10 mM CaCl2 were deposited on freshly cleaved mica substrates affixed to a
89
liquid cell for 3 4 hours at room temperature. Washing with excess 18 M cm Milli Q H2O
followed the incubation.
6.4 Results and Discussion
6.4.1 Lipid Polymer Morphology
The AFM height images of lipid polymer systems consisting of DEC
(dioleoylphosphatidylcholine/egg sphingomyelin/cholesterol) and PS b PEO (polystyrene b
poly(ethylene oxide)) are shown in Fig. 6. This diblock copolymer with hydrophobic PS and
hydrophilic PEO stretches are used in this study to mimic Pluronics (PEO PPO PEO) and to
simulate peptides of varying degree of hydrophobicity and hydrophilicity. Consistent with
results presented in earlier chapters, phase separation of the liquid ordered, Lo domains
(brighter regions) and of the fluid disordered, Ld phase (darker regions) are observed in pure
DEC bilayers (Fig. 6.1 A).8 10 With the addition of 0.05 mol% PS(3.6) b PEO(25), features of
about 6 nm above the Ld phase are seen (Fig. 6.1 B), which were attributed to aggregation of
the added polymer, most likely consisting of PEO. With higher polymer concentration (2
mol%), a more organized molecular assembly of what appears to be as mountain ridges like
features (Fig. 6.1 C) but actually consists of string of pearl like structures (Fig. 6.1 D) are
obtained. When PS(3.6) b PEO(25) is replaced with PS(19) b PEO(6.4), the assembly of
mountain ridges like features disappeared and the resulting AFM height image is similar to that
of a pure DEC bilayer (data not shown). The absence of the more organized molecular assembly
when greater proportion of PS than PEO is added suggests effective incorporation of the
hydrophobic PS block into the hydrophobic bilayer core.
90
Figure 6.1 AFM height images of pure DEC (A), DEC with 0.05 mol% (B) and 2 mol% (C) PS(3.6) bPEO(25), and zoomed in image of C (D).
The molecular assembly in Fig. 6.1 C and D is analogous to worm like micelles first observed by
Won et al. formed from PEO b PB (poly(butadiene) of about 50 weight % PEO in water at a low
diblock concentration, < 5%.24 In a succeeding paper, polymersomes or tough vesicles made
from diblock copolymers has been demonstrated to be tougher and can sustain greater areal
strain before rupture than phospholipid in natural membrane analogs.25 The enhanced
toughness and reduced permeability of the polymersomes is attributed to the increased length
and conformational freedom of polymer chains compared to lipids. In a study that followed,
worm micelles formed from PEO based nonionic copolymers were shown to be extremely
stable in aqueous medium.26 All these results find its application to the use of these amphiphilic
aggregates in drug delivery applications.
In the experiments, lower polymer concentrations were used to prevent the possibility of
micellization, which was shown to occur at polymer concentrations higher than 5%2 and to
avoid significant restructuring of the lipid bilayer.
91
6.4.2 Nanomechanical Stability of the Lipid Polymer Systems
To assess the mechanical stability of the lipid polymer systems in this study, force mapping at
1500 nm/s tip velocity was performed on DEC bilayers with 20% Chol in the presence and
absence of PS b PEO. Figure 6.2 A and B shows the breakthrough force map and the
corresponding histogram of breakthrough forces, respectively. Consistent with the expected
more ordered organization of the Lo domains, higher breakthrough forces are observed in these
regions compared to the Ld phase. This is clearly illustrated in the bimodal distribution of the
histogram in which the higher mean breakthrough force peak, 3.6±0.60 nN corresponds to the
Lo phase while the lower mean breakthrough force peak, 2.8±0.51 nN to the Ld phase. The
breakthrough force map when 0.05 mol% PS(3.6) b PEO(25) was added, is shown in Fig. 6.3 A. It
is also clear from the histogram (Fig. 6.3 B) the significant shift to higher breakthrough forces
upon the addition of the block copolymer. When the PS chain is lengthened and the PEO chain
shortened (PS(19) b PEO(6.4)), much higher breakthrough forces were obtained (Fig. 6.4 A and
B). These results suggest that the presence of the PS b PEO polymer in the DEC bilayer
enhanced its mechanical stability, as reflected in the significant increase in the breakthrough
forces compared to a pure DEC bilayer. The degree of enhancement is dependent on the length
of the hydrophobic PS chain of the polymer.
Figure 6.2 Breakthrough Force Map (A) and corresponding histogram (B) of a DOPC/ESM/Chol bilayer.
92
Figure 6.3 Breakthrough Force Map (A) and corresponding histogram (B) of a DOPC/ESM/Chol
bilayer with 0.05 mol% PS(3.6) b PEO(25).
Figure 6.4 Breakthrough Force Map (A) and corresponding histogram (B) of a DOPC/ESM/Chol bilayerwith 0.05 mol% PS(19) b PEO(6.4).
6.4.3 Proposed Mechanism for the Observed Enhanced Mechanical Stability
The question of how does the presence of the added polymer led to a significant enhancement
in the mechanical stability can be addressed if the mechanism of the polymer incorporation
into the lipid bilayer is elucidated. Based on the AFM imaging and force mapping results, the
observed increase in the breakthrough forces in Figs. 6.3 and 6.4 can be attributed to two
possibilities: (1) the PS moiety incorporates itself in the bilayer running through the defects or
voids in the acyl chain packing resulting to a more ordered organization and (2) the presence of
the polymer, especially the PEO chains could form a film on the bilayer surface which could
serve as steric stabilizer, helps resist rupture, hence increases mechanical stability.
93
Numerous studies in earlier years have provided a number of possible mechanisms on how
Pluronics or PEO PPO PEO�— both in diblock and triblock forms�— act as steric stabilizers.6,12,15,16
Though no definite consensus was reached, among the common points presented is that
stabilization is achieved when the PPO chain length is commensurate with that of the acyl chain
length in the bilayer. The PEO PPO diblock copolymer is compared to PEG lipid conjugates,
wherein the hydrophobic PPO block functions as an anchoring element while the hydrophilic
PEO remains exposed to the aqueous solution, directed away from the headgroup regions.6,13
The number of monomers in the hydrophobic PPO block has been found to be a critical
determinant of the nature of diblock copolymer lipid bilayer association and that optimal
integration of the polymer is achieved when the PPO chain is comparable in length to that of
the acyl chains. It is noted that the PEO chains orient favorably when the PPO block is fully
inserted into the bilayer.
In the results presented in this chapter, much higher breakthrough forces are obtained when
the PS(19) b PEO(6.4) is used over PS(3.6) b PEO(19). Assuming that the lipid bilayer
hydrophobic core is a good solvent for the PS chains, the Flory radius can be approximated
using the equation:
RF aN 3 / 5 Equation 6.1
where a is the effective length of the monomeric unit and N is the number of monomers in the
chain.16 With a = 0.35 nm and N = 35 and 180, we obtain a Flory radius of about 3 and 8 nm, for
PS(3.6) b PEO(25) and PS(19) b PEO(6.4), respectively. From these approximation, we expect
the addition of PS(3.6) b PEO(25), which has a Flory radius more comparable to the lipid acyl
chain length, to lead to a more stable bilayer. However, the contrary is observed in our
experiments. It is speculated that when the PS block incorporates into the lipid bilayer, it takes
a non spherical conformation which could be in a form of a compact cylinder that situates itself
in between and parallel to the acyl chains or in between the two leaflets of the bilayer, as first
94
suggested by Baekmark et al.2 With the first configuration, it is expected that the PEO chains in
PS(19) b PEO(6.4) has a more ordered orientation (chains extended into the water medium,
perpendicular to the bilayer surface) than in PS(3.6) b PEO(25).
It can be argued that this mechanism would increase the disorder in the bilayer, analogous to
that of host guest interactions,14 especially when the added polymer is considered as an
impurity. However, the observed increase in breakthrough forces discounts this possibility. As
previously suggested, the enhanced hydrophobic interaction of PS and the bilayer core may
actually increase the local order as well as the rigidity of the membrane.14
About a decade ago, a series of papers were presented investigating the interactions of
Pluronics with liposomes.15 17 Similarly, a number of possible mechanisms on the incorporation
of the polymer with the liposome were proposed. If the hydrophobic PPO block anchors the
Pluronic molecule to the membrane, the more hydrophilic PEO chains could either stick out on
the same side of the membrane or could assume a membrane spanning configuration.16
Furthermore, simulations show that the PEO chains have different structures at low and high
concentrations. With increasing copolymer concentration, the calculated density profiles reveal
that the PEO chains extends a further distance from the bilayer surface.27
6.4.4 Lipid Peptide System
When the peptide was added, an even higher mechanical stability was observed with
breakthrough forces now in the range of 20 25 nN (Figs. 6.5 A and B). The peptide used in this
study was synthesized based on the properties of a helical Zn binding peptide. Based on the
amino acid sequence, the peptide used has a lot of basic residues with not much hydrophobic
character. Given this, the results then suggest that the peptide adsorbed onto the bilayer
95
surface, creates a film, which repels the AFM tip, leading to higher force needed for rupture to
occur.
Figure 6.5 Breakthrough Force Map (A) and corresponding histogram (B) of a DOPC/ESM/Chol bilayerwith 2 mol% 26 aa Zn binding peptide.
Grasso et al probed two different types of peptide membrane interactions; one type obliges the
peptide to incorporate itself into the lipid bilayer while in the second case, it is interacting with
the external surface of the membrane.14 The transition temperature, from gel liquid phase, as
studied by DSC, was observed to increase and was attributed to the oriented insertion of the
hydrophobic part of the peptide fragment leading to an increase in order of the acyl chains. It is
inferred that in the case studied in this chapter, the minimal hydrophobic character of the
peptide does not allow it to incorporate itself in the bilayer, but rather interact with the
headgroups on the surface. The speculated favorable interaction between the headgroups and
the basic residues of the peptide could explain the observed significant increase in the
mechanical stability of the bilayer.
96
6.4.5 Control PS(3.6) PEO(25)
Figure 6.6 AFM deflection image of control PS(3.6) b PEO(25).
The AFM deflection image of the control PS(3.6) b PEO(25) spun coated onto a mica surface
shows segmented features which is similar to the assembly of the string of pearls like structure
observed in Fig. 6.1 D.
6.4.6 Implications
It is worth noting the numerous studies in literature, on the association of Pluronics with lipid
bilayers and liposomes, which explores the effect of the molecular architecture and
concentration of the copolymer. However, none of these have yet reported the effect of
Pluronics on the stability of phase separated multicomponent lipid bilayers that mimic
biological membrane. The lipid bilayer Pluronic mimic systems presented in this study pose as
an attractive platform for obtaining fundamental understanding on the role of Pluronics in drug
delivery application.
In an in vivo study, it has been shown that the presence of Pluronic as low as 0.05 wt% has led
to a significant increase on mean bursting membrane tension and the mean elastic area
compressibility modulus of the cells, which are critical parameters that determine strength of
97
the cell.28 The significant change in the cell mechanical properties was attributed to adsorption
of the polymer on the surface as well as incorporation into the membrane. The observed
strengthening of the cell is consistent with an earlier work that evaluated membrane fluidity in
the presence of Pluronics.29 Another mechanism suggested on how a Pluronic can strengthened
the cells is because of its ability to intercalate in the plasma membrane, as a consequence of its
amphipatic nature.21 In the work presented in this chapter of the thesis, the polymer
concentration used is comparable to previously used concentrations in in vivo systems,
examining the mechanical properties of cells in the presence of Pluronics.22,30
6.4.7 Current and Future Work
As a work in progress, the nanomechanical stability of raft forming lipid bilayers consisting of
dioleoylphosphatidylcholine, sphingomyelin, and cholesterol in the presence of the
transmembrane (TM) and juxtamembrane (JM) regions of the human epidermal growth factor
(EGFR) through AFM based force mapping are currently being investigated. The results of the
26 amino acid Zn binding peptide upon incorporation with the lipid bilayer described above
would provide a comparison for the model lipid bilayer�’s mechanical stability in the presence of
EGFR.
98
6.5 References
(1) Mouritsen, O. G.; Bloom, M. Annu. Rev. Biophys. Biomol. Struct. 1993, 22, 145.
(2) Baekmark, T. R.; Pedersen, S.; Jorgensen, K.; Mouritsen, O. G. Biophys. J. 1997,73, 1479.
(3) Killian, J. A.; Salemink, I.; dePlanque, M. R. R.; Lindblom, G.; Koeppe, R. E.;Greathouse, D. V. Biochemistry 1996, 35, 1037.
(4) Shaw, J. E.; Alattia, J. R.; Verity, J. E.; Privé, G. G.; C.M., Y. J. Struct. Biol. 2006,154, 42.
(5) Ganchev, D. N.; Rijkers, D. T. S.; Snel, M. M. E.; Killian, J. A.; de Kruijff, B.Biochemistry 2004, 43, 14987.
(6) Firestone, M. A.; Wolf, A. C.; Seifert, S. Biomacromolecules 2003, 4, 1539.
(7) Otoda, K.; Kimura, S.; Imanishi, Y. Bull. Chem. Soc. Jpn 1990, 63, 489.
(8) Sullan, R.; Li, J.; Hao, C.; Walker, G.; Zou, S. Biophys. J. 2010, 99, 507.
(9) Sullan, R.; Li, J.; Zou, S. Langmuir 2009, 25, 12874.
(10) Sullan, R. M. A.; Li, J. K.; Zou, S. Langmuir 2009, 25, 7471.
(11) Barreiro Iglesias, R.; Bromberg, L.; Temchenko, M.; Hatton, T. A.; AlvarezLorenzo, C.; Concheiro, A. Eur. J. Pharm. Sci. 2005, 26, 374.
(12) Firestone, M. A.; Seifert, S. Biomacromolecules 2005, 6, 2678.
(13) Firestone, M. A.; Tiede, D. M.; Seifert, S. J. Phys. Chem. B 2000, 104, 2433.
(14) Grasso, D.; Milardi, D.; La Rosa, C.; Rizzarelli, E. New J. Chem. 2001, 25, 1543.
(15) Johnsson, M.; Bergstrand, N.; Edwards, K.; Stalgren, J. J. R. Langmuir 2001, 17,3902.
(16) Johnsson, M.; Silvander, M.; Karlsson, G.; Edwards, K. Langmuir 1999, 15, 6314.
(17) Kostarelos, K.; Tadros, T. F.; Luckham, P. F. Langmuir 1999, 15, 369.
(18) Xiong, X. Y.; Tam, K. C.; Gan, L. H. J. Nanosci. Nanotechnol. 2006, 6, 2638.
(19) Batrakova, E. V.; Kabanov, A. V. J. Controlled Release 2008, 130, 98.
(20) King, A. T.; Davey, M. R.; Mulligan, B. J.; Lowe, K. C. Biotechnol. Lett. 1990, 12, 29.
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7 Nanoscale Structures and Mechanics of Barnacle Cement
7.1 Overview
Polymerized barnacle glue was studied by atomic force microscopy (AFM), scanning electron
microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and chemical staining.
Nanoscale structures exhibiting rod shaped, globular, and irregularly shaped morphologies
were observed in the bulk cement of barnacle Amphibalanus amphitrite (=Balanus amphitrite)
by AFM. SEM coupled with energy dispersive x ray (EDX) provided chemical composition
information, making evident the organic nature of the rod shaped nanoscale structures. FTIR
spectroscopy of the bulk cement gave signatures of sheet and random coil conformations.
The mechanical properties of these nanoscale structures were also probed using force
spectroscopy and indentation with AFM. Indentation data yielded higher elastic moduli for the
rod shaped structures as compared to the other structures in the bulk cement. Single molecule
AFM force extension curves on the matrix of the bulk cement often exhibited a periodic
sawtooth like profile, observed in both extend and retract portions of the force curve. Rod
shaped structures stained with amyloid protein selective dyes (Congo Red and Thioflavin T)
This chapter was reproduced in part with permission from Sullan, R.M.A., Gunari, N., Tanur, A.E., Chan, Y.,Dickinson, G.H., Orihuela, B., Rittschof, D., Walker, G.C. Biofouling, 25 (3), 263 275. © 2009 Taylor and Francis.
101
revealed that about 5% of the bulk cement are amyloids. A dominant 100 kDa cement protein
was found to be mechanically agile, using repeating hydrophobic structures that apparently
associate within the same protein or with neighbors, creating toughness on the 1 100 nm
length scale.
7.2 Introduction
7.2.1 Biofouling
Barnacles are common and dominant fouling marine organisms. Fouling assessment studies
have shown that when a ship is heavily fouled, more than 60 % of the fouled areas are covered
by barnacles.1,2 Furthermore, heavy calcareous fouling results in powering penalties of up to
86% at cruising speed.3 The cement barnacles secrete provides one of the toughest and most
durable adhesives in the living aquatic world.4 Removal of barnacles is a practical challenge in
marine ship hull husbandry.5,6 Hydrodynamic forces are typically not enough to detach the
barnacles from a ship�’s hull coated with a biocidal antifouling paint. However, the release of
fouling from coatings based on polydimethylsiloxane, the so called fouling release coatings, is
possible if hydrodynamic conditions are suitable.7 9 The release of fouling is a consequence of
poor adhesion between the adhesives secreted by the fouling organisms and the coating.10
7.2.2 Barnacle removal
As mentioned in chapter 1, biofouling is an ongoing problem and barnacles are among the
predominant culprit and most difficult to eradicate. Understanding the structures, mechanical
properties, and functions of the cement components is therefore crucial in elucidating the
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mechanisms involved in barnacle adhesion. Theoretically, the forced removal of a barnacle
from a surface is typically described as a fracture process, with the parameters contributing to
its detachment from a substrate described by Griffith�’s theorem.11 While Griffith�’s theory was
originally cast in terms of a chemically homogeneous material, where brittle solids fail by
incremental propagation of pre existing cracks, it has also been used for heterogeneous
materials, where cracks initiate at internal joints or faults.11,12 Sun et al. showed that the
fracture stress elastic modulus relation was found to apply for the detachment of barnacles.13
They demonstrated that high barnacle adhesion strength correlates with a high mean adhesive
plaque modulus; they also predicted that the characteristic crack lengths for barnacle adhesive
fracture can be sub microscale, although they did not examine which molecular and
supramolecular structural features of the cement gave rise to glue toughness. It is of interest
therefore to explore the microscale morphology of the adhesive.
7.2.3 Barnacle cement
Barnacle cement is mainly proteinaceous.14 A number of studies report distinct protein
components of the cement of Balanus crenatus and Amphibalanus eburneus (=Balanus
eburneus),14 17 Balanus hameri,14 and Megabalanus rosa.18 The mechanism of adhesion,
however, is a topic of considerable research activity. It has been suggested that underwater
attachment may be due to the multifunctionality of the structural proteins present in the
cement.18 20 Structural proteins could act as either the matrix or reinforcement, similar to that
of a composite material. Protein based adhesives with these kinds of structures require
enormous energy to fracture.21 23 It was previously shown that barnacle cement displays
different morphologies on different silicone surfaces24,25 and two morphologies, opaque and
transparent, sometimes termed gummy and hard, respectively, are known to have a genetic
origin.26 The gummy and hard morphologies exhibit different composite moduli but no
difference in their flexural rigidity.10
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Heterogeneous cements are common in natural adhesives such as those of mussels, Ulva spore,
diatoms, oysters, tubeworms, and some gastropods.27 Natural adhesives that have been
studied previously for both their biological/biochemical and physical/mechanical properties
include the Ulva spore,28,29 the diatoms,30 34 and that of the cyprids (early stage of the barnacle
life cycle).35 38 Different components could contribute distinctive desirable properties for tough
cement. This work examines both the composition and the mechanical properties of the
cement of barnacle Amphibalanus amphitrite (=Balanus amphitrite).39 42 Specifically, the link
between composition and toughness of the cement was explored. A broad range of analytical
tools, including staining by dyes, FTIR, AFM, and SEM with EDX, were employed.
7.3 Materials and methods
7.3.1 Barnacle rearing
Adult Amphibalanus amphitrite (=Balanus amphitrite) on silicone (Dow Corning Silastic T2 or
International Veridian) panels were prepared at Duke Marine Laboratory,26,43 and shipped
overnight to the University of Toronto. The panels were placed in a 10 gallon fish tank filled
with 35 ppt (parts per thousand�— 35 g of ocean mix/1 L of distilled H2O) artificial seawater
(Marineland, Instant Ocean Mix, Ohio) at room temperature under a constant 12 hr light/dark
cycle. Nauplius larvae of the brine shrimp (1.5 to 2 mL) Artemia sp. were fed to the barnacles
daily. Prior to performing experiments, the barnacles were removed from the silicone panels in
shear using a mechanical force gauge (Imada, Northbrook, Illinois), following the procedure in
ASTM D 5618 94,44 to ensure future reference capabilities.
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7.3.2 AFM imaging, force spectroscopy, and indentation
For all measurements, barnacles with opaque white glue were resettled on glass coverslips for
up to two days after being detached from the silicone panels and were kept in 35 ppt seawater
at room temperature. Tapping mode AFM (Asylum Research, MFP 3D, Santa Barbara, CA)
images and force spectroscopy measurements in artificial seawater were made on the cement
remaining on the coverslip after the barnacle was removed in shear. AFM images were
obtained using V shaped silicon nitride cantilevers (Veeco, MLCT AUNM, California) with spring
constants of ~ 0.5 N/m.
For force spectroscopy under tension, V shaped silicon nitride cantilevers (Veeco, MLCT AUNM,
California) exhibiting a nominal spring constant of 0.02 0.03 Nm 1 were used. Force spectra
were recorded at 20 ± 2 ° C. The spring constants were determined by the thermal noise
method45 prior to each experiment. Raw data was processed and expressed as force extension
curves using the Igor Pro software (Wavemetrics, Portland, OR). The �“fly fishing�” method46 of
single molecule force spectroscopy was performed on cement left on the glass substrates after
barnacle removal. In this method, the AFM tip was first lowered close to the sample (without
indentation) and slowly retracted until a binding occurrence was observed. The bridging protein
strand was then repeatedly stretched and relaxed. The resulting force curves were analyzed
with the same software as above. In the fly fishing method, macromolecules extending into
solution, if only temporarily, are studied.
In the indentation experiments, AFM topographic images were first obtained in intermittent
contact or tapping mode to select individual morphologies prior to indentation measurements
in contact mode. An AFM silicon nitride probe (Veeco, DNP, California) with a force constant of
0.58 Nm 1 and a tip radius of ~25 nm was applied in force mode. Force indentation curves were
105
obtained from the deflection of the AFM tip as a function of the piezo vertical displacement
plots. The same software as above was used to extract the elastic modulus of the bulk cement.
7.3.3 Scanning electron microscopy energy dispersive x ray (SEM EDX)
Barnacles with opaque glue were removed from the silicone panels and reattached onto
aluminum foil for more than one week, still being fed with nauplius larvae of the brine shrimp
Artemia sp. daily. The barnacles were detached and the remaining cement on the foil was
coated with carbon for imaging with the SEM (S 570, Hitachi, Japan). EDX spectra (Oxford
Instruments, Inca Systems, United Kingdom) were then obtained on the fibrillar features
observed under the SEM. In all SEM EDX measurements, a 5 keV beam energy was used,
resulting in an interaction volume of ~ 1 um3.
7.3.4 Fourier transform infrared (FTIR) spectroscopy
Barnacles with opaque glue were detached from Veridian panels and reattached onto CaF2
windows for two days and kept in 35 ppt seawater at room temperature. Reattached barnacles
were forced off the CaF2 windows before IR spectroscopy was performed on the remaining
cement. IR spectra at 2 cm 1 resolution on the cement were collected using an FTIR
spectrometer equipped with a liquid nitrogen cooled MCT detector and dry air purge (Nicolet,
Nexus 470, Minnesota). The region from 1600 to 1700 cm 1 (corresponding to the amide I band)
was examined in detail. A clean CaF2 window with no cement sample was scanned to provide a
background spectrum. The resulting spectra were processed via Fourier self deconvolution,
followed by peak fitting, as per the well established method of Byler and Susi.47 Fourier self
deconvolution using the Griffiths Pariente method (GRAMS, Salem, NH) was used in order to
resolve the multiple secondary structure bands within the amide I band. Peak fitting using the
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Levenberg Marquadt method (GRAMS, Salem, NH), assuming a Lorentzian line shape for the
deconvoluted spectrum and Gaussian peak shapes for the fit, was performed in order to
determine the integrated areas of the resolved bands. The integrated areas were then used to
estimate the percentage of each major type of secondary structure (e.g. helix, random coil,
sheet, turns) found in the barnacle protein. Uncertainties when using this approach are
typically 10%.48 Bands were assigned to their respective secondary structures based on the
summary of amide I band IR spectral features in H2O by Barth.49
7.3.5 Chemical staining with amyloid selective dyes
Thioflavin T (ThT) and Congo Red were used as purchased (Sigma Aldrich, Madison). ThT
binding was carried out at room temperature as described in Mostaert et al.50 Barnacles with
opaque glue were detached from Veridian panels and reattached onto CaF2 windows for a
maximum of two days and kept in artificial seawater at room temperature. The cement
remaining on a CaF2 window after the barnacle was removed was stained with 10 µM ThT for 5
minutes. It was then rinsed with excess pure water (18 M cm) (Millipore, Milli Q), and air
dried. Images were then taken with a laser scanning confocal microscope (Leica, TCS SP2,
Germany). For Congo Red staining, the barnacles were reattached to glass coverslips for 2 days
and then removed; glass was used instead of CaF2 due to the birefringence of CaF2. The cement
remaining on the coverslip was stained with 0.5 % Congo Red in 50 % ethanol in water for 5
minutes. It was then rinsed with excess 18 M cm MilliQ water and blown dry with N2 gas prior
to examination using the same microscope as above, now equipped with cross polarizers.
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7.4 Results
7.4.1 AFM imaging and force spectroscopy
Barnacles that were resettled on glass substrates tended to leave ~1 mm wide glue deposits
below the periphery of their baseplate, consistent with the location of the glue ducts. After
pushing these barnacles off the substrate, the residual glue on the substrate was imaged.
Typical topography images are shown in Fig. 7.1. Mesh structures such as those reported earlier
by Weigemann and Watermann25 were observed. AFM images of the barnacle cement in both
35 ppt artificial seawater (Fig. 7.1 A) and in air (Fig. 7.1 B) showed this mesh morphology.
Figure 7.1 AFM topographic images of the barnacle cement in 35 ppt sea water (A) and in air (B). Grayscale provides height reference (right hand of each image). Images are 15 x 15 m2.
The mesh structure in Fig. 7.1 is the general picture of the adhesive when scanned in a larger
area. Upon zooming in, the mesh in Fig. 7.1 A is composed of a mixture of different structures
such as those shown in Fig. 7.2 A E. Fig. 7. 2 A shows clusters of globular structures with
diameters ranging from 60 100 nm. Pearl like arrangements (indicated by the ellipse) of the
globular aggregates are clearly seen. Smaller globular structures (10 30 nm) and a small, rod
like structure of 11 nm in diameter and ~300 nm length were also observed (Fig. 7.2 B). Fig. 7.2
C shows a larger, more regular rod shaped structure. An unstructured aggregate (Fig. 7.2 D) is
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also seen in the matrix (Fig. 7.2 E). Thus, the bulk cement is composed of the mesh comprised
of the structures in Fig. 7.2 A C and of the matrix (Fig. 7.2 E) with some unstructured aggregates
(Fig. 7.2 D).
Figure 7.2 The bulk barnacle cement is composed of both the mesh structure and the matrix. Globularaggregates (A), smaller rod like and smaller globular features (B), and larger, more regular rod shapedstructure comprise the mesh (C). An unstructured aggregate (D) is seen in the matrix (E). The dot in Fig.7.2 C E indicates the point of indentation.
There is no specific pattern or localization of those structures. The relative fraction of those
occurring is variable from one barnacle sample to another, even from within the same species.
Figure 7.3 Force extension profiles obtained when pulling on the bulk cement of Amphibalanusamphitrite (=Balanus amphitrite) showing complex and irregular force extension profile with ruptureforces as high as 3.5 nN.
109
We examined the cement nature of the glue in two ways using AFM. In the first type of
measurement, the AFM tip was indented into the matrix (Fig. 7.2 E), in between the fiber like
structures of the mesh and was pulled out. When the AFM tip was pulled away from the
matrix, the force extension profile resembled a sawtooth pattern. The sawtooth pattern was
not periodic when pulling on these molecular aggregates (Fig. 7.3). Therefore, we undertook a
second type of pulling experiment, AFM single molecule manipulation. The �“fly fishing�”
method46 was used to avoid picking up multiple molecules in the extension experiment,
without indenting between pulls. A representative force extension profile is shown in Fig. 7.4 A.
Regular sawtooth like features were observed, demonstrating that the molecule can be
repeatedly unfolded. Fit of such a sawtooth curve to the worm like chain model gave a
persistence length (lp) of 0.35 0.05 nm, comparable to the value of pulling an individual titin
protein (lp = 0.4 nm).46 The histogram in Fig. 7.4 B shows a bimodal distribution in the gap
lengths between the rupture events in Fig. 7.4 A, with average separation lengths of 35 8 nm
and 56 9 nm.
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Figure 7.4 Single molecule pulling on the cement sample (in between the fiber like structures of themesh (Fig. 7.2 E). Force extension fly fish curve showing the fit to the worm like chain model giving apersistence length of 0.35 0.05 nm (A). The corresponding histogram of the observed separationlengths show strong peaks at 35±8 nm and 56±9 nm (B). A schematic showing the extra work needed(shaded area) to rupture the bonds due to the modular nature of a single protein, as compared to asingle protein chain without a modular nature (lower curve) and follows a worm like chain behavior (C).
The ability of the non covalent bonds to reform, and of the cement units to reassemble, is
evident in the force extension profile of the matrix in Fig. 7.5 (only the extend curve is shown
for clarity). A regular sawtooth like pattern observed in the particular extend curve signifies
reassembly of regularly spaced units of the macromolecule. In this measurement of pulling on
the matrix after indentation, a single molecule was coincidentally observed, as evidenced by
the regular spacing in the force extension profile.
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Figure 7.5 Identification of self assembly property of the barnacle cement. Force extension profileshowing refolding of the protein after unfolding and shearing; only the extend curve was shown forclarity.
7.4.2 Elastic moduli of the cement
AFM nanoindentation was performed on the rod shaped structure (Fig. 7.6 A), the unstructured
aggregate (Fig. 7.6 B), and the matrix (Fig. 7.6 C), to determine and compare their elastic
moduli. The spot indicates the point of indentation. The values of elastic moduli were obtained
by considering load indentation dependence for a paraboloidal shaped tip given by equation
7.1, which is an extension of the Hertz model:
2/32 )1(3
4vREF Equation 7.1
where F is the loading force in N, E is Young�’s modulus in Pa, R is the radius of the curvature of
the tip in m, is the indentation in m, and is the Poisson�’s ratio, taken here as 0.5.13,51
Individual fits for the larger, more regular rod shaped structure, the unstructured aggregate,
and the matrix and the corresponding histogram of elastic modulus are shown below the
images of each structure in Fig. 7.6 A C. The corresponding force indentation plots were fitted
112
by Sneddon mechanics.51 The solid line represents the fit to indentation data by the
paraboloidal tip model (Eqn. 7.1). The larger, more regular rod shaped structure (Fig. 7.6 A) was
found to have an elastic modulus in the range of 20 90 MPa; the unstructured aggregate, 0.20 2
MPa; and the matrix, 1 10 MPa. Insets in Fig. 7.6 A and B are the linescans of the larger, more
regular rod shaped structure and unstructured aggregate, respectively. For both rod shaped
and unstructured aggregate structures, the height of the indented feature is ~250 nm and the
rod has a diameter of ~600 nm. Indentations were less than 20% of the sample thickness so
finite thickness effects that can affect the predicted modulus are minimal.52
Figure 7.6 In situ determination of the elastic modulus of the barnacle cement. Elastic modulusdistribution of individual nanostructures/components as observed by AFM (topmost row). Fit to theforce indentation plot (middle row) with the corresponding histogram (third row) of the elastic modulusfor larger, more regular rod shaped structure (A), the unstructured aggregate (B), and the matrix (C).Insets in Fig. 6B and 6C are the line profile of the larger, more regular rod shaped structure andunstructured aggregate, respectively. For both rod shaped and unstructured aggregate, the height ofthe feature is ~250 nm.
113
7.4.3 Scanning electron microscopy energy dispersive x ray (SEM with EDX)
In order to determine the composition of the rod shaped features observed in the AFM, SEM
images coupled to EDX were also obtained. The rod shaped features observed under SEM (Fig.
7.7 A) showed high counts of carbon, nitrogen, and oxygen in the EDX linescan spectrum (Fig.
7.7 B) suggestive of the organic nature of the components. Fig. 7.7 C D shows an area mapping,
indicating the spatial location of the elements in a given region. Consistent with the EDX
linescan in Fig. 7.7 A B, the rod shaped features in Fig. 7.7 C showed fair signals of C, N, and O
(Fig 7.7 D) coming from the rod shaped structures, again indicative of the organic nature of the
components. The high counts of Al for both the EDX linescan and area mapping is attributed to
the background aluminum foil where the cement is attached. The Mg background is similar to
Al. The F elemental map is shown as a control. The elemental x ray lines are indicated for each
line scan and map (a = ).
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Figure 7.7 SEM images and EDX spectra of the barnacle cement resettled on aluminum foil. SEM imageshowing the rod shaped structures and the corresponding (A), EDX linescan spectra showing high countsof elemental carbon, nitrogen, and oxygen coming from the rod shaped structures (B). SEM images ofthe rod shaped structures (C) and corresponding elemental maps of the elements (D). Fair signals of C,N, and O coming from the rod shaped structures are obtained. The elemental x ray lines from which theEDX signals were derived are indicated for each line scan and map (a = ).
7.4.4 Fourier transform infrared (FTIR) spectroscopy
Transmission IR spectra of bulk barnacle cement on CaF2 gave signatures of sheet and a large
fraction of random coil conformations. Figure 7.8 shows the deconvoluted FTIR spectrum of
barnacle Amphibalanus amphitrite (=Balanus amphitrite), fit with 4 bands (standard error:
0.0003, correlation value R2 = 0.999). To check the validity of this fit, multiple fits were
performed on the same deconvoluted FTIR spectrum, from 2 to 11 bands. The standard error
did not undergo significant improvement for fits with more than 4 bands. Assignment of the 4
bands to their corresponding secondary structure resulted in the identification of a large
random coil component at 1655 cm 1 (79%), and smaller low and high frequency sheet
115
components, at 1623, 1637, and 1692 cm 1. The distribution of secondary structures obtained
from the FTIR spectra by the peak fitting algorithm is summarized in Table 7.1. Note that this
distribution applies to the barnacle cement as a whole, which is made up of a number of
different proteins in unknown proportions.
Figure 7.8 FTIR spectra of the bulk cement from Amphibalanus amphitrite (=Balanus amphitrite). Topspectrum is the original data prior to processing, offset for clarity. Bottom spectrum is the deconvoluteddata, with a 4 band peak fit showing peaks corresponding to random coil structure (1655 cm 1) and low(1623, 1637 cm 1) and high (1692 cm 1) frequency sheet components.
Secondary structure band positions in H2O (cm 1) Relative Peak Area
Fraction (%)
Low frequency sheet components (1623, 1637) 17
Random coil (1655) 79
High frequency sheet component (1692) 4
Table 7.1 IR peaks and the corresponding fraction of the observed secondary structures found in agummy barnacle cement sample. Almost 80% of the cement exhibits a random coil structure signature,while the remaining 20% correspond to sheet components.
116
7.4.5 Chemical staining with amyloid selective dyes
Chemical staining of the barnacle cement with amyloid selective dyes was done to check the
possible presence of amyloids in the cement proteins. Fluorescence of the rod shaped
structures was evident in the presence of ThT (Fig. 7.9 B), and an apple green birefringence was
observed in the cement stained with Congo Red (Fig. 7.9 D) under cross polarized light. Images
of the barnacle cement in the absence of ThT (Fig. 7.9 A) and the cement stained with Congo
Red in the bright field (Fig. 7.9 C) were shown for comparison.
Figure 7.9 Chemical staining images of the barnacle cement with amyloid selective dyes. Confocalimages of the barnacle cement on a CaF2 window without (A) and stained with (B) ThT. Images of thebarnacle cement with Congo Red in bright field (C) and in polarized light (D). Fluorescence and applegreen birefringence of the rod shaped structures is observed when the barnacle cement is stained withThT and Congo Red, respectively.
117
7.5 Discussion
7.5.1 Adhesivity of the barnacle cement
AFM pulling data on the matrix of the barnacle cement revealed a complex and irregular profile
with rupture forces as high as 3.5 nN (Fig. 7.3). Subsequent curves (3rd, 15th) further illustrate
the irregularity of the force extension profiles. The complex and irregular force�–extension
profiles also illustrate the heterogeneous nature of barnacle cement. Barnacle cement is
composed of several proteins, six of which have been identified, ranging in size from 7 165
kDa.16 19 Based on Kamino�’s non proteolytic method in rendering the cement of Megabalanus
rosa soluble, the two major proteins which have bulk functions are the 52 and 100 kDa
proteins.19 These major proteins were found to be highly hydrophobic and what gives the
cement its extremely insoluble nature. The 100 kDa protein in particular has been found in
several other barnacle species.
Based on the extension length in Fig. 7.3, we conclude that there are multiple molecules being
pulled (a 100 kDa protein pulled at 80% of the contour length would extend ~285 nm). To avoid
picking up several molecules in a single pull, a �“fly fishing�” method, as described in section 7.3
was conducted. Occasionally for shorter pulls, regularly spaced sawtooth profiles were
obtained. The sawtooth like feature observed in Fig. 7.4 suggests a multi domain structure of
the proteins, comprised of at least one non fibrillar component (e.g. random coil component).
A multi domain structure has been previously suggested21,22 and multiple polyproteins giving
rise to peaks in register has been demonstrated.53
Kamino et al. reported a repeating hydrophobic/hydrophilic pattern within the 100 kDa protein
of Megabalanus rosa.18 Fig. 7.10 A (after ref 18), illustrates the proportion of hydrophilic (Hp),
118
hydrophobic (Hb), and neutral (N) regions along each segment of the 100 kDa protein. Ten
repeats (r1 r10) were observed. The contour length of the 100 kDa protein (~357 nm) divided
into ten segments can explain the ~35 nm periodicity we observed in the force spectra. Figure
7.10 B is a representation of one segment showing the three regions with the hydrophobic
region approximately twice as long as both hydrophilic and neutral regions. The set of rupture
forces with two recurring separation lengths at 35 and 56 nm in Fig. 7.4 A could correspond to
the rupture of a hydrophobic region associating with its nearest neighbor and with its next
nearest neighbor, respectively. Figures 7.10 C D illustrate this, providing a novel mechanism for
strong, adaptable glue. Figure 7.10 C shows the rupture of a hydrophobic region (Hb,
represented by the solid circles) in a segment associating with its nearest neighbor (e.g., 2 3 in
Fig. 7.10 C), giving rise to the 35 nm gap length observed in the force extension profile. Figure
7.10 D illustrates another type of rupture, coming from the association of a hydrophobic region
with its next nearest neighbor (e.g., 2 4). Given that the longer gap between force transitions in
Fig. 7.4 A that we observe (56 nm) is less than the distance of two �“Hb�” regions (~ 70 nm), we
infer that the length of protein folded in such an isolated hydrophobic region (e.g., 3 in Fig. 7.10
D) is ~ 14 nm. If two chains are pulled in parallel or if the self association within a chain does
not occur at regular intervals or if domains do not follow a two state model for unfolding then
the ruptures of interchain bonds would appear irregularly in reference to the regular transitions
seen for a single chain. The histogram shows evidence of such effects. Nonetheless, strong
peaks at 35 and 56 nm are observed.
119
Figure 7.10 Novel mechanism for providing strong, adaptable glue. Figure is after Kamino (JBC, 275 (35),27360 27365) showing the proportions of hydrophilic (Hp), hydrophobic (Hb), and neutral (N) regionsalong each segment of the 100 kDa protein of Megabalanus rosa (A). A representation of one segmentshowing the three regions (Hp, Hb, N); the hydrophobic region is approximately twice as long as theother two regions (B). A schematic illustrating the rupture of a hydrophobic region (Hb) in a segmentassociating with its nearest neighbor (2 3), giving rise to the 35 nm gap length observed in the forceextension profile (C) and rupture of the association of a hydrophobic region with its next nearestneighbor (2 4) giving a longer gap length (56 nm) but less than the distance of two �“Hb�” regions (~ 70nm) (D). It can be inferred from here that the length of protein folded in such an isolated hydrophobicregion (e.g. 3 in Fig. 10 D) is ~ 14 nm. The solid circles represent a hydrophobic region in a proteinsegment.
According to Kamino,20 the hydrophobic interactions are responsible for the extremely
insoluble nature of the cement and not the other types of cross linking. This was based on the
non proteolytic treatment (i.e. heat denaturation and treatment with high concentrations of
urea and guanidinium hydrochloride) of the barnacle cement that led to a complete loss of its
adhesive strength. In a recent study, amyloid like peptides are found in the primary structure of
120
the bulk cement.54 It was suggested that the control of hydrophobic interactions by
conformational change of the protein is a possible mechanism for the self assembly of the
barnacle cement. The major proteins in the barnacle cement are hydrophobic and as such are
thought to provide a stable molecular structure by forming a hydrophobic core in the molecule.
Prediction of the secondary structure of the 100 kDa protein of Megabalanus rosa by Kamino
indicates an 87% of the protein exists in a sheet conformation.18 sheet structures are well
known to resist shear and give rise to force transitions. The magnitude of the rupture forces
that we observed were low (~ 40 100 pN), compared to transitions of, for example the type 3
subunit of fibronectin (~ 90 200 pN),55 which assumes barrel structure. The force transitions
observed in Fig. 7.4 A do not occur with ever increasing force, but instead, occasionally a high
force transition is followed by a low force transition and vice versa. Although this could arise
from the stochastic nature of domain unfolding dynamics, quite likely it is in part due to
cooperative effects between domains. The domains within a chain can associate with each
other and thereby influence each other�’s unfolding force. The stochastic nature and low force
of unfolding also partly explains the widths of separation length peaks in Fig. 7.4 B, ~35 nm and
~56 nm, respectively.
We now address the question, which part of the chain attaches to the AFM tip? Kamino noted a
trend in the isoelectric point (pI) of the 100 kDa protein of Megabalanus rosa; it has a high pI
(pI=12) in the C terminus region and a low pI (pI=5) in the N terminus region.18 The AFM tip that
we used, Si2N3, is negatively charged at pH = 7.7 ± 0.2, which was characteristic of the artificial
seawater solution. Therefore, it is reasonable to assume that the tip is more likely to attach to
the positively charged N terminal end of the 100 kDa protein and significantly less likely to
attach to the slightly anionic C terminal region. We suggest that the overall trend in the rupture
forces could also be affected by the variation in pI along the 100 kDa protein, where the extent
of charging affects how much the protein is pre stressed from like charge repulsion in a given
region, which reduces the applied force necessary to unfold that region.
121
Fig. 7.4 C is a schematic showing the extra work needed (shaded area) to rupture the bonds
when the protein has a modular nature, as compared to a single protein chain (lower curve),
which follows a worm like chain behavior. The unfolding process then represents a non elastic
dissipative process that enhances the material strength. The sawtooth profiles observed in the
force extension curves of the barnacle cement were also observed by Fantner et al in human
bone studies.21 In their work, the fracture of the bone is said to follow a sacrificial bond
mechanism. Briefly, when the molecule is stretched, energy is dissipated in the cement through
the rupturing of the sacrificial bonds, before the main structural link is broken. The presence of
these sacrificial bonds increases the energy needed to rupture the material.
Another interesting feature of the cement matrix studied here is the occurrence of the
sawtooth pattern in the extend portion of the curve as well (Fig. 7.5). This suggests self
assembly property of the cement.23 The process would serve to reform a portion of the
cement's strength and is an important mechanical feature of natural adhesives56 and most
tough materials. The regular gaps shown in Fig. 7.5 indicate a reformation of a regular
structure.
The AFM indentation on some of the morphologies seen in Fig. 7.2 A E gave a wide range of
elastic modulus values as shown in Fig. 7.6. The elasticity of the cement is heterogeneous. From
the force indentation plot of the rod shaped structure in Fig. 7.6 A, the depth of indentation
changes only slightly as the force is increased. In the case of the unstructured aggregate and of
the matrix Fig. 7.6 B C, the depth of indentation increases as the force is increased. This is
reflective of the more compliant nature of these morphologies. These results indicate the stiffer
nature of the larger, more regular rod shaped structure than both the unstructured aggregate
and the matrix. This is also evident from the magnitude of their elastic moduli. The rod shaped
122
structure has an elastic modulus in the range of 20 90 MPa, which is similar in range to what
has been observed for the amyloid fibrils in insulin (5 50 MPa).57
Materials with higher elastic modulus typically require more stress to fracture.11 The higher
elastic modulus value for the rod shaped structure suggests that it has a stiffer nature with
higher cohesive forces. This causes the load applied to it at any point to be distributed across its
length, making a composite more resistant to fracture. A simple way to understand how the
rod shaped structures could contribute to the cement strength is to consider that they
distribute load across a relatively wide region of the bulk cement. Our results do not allow us to
determine how important this effect is for barnacle cement toughness, but their presence
needs to be considered in a detailed model for fracture.
Kamino et al. previously suggested that the 100 kDa protein present in the barnacle cement of
Megabalanus rosa may be similar to the proteins involved in the formation of amyloid fibrils.18
This was based on the abundance of sheet structures in the 100kDa barnacle protein, its
alternating hydrophobic and hydrophilic profile, and its very insoluble nature. It was reported
that the pattern of alternating polar and nonpolar residues in a cross structure is essential in
the formation of the insoluble amyloid fibrils.58 Recently, amyloid like sequences has been
found in the primary structure of the protein in the bulk cement of the Megabalanus rosa.20
Moreover, the 100kDa cement protein of Megabalanus rosa is particularly rich in Isoleucine
(Ile), Valine (Val) and Threonine (Thr) residues, which are the three amino acid residues
reported to have the highest propensity to form the sheet structure.59
Chemical staining of the barnacle cement with amyloid selective dyes was done to check for the
possible presence of amyloids in the cement proteins of Amphibalanus amphitrite (=Balanus
amphitrite). The apple green birefringence under polarized light when stained with Congo Red
(Fig. 7.9 D), and fluorescence of ThT (Fig. 7.9 B) indicate the presence of an amyloid fibril
123
structure in the barnacle cement. Both ThT and Congo Red dyes are known to be amyloid
selective.60 63 The fluorescence observed in the presence of ThT and the apple green
birefringence with Congo Red are related to the cross core structure, both of which are
indications of the binding of the dyes to an amyloid fibril structure in the barnacle cement.
A variety of rod shaped structures (or fiber like features) were observed under the optical
microscope, but only a small portion gave an apple green birefringence and fluorescence when
stained with Congo Red and ThT, respectively. This indicates that the amyloid fibrils in the
barnacle cement only comprise a small fraction of the bulk cement. Such a small fraction is still
significant since, in most fiber reinforced composite materials, fibers need to be present in only
a minute fraction in order to lead to a considerable increase in the toughness of the material.
Non amyloid fibers could also play a similar mechanical role.64,65
7.6 Conclusion
Different nanoscale structures were observed in the bulk cement of barnacle Amphibalanus
amphitrite (=Balanus amphitrite) through AFM; a mesh composed of rod shaped, threadlike,
and globular structures and a matrix with unstructured aggregates. Indentation data suggested
a stiffer nature of the rod shaped structure over the other components of the bulk cement. The
EDX spectrum of those rod shaped structures is suggestive of its organic nature. The FTIR
spectra supported the presence of a sheet conformation while results of chemical staining
with both ThT and Congo Red confirmed the presence of a small fraction of amyloid fibrils in
the bulk cement. Molecular pulling experiments on the matrix indicate a modular nature of
some of the proteins (e.g. 100 kDa protein), which also contributes to an increase in the
cement�’s resistance to fracture. A model on how hydrophobic interactions contribute to the
tough nature of this protein component of the barnacle cement is presented.
124
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127
8 Conclusions and Recommendations
Since its inception, the use of atomic force microscopy as a probe of nanomechanics, has found
a wide range of applications in the fields of material science, nanotechnology, and life science.
This work has shown that AFM indentation, AFM based force mapping and single molecule
pulling experiments have helped address important questions in membrane biophysics as well
as in biofouling. The direct correlation of bilayer structure with its nanomechanical properties
presented in this thesis is the first of such report that involves multicomponent lipid mixture of
biological importance. The work pointed out how crucial nanoscale mechanoelastic properties
of the membrane can be in understanding its complex biological function. As such, it represents
a significant contribution to the biophysics of lipid membranes. The study on barnacles
provided answers on how micro and nano scale assembly of proteins resulted to highly
organized structures in the barnacle cement. This work also shed light on how protein assembly
contributes to the unique properties of this highly adhesive material.
8.1 Multicomponent Lipid Bilayers
Knowledge of the structures as well as of the mechanical stability of physiologically relevant
lipid bilayers is important in understanding the functions of biological membranes. In chapter 3
of this thesis, high resolution force mapping coupled with AFM imaging has quantified the
nanomechanical properties of the coexisting phases in a supported planar multicomponent
lipid bilayer of biological relevance and have provided a direct correlation of these properties
with the bilayer structure. The 2D visual maps of the intrinsic breakthrough forces, elastic
moduli, and adhesion generated from the custom made batch analysis code have enriched
what is known on the mechanical stability of the PC/SM/Chol bilayers, which are raft mimics.
128
Insights into the mechanical stability of the coexisting phases as well as heterogeneity on the
nanoscale, not apparent from AFM images, were obtained. This work has demonstrated the
effectiveness of AFM based force mapping as a valuable complement to other biophysical
techniques such as imaging and spectroscopy, as it provides unprecedented insight into lipid
membrane mechanical properties and functions.
The AFM tip induced rupture platform can be used to study several other aspects of model
membranes. For one, changes in the phase organization in a localized area can be probed by
not breaking all the way through the membrane but by only indenting into it (e.g. holding the
AFM tip at a constant extension away from the bilayer surface). To aid in probing this localized
transformations, AFM coupled with Fourier transform infrared (FTIR) spectroscopy can be
employed, as has been demonstrated in a recent study.1 This technique could help verify the
hypothesis that the Lo phase transforms into an Ld before rupture. This will also be an excellent
way to track the conformational changes of proteins when incorporated/reconstituted in model
membranes.1
Another interesting direction using the experiments described in this thesis is to compare
breakthrough forces on model lipid bilayers which typically used paraboloidal shaped silicon
nitride probes with rupture forces on living cells using AFM nanoneedles2 or AFM tips
terminated with carbon nanotubes3. For an in depth analysis, differences on the two systems
such as the following must be considered: (1) thickness/diameter of the sample being probed;
Model lipid bilayers are about ~4 6 nm in height (including the water layer) whereas living cells
range from 3 10 m. This is particularly important when Hertz model is used to fit the
indentation region. (2) heterogeneity of the samples; While model lipid bilayers also exhibit
heterogeneities on the nanoscale4, the anisotropy exhibited by live cells is unequivocally
greater. (3) substrate effect; Due to the smaller height of the lipid bilayer, the effect of
substrate can be more pronounced compared to that of a live cell. Existing studies showed that
the force needed in rupturing a live cell membrane is orders of magnitude smaller than
breaking through a lipid bilayer.2,3 Live cells probed with ultrathin needles (sharpened AFM tips)
129
of 200 300 nm in diameter gave rupture forces of about a nN2 while 100 200 pN penetration
forces were observed when fortified carbon nanotube tips of 30 40 nm diameters3 are used.
Correlating the bilayer�’s line tension with the magnitude of the rupture force is also another
track that the experiments in this thesis can take as a future experiment. On longer timescales
commencing formation of phase segregated lipid bilayers, line tension becomes an important
factor in determining the domain size distribution.5 Elucidating the relation of the membrane
mechanical stability as a function of the line tension may provide a better understanding of lipid
organization in cellular membranes.
In chapter 4, results of force mapping experiments provide quantitative evidence of the
robustness of the ceramide enriched domains as well as the competitive binding of ceramide
for cholesterol enriched liquid ordered phase. An interesting finding in this work is that
ceramide enriched domains exhibit a packing behavior not typical of gel phases. This study can
provide further insights on the roles that ceramide enriched domains have played in a wide
range of cellular processes. Ceramide�’s highly stable nature can be correlated with its proposed
function in the cell in facilitating the assembly of smaller domains to form larger rafts.
Sphingomyelin in cells undergoes hydrolysis through the action of Sphingomyelinase which
consequently generates ceramide. Studying the nenomechanical stability of the model
membrane with this enzymatic generation of ceramide is a good comparison to the direct
incorporation (of ceramide) study described in this chapter. The breakthrough forces obtained
from these two different modes (ceramide direct incorporation vs. in situ generation) might
provide a good comparison on the nature of these ceramide enriched domains.
130
In chapter 5, the influence of different cholesterol content on the morphology, lateral
organization, and nanomechanical stability of multicomponent lipid bilayers was examined.
Force mapping on these bilayers reveal decreasing nanomechanical stability of the coexisting
phases with increasing Chol content, confirming the fluidizing effect of Chol in membranes. The
obtained Ea values agree well with reported values for biomembrane and model membrane
fusion processes. Results of this AFM tip induced rupture of bilayers study can serve as a model
platform for studying relevant cellular processes, such as membrane fusion or poration where
the AFM tip could represent proteins, viruses, or other trigger particles that mediate pore
formation and bilayer rupture. For instance, a model of how viruses go into the cell can be
developed.
In chapter 6, the addition of model polymers to the phase segregated lipid bilayers led to an
increase in the bilayer stability. Significant increase in breakthrough forces was observed when
PS b PEO were incorporated into DEC bilayers. The increase was mainly attributed to the
degree of incorporation of the hydrophobic PS chain into the bilayer core. This study has shed
light on the long standing argument and the inconsistencies on the mechanisms presented on
how Pluronics alter membrane permeability and stability.
8.2 Barnacle Proteins
From our study of the polymerized glue of the barnacle adhesive of Balanus amphitrite, we
observed nanoscale structures with robust mechanical properties (stiff nature), which we
attribute to be a possible source of the cement�’s strong adhesion on the molecular scale.
Presence of a small fraction of amyloid like rod shaped structures, which may provide a specific
insight on the toughness of the barnacle cement, was also identified. Results of the molecular
131
pulling experiment revealed a modular nature of the cement�’s matrix that could contribute to
an increase in the cement�’s resistance to fracture.
A possible future work that can be done utilizing results of this study is to perform force
mapping on barnacle adhesive samples in order to obtain a spatial distribution of amyloid like
structures as well as to determine how local mechanical properties vary on a given barnacle
sample. Also, a macroscopic model for fracture mechanics of barnacle adhesive can be
developed.
8.3 References
(1) Verity, J. E.; Chhabra, N.; Sinnathamby, K.; Yip, C. M. Biophys. J. 2009, 97, 1225.
(2) Obataya, I.; Nakamura, C.; Han, S.; Nakamura, N.; Miyake, J. Nano Lett. 2005, 5,27.
(3) Vakarelski, I. U.; Brown, S. C.; Higashitani, K.; Moudgil, B. M. Langmuir 2007, 23,10893.
(4) Sullan, R. M. A.; Li, J. K.; Zou, S. Langmuir 2009, 25, 7471.
(5) Johnston, L. J. Langmuir 2007, 23, 5886.
132
9 Appendix
9.1 Rupture Activation Energy Calculation
The universal rupture kinetics model introduced by Butt et al. was used to calculate the
activation energy ( Ea) of rupturing lipid membranes.1,2 Below is a brief summary of the
calculations.
An ensemble of N0 simultaneous, identical AFM tip film indentation experiments occurring at a
constant tip loading rate v (loading rate is calculated using the approaching velocity of the base
of the cantilever) is considered. N is the number of films that remain intact after time t has
elapsed. Time is referenced to t=0, the point at which tip sample contact begins. The change in
number of intact layers dN after time dt can be expressed as:
dN k r Ndt Equation 9.1
where kr is a time dependent constant. Dividing by N0, Equation 9.1 is converted to:
dP kr(t)Pdt Equation 9.2
where P=N/N0 is the probability of finding a tip on top of the intact film. Integrating Equation
9.2,
lnP(t) kr(t )dt 0
tEquation 9.3
133
Assuming that the rate constant is associated with an activated process following an Arrhenius
Law:
TkFE
rB
a
Aetk)(
)( Equation 9.4
where Ea is the activation energy necessary for the formation of a hole or fracture in the film
that is large enough to initiate rupture and let the tip break through, F is the yield force, kB is
Boltzmann�’s constant, T is the Kelvin temperature, and A is the frequency at which the tip
attempts to penetrate the film.
B
Time is transformed to force, using the latter�’s relation with loading rate
F=kvt Equation 9.5
where k is the tip spring constant. Combining Equation 9.3 9.5
lnP(F)Akv
eEa (F )kBT dF
0
FEquation 9.6
The probability distribution that this creates represents the distribution of yield forces. If this
distribution is narrow, then at the mean yield force F0, the probability P(F)=0.5. With ln(0.5)=
0.693, Equation 9.6 can be rewritten:
vA
0.693ke
Ea (F )kBT dF
0
F0Equation 9.7
Ea (F0) kBT ln0.693k
AdvdF0
Equation 9.8
If the v(F0) relationship is known then its derivative can be calculated to obtain activation
energy�’s dependence on yield force. This is a useful equation, as v and F0 are experimental
observables.
In the case where the distribution of mean breakthrough forces is relatively narrow ( F/F0 << 1,
where F is the half width of the yield force distribution), F0 can be obtained from the
134
histogram of breakthrough forces; the loading rate demonstrates a logarithmic dependence on
the breakthrough force, namely,
F0 a b lnv Equation 9.9
where a and b can be derived from geometric parameters and mechanics arguments3, and
more importantly are parameters obtained from fits to experimental data, the dependence of
activation energy on loading rate can be explicitly expressed as:
Abk
bFaTkFE Ba
60.1ln30.2)( 00 Equation 9.10
When this relation is extrapolated to zero mean yield force (F0 = 0), it provides the intrinsic
activation energy of the lipid membranes.
In the experiments, the probability distribution of forces is the breakthrough force histogram,
which shows bimodality corresponding to the two phases (liquid ordered domains, Lo and fluid
disordered phase, Ld), in a series of loading rates. In the calculations, a k of ~0.25 N/m, a
temperature of 296.2 K, and A equal to the cantilever�’s resonant frequency under water (~15 ×
103 Hz) were used.
9.2 References
(1) Butt, H. J.; Franz, V. Phys. Rev. E 2002, 66, 031601.
(2) Loi, S.; Sun, G.; Franz, V.; Butt, H. J. Phys. Rev. E 2002, 66, 031602.
(3) Franz, V.; Loi, S.; Muller, H.; Bamberg, E.; Butt, H. H. Colloids Surf. B 2002, 23,191.
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