Doctoral_Thesis_slides

Structural changes of the cell adhesion protein, fibronectin,in cell culture and on surfaces measured using

fluorescence resonance energy transfer

Loren Baugh

Final ExamSeptember 4, 2003

University of WashingtonDepartment of Bioengineering

Advisor: Viola Vogel Supervisory Committee: Thomas A. Horbett

Patrick S. StaytonFrancois Baneyx

Graduate Student Representative: Mohamed A. El-Sharkawi

Background – The Problem

Proteins in crystals or in solution

Lack of techniques to measure protein structure in physiological environments Lack of knowledge of how proteins work


Mechanically-regulated proteins

Mechanical force Biological response

Macroscopic level – Tissue maintenance, diseaseCellular level – Changes in gene expressionMolecular level – How is mechanical force converted to a biochemical signal?

On Off

Protein

Cell




Affect the performance of surgical implants, tissue culture substrates, biosensors

How do surfaces control protein activity?

Protein-biomaterial interactions

AdsorptionOn Off

Cell

Protein

Surface A Surface B



Background – Fibronectin

Polymeric fiber, cell adhesive

Cell adhesion, migration, proliferationDevelopment, wound healing

Unregulated fibronectin Fibrotic diseasesLack of fibronectin Cancer metastasis

Cell attachment and growth on biomaterials

Primary biological function: Mediates cell attachment to surrounding environment

Soluble, inactiveBlood, body fluids

Fibronectin

Fibronectin matrix

450 kDa

Single-molecule elasticityModule unfolding / refolding

Background – Fibronectin

Cell

How do structural changes affect the presentation of binding sites?

Unknowns: Structure of Fn in fibers, mechanisms of polymerization, elasticity

Binding sites buried

Binding sites exposed

Fibronectin fibers and single molecules

are elastic

Fibronectin

Overview

Fluorescence resonanceenergy transfer (FRET)

OVERALL GOAL: Study structural changes of fibronectin that control its activity

(1) Calibrate and test energy transfer in solution

(2) Apply to study changes in protein structure in cell culture and on solid surfaces

Insight into how proteins are regulated by mechanical forces and by interactions with biomaterials

Outline

I. Calibration and testing of FRET in solution

II. FRET to measure changes in fibronectin structure in cell culture

III. FRET to measure changes in fibronectin structure on surfaces

D = DonorA = Acceptor

Background – Fluorescence resonance energy transferWhat is FRET?



Normal fluorescence





FRET

Dipole-dipole interaction~ Through-space NOESY in NMR~ Two antennae

FRET is sensitive to nanometer-scale changes in donor-acceptor distance

BackgroundWhy is FRET useful?

“Spectroscopic ruler”

BackgroundWhy is FRET useful?

Sensitive range

Typical Ro = 2-7 nmShown: Ro = 6 nm

dos Remedios, Moens.J Struct Biol (1995)115(2): 175-185

Background – FRET between a single donor-acceptor pair

(Ro = 2 - 7 nm)

Structural changes undetected

Background – FRET between a single donor-acceptor pairis not sensitive to full range of structural changes of a LARGE protein

(Ro = 2 - 7 nm)

Background – FRET between a single donor-acceptor pairis not sensitive to full range of structural changes of a LARGE protein

Structural changes undetected

50:50 mixturedonors and acceptors

buffer, pH 7, RT, 4 h

25%

25%

25%

25%

~ 100% efficient 2 D, 2 A per Fn

MethodsLabel fibronectin so FRET is sensitive to its structural changes

Labeling Scheme 1 : Donors + acceptors on free cysteines (D/Acys)

Fibronectin

~ 160 nm

SH SH

SH SH

buffer, pH 7, RT, 4 h

25%

25%

25%

25%

> 10 nm~ 100% efficient 2 D, 2 A per Fn

50:50 mixturedonors and acceptors

Outside range of FRET


Labeling Scheme 1 : Donors + acceptors on free cysteines (D/Acys)

~ 160 nm

SH SH

SH SH

Step 1: Cysteine-reactive donors, pH 7, 4 h

4 D, 7 A per FnStep 2: Amine-reactive acceptors, pH 8.3, 4 h

Increase number of donor-acceptor pairs per protein

Increase sensitivity of FRET


Labeling Scheme 2 : Donors on free cysteines, acceptors on random amines (Dcys Aamine)

Fibronectin

~ 160 nm

SH SH

SH SH


4 D, 7 A per FnStep 2: Amine-reactive acceptors, pH 8.3, 4 hFibronectin

~ 160 nm

Oregon Green 488 Tetramethylrhodamine

Donor Acceptor

1.1 nm 1.5 nm

Largest fluorophore and Fndrawn to scale



SH SH

SH SH


4 D, 7 A per FnStep 2: Amine-reactive acceptors, pH 8.3, 4 h

Alexa Fluor 488 Alexa Fluor 594

Donor Acceptor

1.0 nm 1.8 nm

Largest fluorophore and Fndrawn to scale



SH SH

SH SHFibronectin

~ 160 nm

Secondary structure (-sheet)

Test FRET vs. known structural changes

Denaturant

[Guanidine-HCl] (M)0 1 2 8

Fn

Circular dichroism spectroscopy

Part I : Calibration and testing of FRET - Results

[Guanidine-HCl] (M)1 2 80



Labeling scheme 2: Dcys Aamine

1 2 80



[Guanidine-HCl] (M)

Labeling scheme 2: Dcys Aamine



FRET

Secondary structure (-sheet)

D/Acys

1 2 80

Dcys Aamine

FRET is sensitiveto a larger rangeof structural changes

[Guanidine-HCl] (M)

Dcys Aamine



NaCl

Dithiothreitol

Thermolysin

1 M, RT, 1 h

0.5 M, RT, 1 h

1 mg/mL, 70° C, 4 h

Changes in FRET are consistent with known changes in fibronectin structure

D/AcysDcys Aamine

Part II : FRET to measure fibronectin structure in cell cultureMethods

Rinse, Fix cells,

Mount samples

0.5 h 1-24 h

37° C 37° C

FRET microscopy / spectroscopy

+

Plate cells

NIH 3T3 fibroblasts

Add labeledprotein

Excessunlabeled protein

to preventinter-molecularenergy transfer

10 g/mL Fn**90 g/mL Fn

Plate cells

NIH 3T3 fibroblasts

0.5 h 1-24 h

37° C 37° C

Add labeledprotein

Excessunlabeled protein

to preventinter-molecularenergy transfer

10 g/mL Fn**90 g/mL Fn



Disrupt celltension

Cytochalasin D, 10 M

1 h

37° C

Rinse,Fix,

Mount

Part II : FRET to measure fibronectin structure in cell cultureMethods

+

Slit, Mirror Slit, Grating

Wavelength

Pos

ition

Window, Mirror

~ 1 m

D A

Color Structure

Excite donors,collect donor (green) and acceptor (red)emission

Methods – FRET microscopy and spectroscopy

Imaging Spectroscopy

Results – FRET microscopy

Red High IA / ID High FRET Compact

Green Low IA / ID Low FRET Extended

Excite donors, collect donor and acceptor emission

Fibronectin is more extended in fibers than on the cell surface

D

A

I

Cell surface (red)

Fibers (green – range of values)

10 m

Color ofemissionin image

Proteinstructure

Baneyx, Baugh, Vogel. PNAS (2001) 98(25): 14464-14468.

D

A

I

Cell surface (red)

Fibers (green, range of values)

10 m

Results – FRET spectroscopy

Fibronectin is compact on the cell surface

Fibronectin exists in a range of extended conformations in fibers

Spectra in black: Labeled Fn in 0, 1, 8 M guanidine-HCl solutions

Fibronectin is converted by cells into an extended structure in early stages of polymerization

Red Compact

Green Extended

Structure changes during polymerization

Populations ofprotein

Results – Time-lapse FRET imaging

Extension of Fnby cell receptors exposes Fn-Fn binding sites

Schwarzbauer, Sechler.Curr Opin Cell Biol (1999)11(5): 622-6270.

Fibronectin Polymerization

Fibers are pulled out of stationary fibronectin clusters by cell integrins, driven by the actin cytoskeleton

Pankov et al. J Cell Biol(2000) 148(5): 1075-1090.

Fibronectin Polymerization

Role of mechanical force in polymerization

Cytochalasin D

10 M

1 h37° C

Disrupt cell tension

I Fibers Range of FRET

Measure FRET from 1 m segments along fibers of treated and untreated cells

FRET to study the structure of fibronectin in fibers

Test effect of cell tension on conformation of fibronectin in fibers

Range of conformations

IAID

Distance along fiber (m) • FRET was uniform along some fibers, varied along others

• Mean FRET level in fibers was low fibronectin extended

Cytochalasin D



Conformation of fibronectin in fibers in untreated cells

Untreated

10 m

IAID

Distance along fiber (m)

Buffer

8 M Guanidine

IAID


IAID


• Treatment with cytochalasin D caused an increase in FRET in fibers

Release of tension allowed fibronectin in fibers to refold

Untreated Cytochalasin D

C

Buffer

8 MGuanidine

Cyto-chalasin D

10 m

Untreated



Conformation of fibronectin in fibers in cells treated with cytochalasin D

Intermediate FRET

Low FRET

Fibronectin fiber elasticityModule unfolding / refolding

Single-molecule AFM force-spectroscopy

Fn-Fn sliding

Fibronectin un-bending / re-bending

Module unfolding / refolding

Intermediate FRET

Low FRET

High FRET

Intermediate FRET

Intermediate FRET

Intermediate FRET

Fibronectin fiber elasticityModels of elasticity

Flattening of the RGD loop and loss of cell integrin binding activity

Fibronectin as a force-regulated adhesive switch

Appliedforce

Fixed

Steeredmoleculardynamicssimulations

Functional consequences of module unfolding / refolding

Krammer et al.PNAS (1999) 96(4): 1351-1356.

FnIII-10FnIII-9

Appliedforce

Fixed

Regulation of proteins by mechanical force

• 2% of all animal proteins share fibronectin’s force-sensitive FnIII modules

• Many adhesion proteins share the RGD loop

Module unfolding and refolding – Common mechanism of elasticity and force-regulated protein function?

FnIII-10FnIII-9

Proteins adsorbed to biomaterialsOverall picture of protein structure on surfaces is limited

Adsorption

Antibody binding

ElectronMicroscopy

Fibronectin

Circular dichroism spectroscopyDifferential scanning calorimetry Tryptophan fluorescenceAtomic force microscopy

Other techniques

Artifacts:Protein sprayed

from glycerolsolution

FRET spectroscopy

1 h, RT

Add labeledand unlabeled

fibronectin

Rinse by serial dilution Measure FRET, Total fluorescence

intensity

0.1 – 25 g/mL (total)

FRET to measure fibronectin structure adsorbed to surfacesMethods

SurfacePreparation

Adsorption

Glasscoverslips Sulfuric acid

No-ChromixFluorosilanedeposition

SiO- CF3(CF2)5(CH2)2Si

Eliminate inter-molecular energy transfer

adv = 5adv = 110

FRET to measure fibronectin structure adsorbed to surfacesResults

(Density)Fn on fluorosilane > (Density)Fn on glass

Amount offibronectinadsorbed

Undesired energy transfer between adjacent adsorbed proteins

Labeled Fn + unlabeled Fn = 25 g/mL

Labeled fibronectin

only


Labeled Fn + unlabeled Fn = 25 g/mL

Labeled fibronectin

only

Inter-molecular energytransfer eliminated

Lower FRET on glass

Fibronectin is more extended on glass


Adsorption

Glass Fluorosilane

Strong hydrophobic interactions trap Fn in a compact state

Fibronectin-surface interactions

SiO- CF3(CF2)5(CH2)2Si

FRET results Fibronectin is more extended on glass than fluorosilane

Hydrophilic, negatively charged Hydrophobic, neutral

Charge-charge interactions disrupt Fn’s compact state

Previous studies Antibody binding Secondary structure – circular dichroism, calorimetry

How do surface interactions affect fibronectin’s cell adhesion activity?

Adsorb labeledfibronectin

1, 25 g/mL

1 hRT

Rinse Plate cells45 min37C

Remove unbound cells

NIH 3T3 fibroblasts

Serum-free medium, 10 mg/mL albumin

(block non-specific adhesion)

Count

Cell attachment – MethodsCompare Fn’s cell adhesion activity on glass and fluorosilane

Glass1 g/mL, Labeled Fn

Fluorosilane1 g/mL, Labeled Fn

Imageof cells

Cell attachment – Results

Why was cell attachment lower on fluorosilanedespite a greater density of adsorbed Fn than on glass?

Why the concentration-dependence?

AdsorptionCell

Glass Fluorosilane

Fibronectin’s conformation on glass was favorable for cell attachment

Above

Above Above

Below

Density of exposed sites (above/below threshold for attachment)

• Minimum density of exposed integrin binding sites required for cell attachmentMassia, Hubbell. J Cell Biol (1991) 114(5):1089-100.

• Fibronectin more extended on glass Greater avg exposure of RGD sites

• (Density)Fn on fluorosilane > (Density)Fn on glass

Summary of Results

IAID

Cell surfaces

Synthetic materials

Extracellular matrix fibers

Solution

PBS NaCl 1 MGdn

DTT 8 MGdn

4 MGdn

Future Directions

• FRET microscopy and spectroscopy applied to other proteins

Spatial resolution of different protein conformations

• FRET combined with site-directed mutagenesis

Novel labeling sites in strategic locations

Conclusions

Native structures Structures on biomaterials

Force - regulated Unregulated

Compact,Inactive

Extended,Adhesive

IntermediateConformations

Acknowledgements

Viola Vogel

Tom HorbettPat StaytonMohamed El-Sharkawi

Vogel LabGretchen BaneyxMeher AntiaJohn ClemmensMichael HalterBilly LittleLichia FengEmilie Clemmens

Funding - NIH

Family and friends

Thank you

Documents

Doctoral_Thesis_slides