Molecular Biomechanics: The Molecular Basis of How Forces ...Molecular Biomechanics: The Molecular Basis of How Forces Regulate Cellular Function GANG BAO, 1 ROGER D. KAMM,2 WENDY

Molecular Biomechanics: The Molecular Basis of How Forces

Regulate Cellular Function

GANG BAO,1 ROGER D. KAMM,2 WENDY THOMAS,3 WONMUK HWANG,4 DANIEL A. FLETCHER,5

ALAN J. GRODZINSKY,2 CHENG ZHU,1 and MOHAMMAD R. K. MOFRAD5

1Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA;2Departments of Mechanical and Biological Engineering, MIT, Cambridge, MA 02139, USA; 3Department of Bioengineering,University of Washington, Seattle, WA 98195, USA; 4Department of Biomedical Engineering, Texas A&M University, College

Station, TX 77843, USA; and 5Department of Bioengineering, University of California, Berkeley, CA 94720, USA

(Received 6 September 2009; accepted 16 February 2010; published online 2 March 2010)

Abstract—Recent advances have led to the emergence ofmolecular biomechanics as an essential element of modernbiology. These efforts focus on theoretical and experimentalstudies of the mechanics of proteins and nucleic acids, andthe understanding of the molecular mechanisms of stresstransmission, mechanosensing and mechanotransduction inliving cells. In particular, single-molecule biomechanicsstudies of proteins and DNA, and mechanochemical cou-pling in biomolecular motors have demonstrated the criticalimportance of molecular mechanics as a new frontier inbioengineering and life sciences. To stimulate a moresystematic study of the basic issues in molecular biomechan-ics, and attract a broader range of researchers to enter thisemerging field, here we discuss its significance and relevance,describe the important issues to be addressed and the mostcritical questions to be answered, summarize both experi-mental and theoretical/computational challenges, and iden-tify some short-term and long-term goals for the field. Theneeds to train young researchers in molecular biomechanicswith a broader knowledge base, and to bridge and integratemolecular, subcellular and cellular level studies of biome-chanics are articulated.

Keywords—Mechanobiology, Force, Cytoskeleton, Mecha-

notransduction, Protein conformational change, Molecular

motors.

MOLECULAR BIOMECHANICS:

AN EMERGENT FIELD

Living cells are dynamic systems that performintegrated functions including metabolism, control,sensing, communication, growth, remodeling, repro-duction and apoptosis (programed cell death). Duringthe past few decades, extensive studies have elucidated

the structure, mechanical responses and biologicalfunctions of cells in different organs and tissuesincluding, for example, lung, bone, cartilage, bloodvessels, and skeletal and cardiac muscles. These studieshave led to a better understanding of how the biolog-ical functions of a cell are regulated by mechanicalforces or deformation. They have also demonstratedthe central role that forces play in the initiation andprogression of numerous diseases such as atheroscle-rosis, arthritis, asthma, to name a few. However, todecipher the fundamental mechanisms of force-induced control of cellular function, more systematicstudies of deformation, structural dynamics andmechanochemical transduction in living cells and bio-molecules are needed.

As the basic unit of life, living cells perform anenormous variety of functions through synthesis,sorting, storage and transport of biomolecules;expression of genetic information; recognition, trans-mission and transduction of signals; and conversionbetween different forms of energy. Many of these cel-lular processes involve mechanical force, or deforma-tion, at the cellular, subcellular and molecular levels.For example, biomolecular motors and machinesconvert chemical energy into mechanical work in per-forming their diverse range of functions. During cellmigration, contractile forces are generated within thecell in order for the cell body to move forward. Thesecontractile forces, in combination with the adhesion ofcells to extracellular matrix (ECM) through focaladhesion complexes enable cells to sense the stiffness ofthe surrounding substrate and respond to it. Manynormal and pathological conditions are dependentupon or regulated by their mechanical environment.Some cells, such as osteoblasts and vascular cells, aresubjected to specific forces as part of their ‘native’physiological environment. Others, such as muscle andcochlear outer hair cells,61 perform their mechanical

Address correspondence to Gang Bao, Department of Biomedi-

cal Engineering, Georgia Institute of Technology and Emory Uni-

versity, Atlanta, GA 30332, USA. Electronic mail: gang.bao@bme.

gatech.edu

Cellular and Molecular Bioengineering, Vol. 3, No. 2, June 2010 (� 2010) pp. 91–105

DOI: 10.1007/s12195-010-0109-z

1865-5025/10/0600-0091/0 � 2010 Biomedical Engineering Society

91

function either by converting an electrical or chemicalstimulus into mechanical motion or vice versa.

Of particular importance is the ability of cells tosense mechanical force or deformation, and transducethese mechanical signals into a biological response.96

For example, endothelial cells can recognize the mag-nitude, mode (steady or pulsatile), type (laminar orturbulent) and duration of applied shear flow, andrespond accordingly, maintaining healthy endotheliumor leading to vascular diseases including thrombosisand atherosclerosis.17,54 Vascular smooth muscle cellsin the arterial wall remodel when subjected to pressure-induced wall stress. Fibroblast cells ‘crawl’ like aninchworm by pulling the cell body forward usingcontractile forces. Bone alters its structure to adapt tochanges in mechanical environment as occurs, forexample, in spaceflight. Stem cells sense the elasticityof the surrounding substrate and differentiate intodifferent phenotypes accordingly.22 These and otherexamples vividly demonstrate the ability of cells tosense and respond to their local mechanical environ-ment. However, little is currently known about thefundamental molecular mechanisms by which cellssense mechanical force or deformation, and transducethe mechanical signal into a biological response.Ample evidence suggest that there are specialized‘force sensors’ on the cell membrane that detect the

mechanical signal, however little is known about howthese sensors work (see reviews in Kamm andKaazempur-Mofrad46 and Vogel and Sheetz88). Asillustrated in Fig. 1 using numerical simulation of amechanosensitive ion channel, one class of mem-brane-localized mechanosensors is stretch-activatedion channels on the cell membrane that change theirconductance in response to forces.29 These ion chan-nels could sense increases in membrane tension as aresult of applied mechanical load and open, thusconverting mechanical force exerted on the cell mem-brane into electrical or biochemical signals.39,78 How-ever, it is not yet clear if the force-induced activation ofion channels is the dominant mechanism for mecha-notransduction. Integrin molecules or other proteinssuch as vinculin and talin in the focal adhesion com-plex may serve as force sensors and transducers41,53,60

as illustrated in Fig. 2 in which the molecular dynamicssimulation of the force-induced activation of talin’svinculin binding site is shown. Talin, an essentialstructural protein in the focal adhesion complex, con-tains the N-terminal five-helix bundle in the roddomain with a known cryptic vinculin binding site 1(VBS1). The perturbation of this stable structurethrough elevated temperature, destabilizing mutationand mechanical force activates vinculin binding.52

However the underlying molecular mechanism is only

FIGURE 1. Schematic of numerical simulation of a mechanosensitive ion channel with (a) small and (b) large opening under theaction of membrane tension. Left: side view showing the lipid bilayer (white region with red phospholipid head groups) in anaqueous (blue) environment. Right: End view showing the membrane pore. Reproduced with permission from Yefimov et al.93

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beginning to be explored. Yet another possibility isthat forces are transmitted via the cytoskeleton toremote sites within the cell or nucleus where they elicita response.35,42 There is a critical need to identifydefinitively the molecular mechanisms of mechano-transduction in living cells that are common to most, ifnot all, cell types.

As an emerging field, molecular biomechanicsintegrates mechanics, molecular biology, biophysics,biochemistry and biomolecular engineering, andencompasses three broadly defined and inter-relatedareas: (1) the molecular mechanisms of mechanosen-sing and mechanotransduction; (2) the mechanics ofbiomolecules, either individually or as part of a largercomplex; (3) molecular motors or machines and theiruse in bio/nano-devices, including mechanochemical

coupling and interfacing issues. There is no doubt thatprogress in these areas will have a tremendous impacton the life sciences and our understanding of diseaseprocesses.

Studies of force sensing and mechanotransductionin cells are inevitably related to the mechanics of bio-molecules (proteins and nucleic acids), and theseinclude the molecular analysis and constitutive modelingof single biomolecules and multi-molecular complexes.Specifically, it is important to characterize how thethree-dimensional structural rigidity of DNA, RNAand proteins control their deformation under variousloading conditions—stretching, twisting, bending andshear conditions—and how such deformation altersDNA condensation, gene replication and transcrip-tion, DNA–protein/RNA–protein interactions, protein

FIGURE 2. Molecular dynamic simulation of the force-induced activation of talin’s vinculin binding site (VBS1) (red ribbon withorgan spheres). Helix H1 (blue ribbon), H2 (transparent yellow), H3 (transparent tan), VBS1 (red ribbon), H5 (green ribbon),hydrophobic residues of VBS1 (orange VDW; also the vinculin-binding residues), hydrophobic residues of H5 (white VDW), andsome important polar residues (stick representation with color denoting the atom type). Polar residues are labeled on the figures.(a, b) side view; (b, d) top view. (a and c) Before the conformational transition, the hydrophobic residues of VBS1 are hidden in thehydrophobic core. (b and d) At increased force, the hydrophobic residues rotate and become exposed to solvent. Hydrogen bondsbetween H5 and VBS1 are broken. The hydrophobic residues, or the vinculin binding residues, point into the page in (a) and pointto left in (b). (c) Conformation at t = 0.86 ns viewed from top. The V-shaped VBS1 hydrophobic residues are packed within thehydrophobic core of TAL5 (cyan dotted lines). (d) Conformation at t = 9.24 ns showing VBS1 rotation. The hydrophobic residuesH5 (white VDW) fit into the ‘V’ of the VBS1 hydrophobic residues (orange VDW). Reproduced with permission from Lee et al.52

Molecular Biomechanics 93

function, protein–protein interaction, and protein–ligand interaction.5–7,14,48,51,53,71 These research topicsencompass bond formation, reaction rates, and thethermodynamics and kinetics of biomolecular interac-tions. While there have been extensive single-moleculestudies of the mechanics of DNA, to date only verylimited theoretical and experimental studies of themechanics of proteins have been conducted. A betterunderstanding of the mechanical behavior of proteins,nucleic acids and other macromolecules will providethe opportunity to decipher the fundamental mecha-nisms of mechanotransduction, and to predictmechanical function of protein complexes (includingfilaments, focal adhesion complexes, and subcellularstructures) in living cells.

With the recent advent of biotechnology andnanomedicine, there is an increasing need tounderstand mechanochemical coupling in biomolecu-lar motors and enzymes, and to uncover the engi-neering design principles of proteins as nanomachines.Since DNA and proteins may be used as componentsof hybrid nanobiosystems, the optimal design of suchsystems inevitably requires an understanding of themechanics of biomolecules, as well as how the indi-vidual components interact with each other. A specificexample is the development of nanodevices utilizing, orpowered by, biomolecular machines that can producelinear, rotary, or even reciprocating motions such askinesin, dynein and F1-ATPase25 (Fig. 3). The use ofcurrent technologies (e.g., batteries, electromagneticmotors or hydraulic energy sources) are often unableto meet the needs of functionalized nanoscale inor-ganic machines with moving parts, especially when thenanodevices are used in vivo. On the other hand, pro-gress in molecular biology has revealed intriguingfeatures of the structures, mechanisms and functions ofmany biomolecular motors, including kinesin, myosin,dynein, ATP synthase, DNA polymerase and RNA.37

These biomotors directly convert chemical energyderived from ATPase activities into mechanical forceor motion with high efficiency. With their nanometersize, they have the potential for use in multifunctional,self-powered nanosystems.76 How to integrate biomo-lecular motors with other nanodevices to performspecific mechanical, biochemical or biological func-tions in a controlled fashion remains a significantchallenge.

MAJOR ISSUES AND NEW OPPORTUNITIES

It is now widely accepted that mechanical and bio-chemical functions of living cells are highly integratedin controlling the phenotype of the cell in healthand disease. How cells sense mechanical forces and

deformation, and convert and combine such signalswith biochemical responses are not well understood.Living cells are dynamic and their structures canchange in response to mechanical load. This raisesmany fundamental questions essential to biomechan-ics: How do forces applied to a cell, either directly orthrough cell–cell or cell–matrix adhesion sites, inducereorganization of the cytoskeleton, thus changing itsmechanical properties? How do the dynamics ofcytoskeleton affect cell spreading, rounding, crawling,and adhesion? How does the interaction between ECMand focal adhesion complexes transduce a mechanicalsignal (force or deformation) into cells? Answeringthese and other questions will be crucial inunderstanding the structural basis of cellular function.To date, the molecular mechanisms of force sensing andmechanotransduction remain elusive. While numerousmechanisms have been proposed, a strong candidatefor the molecular mechanism of mechanosensing and

FIGURE 3. Two types of molecular motors. (a) Linear motor:Two linear molecular motors, kinesin and dynein, are shown.Both walk along a microtubule, but in opposite directions. Inthis schematic the motors are shown attached to vesicles forintracellular transport along a neuronal process (reproducedwith permission from Vale85). (b) Rotary motor: Schematic of abacterial flagellar rotary motor. Rotational speed is over100,000 rpm and it measures just 50 nm across (Image pro-vided courtesy of A. Ishijima, Tohoku University, Japan).

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mechanotransduction is protein deformation, or proteinconformational change under force.41,51,53,87,95,96 Theunique three-dimensional structure (i.e., conformation)of a protein largely determines its function. However,proteins in a cell are deformable and can assume dif-ferent (altered) conformations under physical forces.Just as proteins can transform from a native or biolog-ically active state to a denatured or inactive state inresponse to small changes in temperature or pH in itsenvironment, the application of mechanical forces canlead to protein domain conformational change and evenunfolding, thus affecting protein–protein and protein–DNA recognition, binding/unbinding, enzymaticactivity, causing changes in downstream biochemicalprocesses that trigger intracellular signaling pathwaysand ultimately control cellular behavior. The underlyingreason is that the three-dimensional geometry and sur-face chemistry local to the binding pocket of a receptor–ligand pair or the protein–DNAbinding site contributessignificantly to the characteristics of their binding.Goodconformationalmatches usually lead to strong and long-lasting bonds. However, the conformational match atthe binding site may change when the protein domainsare deformed or unfolded under mechanical forces. Incertain cases, an applied force can alter the affinity andlifetime of a receptor–ligand pair. In some other cases,protein deformation can expose (or bury) the bindingsite, thus switching between the ‘on’ and ‘off’ states ofprotein, as illustrated by the extension and unfolding offibronectin.28 In vertebrate muscle, the giant elasticprotein titin is involved in strain sensing via its C-ter-minal kinase domain (TK) at the sarcomeric M-bandand contributes to the adaptation of muscle in responseto changes in mechanical strain.69

To understand the essential roles of protein con-formational change in mechanosensing and mechano-transduction, theoretical and experimental studies needto be carried out, first to understand how forces aredistributed throughout the cell via its protein networksand molecular complexes. This information then needsto be coupled with studies to analyze the constitutivebehavior of proteins, including how proteins deformunder different mechanical loads, such as tension,compression, shear, torsion, and their combinations,and how such deformations are related to proteinstructural rigidities. It is necessary to study proteindynamics, including domain motion, the rate effect,and to quantify how the dynamics of proteins, i.e., themodes and time scales of protein motions and defor-mations, are determined by the structural features ofproteins. It is necessary to demonstrate experimentallythat protein deformation (i.e., conformational changeunder force) occurs in live cells under physiologicalconditions, and it is a key event of mechanosensingand mechanotransduction. It is also necessary to study

how protein deformation affects protein–protein,protein-DNA, and receptor–ligand interactions, andhow such deformation is directly related to humandiseases.

In the past 10 years, enormous strides have beenmade in the field of molecular biomechanics, includingmost notably the development of methods to applyforces in the range of 0.001 pN–10 nN to single mol-ecules or small molecular complexes (Table 1). Themagnitude of the forces under consideration inmolecular biomechanics studies is typically in therange of 0.1–1000 pN. During this time, a smallnumber of model systems have been extensively stud-ied, including elastic molecules such as titin56 andfibronectin,64 receptor ligand pairs such as streptavi-din–biotin,94 selectin-PSGL-1 and FimH-mannose andmotor proteins such as kinesin and various myosins.37

These studies have pushed the envelope for methodsdevelopment and for understanding the principlesunderlying molecular biomechanics. This creates theopportunity for the community to apply these methodsand approaches to current medical and technologicalproblems.

Medical and Biological Applications

An opportunity exists to gain a better understandingof diseases in which single molecules or molecularcomplexes change function with the application ofmechanical force. Many diseases in which molecularbiomechanics plays a central role are diseases thatinvolve pathologies inmechanotransduction. For exam-ple, it is well known that atherosclerotic plaques formin regions of low and oscillatory wall shear stress.91

Even in patients who are biochemically susceptible toatherosclerosis (e.g. due to LDL levels), most of theirarteries may be protected from the disease due to theirshear stress environment. Understanding this mecha-nism may enable alternative treatments for the diseaseto be identified by providing targets for new drugs thatwould mimic the protective effects of high steady shearstress. Mechanotransduction may also play a role in thepathobiology of asthma. It has been shown that airwayepithelial cells sense compressive forces and respond by

TABLE 1. Specifications for three methods commonly usedto manipulate single molecules (adapted from Neuman

et al.62).

Specification AFM Magnetic trap Optical trap

Bandwidth (Hz) 1000 10–1000 50–5000

Stiffness (pN nm�1) 1–105 10�6 0.005–1

Spatial resolution (nm) 0.1–1 2–10 0.1–5

Force range (pN) 5–103 10�3–104 0.1–100


activation of several intracellular signaling pathwaysincluding the epithelial growth factor (EGF) receptorpathway.82 Airway smooth muscle cells respond toboth external mechanical stress and their own con-traction by remodeling and stiffening the cytoskeleton,which in turn increases stress. This suggests that apositive feedback loop might exist between stiffeningand increased stress tending to exacerbate asthmaticconditions, and that bronchodilators function bydecreasing contraction to break the cycle.20 Mechano-sensing by organelles such as cilia allow detection offluid flow in the inner ear, kidneys and other locationsand malfunctions in the cilia cause a variety of dis-eases.11 For example, polycystic kidney diseases involvemutations in proteins that localize to the cilia,32 sug-gesting that the detection of fluid flow by the cilia isessential to proper renal function. However, themolecular mechanism(s) of how the mutations affectcilia mechanobiological function is not known. Perhapsthe most widely recognized example of mechanotrans-duction in disease is that bones or connective tissuerequire mechanical forces to heal. Loss of forces due toimmobilization, bed rest or even low gravity can causedisease or limit healing.73 Mechanosensing is alsoinvolved in cancer. First, matrix stiffness regulates theability of cancer cells to invade.2,66 Second, it has beenhypothesized21 that cancer cells may metastasize in partbecause they exhibit aberrant mechanosensing,30 whichallows them to become adhesion independent and ableto travel around the body. A drug that interferes withthe tension-independent signaling by this pathway mayhave fewer side effects than traditional chemotherapydrugs that kill all dividing cells.8 In each disease men-tioned here, the actual mechanotransducers—the pro-teins that sense mechanical force and transduce themechanical signal to a biological response—are gener-ally not known. What is known is that certainmechanical forces lead to the activation of many pro-cesses including signaling pathways, transcription fac-tors and changes in gene expression. However, thesesame processes are also involved in a wide variety ofother regulatory responses and thus would make riskydrug targets due to numerous side effects. In contrast,identifying the actual mechanosensor and the mecha-nism of force sensing and mechanotransduction mightprovide for a drug target that is specific to the me-chanosensing process.

Other diseases involve a wide range of proteins thatare subjected to mechanical forces, often outside thecell, in various bodily compartments. In some cases,the proper functioning of these proteins is needed tomaintain normal function in high-force situations. Oneexample is the involvement of proteoglycans in disease.These molecules contain high molecular weightnegatively charged polysaccharide chains called

glycosaminoglycans (GAGs) attached to a core protein.Large proteoglycans containing 10–100 GAG chainsare found in musculoskeletal and cardiovascular con-nective tissues where compressive or osmotic forcesact.43 Diseases involving these connective tissues ofteninvolve the proteolytic degradation of constituentproteoglycans, resulting in loss of tissue-level mechan-ical function. An example is the ~3 MDa proteoglycan,aggrecan, found in cartilaginous tissues and the inter-vertebral disc, shown in its monomeric form in Fig. 4.63

The high negative charge density of the linear GAGchains results in molecular level electrostatic repulsionforces that significantly contribute to tissue compres-sive stiffness and osmotic fluid retention. Glycosami-noglycans such as hyaluronan are also found insynovial fluid and have been used for orthopedic ther-apy because of their role in joint lubrication.89 It iscurrently unknown how the mechanical properties ofthese molecules and their molecular complexes arechanged in disease states such as osteoarthritis. Animportant step in addressing these issues is to image therelevant molecules, using methods such as atomic forcemicroscopy (AFM) (Fig. 4), and to directly measuremolecular level nanomechanical function, using tech-niques such as optical tweezers or AFM.18

In other cases, proper mechanical functioning ofextracellular proteins is necessary to respond toabnormal force conditions to initiate certain processes.For example, high shear stress induces platelets to bindto the blood serum protein von Willebrand factor(VWF) to mediate adhesion to the substratum and

FIGURE 4. AFM image of a single aggrecan monomerextracted from human articular cartilage, consisting of a coreprotein substituted with almost 100 chondroitin sulfate and10–20 keratan sulfate glycosaminoglycan chains. Scalebar = 300 nm. The globular G1 domain at the left-most(N-terminal) end can bind to hyaluronic acid (HA), stabilizedby co-binding of a 45 kDa link protein, thereby formingsupramolecular aggregates containing as many as 100aggrecan monomers. Enzymatic cleavage of aggrecan byaggrecanase enzymes (e.g., ADAMTS-4, -5) at 5 or more sitesalong the core protein causes degradation and loss of thesemonomers in diseases such as osteoarthritis (Image courtesyof H.-Y. Lee, A.J. Grodzinsky, and C. Ortiz).

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thrombosis.15 This allows blood to clot upon injury toarteries to prevent bleeding but can also causethrombotic occlusions leading to heart attack andstroke in patients with cardiovascular disease. It hasbeen proposed that shear stress changes the 3D struc-ture of the platelet glycoprotein Ib (GPIb), which is thereceptor for VWF,55,72 or that VWF may form catchbonds with GPIb.50 The catch bonds between VWFand GPIb have been demonstrated by AFM experi-ments, their structural mechanism has been explainedusing SMD simulation results, and their relationship tovon Willebrand diseases (a bleeding disorder) has beenproposed.92 Understanding the mechanism of shear-activation may allow novel therapies for both bleedingand thrombotic disorders. Another example is thatpassive forces in muscle are elevated in some diseasestates. This may reflect changes in the elasticity of titin,but while titin elasticity has been well-studied, changesdue to disease have not been addressed.

Technological Applications

Another broad area of application is the incorpo-ration of molecular biomechanics design principlesinto nano- and micro-scale devices. For example, thereare many potential technologies based on the force-modulated adhesion of particles via biomolecules.These include adhesion of nanoparticles in the bloodstream, intestines, or lungs for either drug delivery ormolecular imaging.80 Another example is the captureof viruses, bacteria, or cells in microfluidic devices, allof which must bind when biological bonds are sub-jected to tensile force due to drag on the particle. Thistensile force may weaken adhesion for manybonds,10,23 but strengthens adhesion for a variety ofbonds termed catch bonds.19,81,97 Another applicationof molecular mechanics in adhesion technology is thedevelopment of mechanically smart adhesives thatrespond to mechanical force. For example, there hasbeen a great deal of interest in gecko adhesion4 as astrong reversible adhesive that is mechanically regu-lated and thus appropriate for robotics. Can catchbonds be used to engineer smart nano-adhesives thatrespond to force rather than pH and other signals, foruse in medical microrobotics? A final example of smartadhesives is the need to develop imitation platelets andtheir cofactors that will enable patients with bleedingdisorders to form thrombi in high shear conditions butwill not clot and block arteries at physiological levelsof shear stress.

Molecular biomechanics may also be used todesign novel nanomechanical devices. For example,molecular machines can be used to move particlesinstead of fluid in nanofluidic devices.33,79,86 Smartpolymers may be designed to have actively regulated

mechanical properties, or have specific functionsactivated by mechanical force.59 For example, theymight contract or expand with changes in pH, light,temperature, electrical current, or a chemical or bio-logical compound. The biocatalytic activity of nano-assemblies consisting of polyelectrolyte multilayerstratum loaded with enzymes can be switched on/offreversibly by mechanical stretching.59 One challengeover the next decade will be to design and incorpo-rate force-sensing or force-generating componentsinto larger devices in an oriented fashion. This willrequire many of the concepts of self-assembly thatarise with the engineering and manufacture of allnanoscale devices. However, there will be the addedchallenge of engineering linkages that have the rightmechanical properties to transfer and properly dis-tribute force without breaking. It is even possible thatthe components can be made to reassemble intoalternate complexes as well as change their individualstructure and function in response to mechanicalforce.

Moving the field of molecular biomechanics towardsolving biological, clinical and technological challengeswill have many benefits. It will enhance the field ofresearch by attracting more funding and interest fromindustry and by raising new challenges that will inspirenovel research and methods. More importantly, thenovel solutions provided by this field may benefithuman health and the economy.

COMPUTATIONAL AND EXPERIMENTAL

CHALLENGES

The study of single-cell and molecular biomechanicshas expanded enormously with the advent andincreasingly common usage of more precise experi-mental tools and techniques, including embeddedparticle tracking, micropipette aspiration, atomic forcemicroscopy (AFM) and optical tweezers. Althoughthese experimental techniques have led to significantprogress in single-molecule biomechanics, they stilllack atomic level resolution. Computational tech-niques, including Molecular dynamics (MD), MonteCarlo methods, and Brownian dynamics calculations,have the potential to complement the single-moleculeexperimental investigations, and provide insight intothe underlying mechanisms with atomic resolution.For example, in the case of the motor protein kinesin,its force generation mechanism had been proposed byMD simulation40 which agreed well with subsequentsingle-molecule experiments.47 However, significantchallenges exist in studying the conformational chan-ges of biomolecules under force, including the non-equilibrium nature of conformational dynamics, and


the emergence of collective behavior while individualproteins follow stochastic processes.

Computational techniques have been used to studya variety of issues in molecular biomechanics. Forexample, motivated by experimental studies usingAFM and optical tweezers, MD and Monte Carlocalculations have been used to explore the dynamicproperties of nucleic acids that determine theirbehavior during unfolding and refolding, and thestructural basis underlying these properties, includingthe specific interaction among residues, and secondarystructure rearrangements. Another example is thecomputational simulation of the mechanical propertiesof ATP synthase and other molecular motors. In thecase of ATP synthase, MD simulations have revealedthat the gamma subunit rotates as a consequence oftorsional forces generated by electron transport acrossthe membrane bound domain of the molecule.1 One ofthe most important applications of computationalmolecular biomechanics is the study of mechano-transduction, the process by which cells transducemechanical signal into biological processes. Numerousexperimental investigations have revealed that, whenexternal forces are applied to the surface of a cell,cytoskeletal filaments and associated molecules rear-range to form focal adhesion complexes, which act totransmit mechanical signal into and out of cellsthrough membrane-bound integrin molecules. MDsimulations of locally applied forces to talin, one of themechanically-sensitive molecules in a focal adhesion,have shown activation of its cryptic vinculin bindingsite, which is important to the linkage of actin fila-ments to integrin molecules41,53 (Fig. 2). Other MDsimulations have shown that the interaction betweenintegrin and the arginine–glycine–aspartate (RGD)peptide sequence in ECM filaments is due to a multi-tude of stabilizing hydrogen bonds, which account forthe strength of the interaction between the cell and thesubstrate. Still others have investigated the modulationof transport though ion channels.31,93

Several computational tools have been developedfor molecular biomechanics studies. For example,steered molecular dynamics (SMD) has been used as atechnique for simulating nonequilibrium conforma-tional changes along a molecule’s free energy land-scape by overcoming energy barriers with externallyapplied forces. The umbrella sampling technique cal-culates the potential of mean force along the reactioncoordinates of the system. Normal mode analysis hasadvanced as a technique for determining the naturalvibrational states of molecules. Together these com-putational techniques are used to determine mechani-cal and physical properties of molecules that accountfor their functional roles. Although these computa-tional techniques have demonstrated wide applicability

to the study of molecular biomechanics, there areseveral limitations. The greatest limitation is theavailability of protein structures solved by X-raycrystallography and other experimental techniques.Further, the force fields used in simulations areempirically based and the accuracy of the simulationsis limited by the accuracy of these force fields. Thetreatment of solvent has also been a computationalchallenge: implicit solvent representations are moreefficient but less accurate, whereas explicit solventrepresentations, although they better capture the sol-vent-induced conformational motion, are computa-tionally very demanding.

A common question in MD simulations is how tomimic in vivo conditions. Specifically, how should themechanical signal be applied to the cell, constant dis-placement (velocity) or constant force? What is theappropriate direction in which to apply the force? Howdo steric effects of protein complexes affect thesestudies? What boundary conditions are most appro-priate in studying single protein molecules or multi-molecular complexes? How can one best bridge dif-ferent length scales in molecular biomechanics simu-lations? Since biomechanical phenomena span a largerange of time scales (from femtoseconds to days andeven years) and length scales (from Angstroms tometers), one important challenge is to develop novelalgorithms and modeling approaches that can bridgethese disparate scales. Finally, while most of the bio-logical processes in a living cell occur with a timescalelonger than a few milliseconds, available computingpower prevents simulations over times longer thanmicroseconds. This severely limits the applicability ofMD simulations.

In addition to computational challenges, manyexperimental challenges exist in molecular biome-chanics studies of proteins and nucleic acids. Many ofthe experimental challenges in molecular biomechanicsinvolve the need to study molecular complexes inconditions closer to their in vivo environment. Currentmethods allow researchers to apply forces to simplemolecular complexes in vitro with an AFM,26,70 opticaltraps,12 biomembrane force probe (BFP),24 or othersingle molecule force spectroscopy methods. Ideally,these methods require the complexes to be stronglybound to two surfaces via two distinct sites. Whilemany cross-linkers can achieve covalent binding, theyrecognize functional groups such as cysteines that areincreasingly common as the complexity of macromo-lecular structures grow. Conversely, functional groupssuch as gold binding polypeptide,67 His-tags, andstreptavidin bind noncovalently and may detach underthe applied force before the molecular event to bestudied occurs. Therefore, a more specific method oflinking proteins in an oriented fashion is needed

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depending on the types of macromolecular complexesthat have a large number of components in vitro. It isalso possible to apply forces to cells using beads thatare tethered to cell receptors and manipulated with amagnet36 or optical tweezers,16 or by applying fluidicshear stress,84 or stretching the substratum.68 Finally,we need to improve and apply methods to measure orsense forces quantitatively on target molecules insideliving cells, so that we can determine how forces areapplied to molecules of interest in vivo.58 It is possibleto apply and sense forces using a single tool such asoptical trapping of liquid droplets to characterizemotor protein function in vivo.74 Alternatively, forcesthat are applied by normal physiological processes maybe sensed with a method such as a fluorescent force-sensitive probe58,75 or using cysteine labeling to iden-tify intracellular proteins that change their conforma-tion under force.45 Together, these methods will allowresearchers to apply and measure forces on compli-cated molecular complexes to understand the forceresponse of systems that bridge the length scalebetween single molecules and cells.

ROADMAP: SHORT-TERM AND LONG-TERM

GOALS

Numerous open questions and opportunities existon the roadmap of molecular biomechanics. One mostrelevant question is to specifically identify themechanosensing proteins, i.e. what proteins are likelyto be deformed? What is the extent and importance ofmechanosensing characteristics of different proteins inthe molecular machinery of the cell? How can wecontrol molecular conformational changes and pro-tein’s modes of deformation to ascertain the biome-chanical phenomena under study? To demonstrateexperimentally that protein deformations actuallyoccur in vivo and link them to certain biologicalfunctions, we need to distinguish with specificity thedistinct molecular conformations. We also needto be able to alter the molecular conformation ofmechanosensing proteins or nucleic acids with thresh-old specificity. Given that protein domain motions canoften function as switches between distinct biologicalfunctions, experimental techniques are necessary tocharacterize and quantify how domain deformationand unfolding alter interactions. In order to ascertainrelevance of these localized molecular events to overallbiological phenomena, it is essential to identify howconformational changes at one location may poten-tially change binding affinity at another location. It isalso important to characterize allosteric effects. Opti-cal fluorescent techniques involving green fluorescentproteins (GFP), which can be genetically incorporated

in a robust manner, have recently made major contri-butions to many biomechanical and biological dis-coveries, yet one must note that the GFP proteinsthemselves are large (approximately 5 nm in size),comparable in size to most proteins that are to betagged. This caveat points out the need for less invasiveapproaches for marking proteins in vivo. Other newexperimental techniques are required for mechanicalregulation of proteins and other biomolecules. Similarto computational approaches, multiscale experimentalsettings are also essential to the study of molecularbasis of macroscale mechanical behavior of the cells.Taking into account the spatial organization of pro-teins and cellular complexes, and how forces are dis-tributed in molecular complexes proves critical here.Many of these phenomena require breakthroughthinking as in many cases one needs to identify, devise,and build the appropriate building blocks for suchcomplexes and incorporate the suitable interactionsand force fields, similar to what is needed for thecomputational approaches. It is vitally important todevise surrogate cell models in vitro where we can addone component at a time and study that by applyingmechanical stimuli. Such cases rapidly becomeextremely complex, and will likely require a ‘‘systemsbiomechanics’’ approach.

New computational modeling techniques are neededto model protein clusters and apply forces that realis-tically mimic biologically meaningful settings. How dowe discover alternative protein states and identify theeffect of force on them? New computational methodsare necessary to show (slow) allosteric changes and weneed to address the critical role of allosteric regulationby force. Experimental methods are clearly essential.We need experimental methods to better elucidate howthe structure is related to mechanical properties. Infact, it was revealed recently that integrin catch bondsprovide a mechanism for mechanosensing and thatforce is required for outside-in signaling.49 Mutationexperiments represent a valuable tool to ascertain theeffect of localized changes in the molecular constructsof the protein machinery on their overall mechanicalbehavior. Perhaps the most subtle step is to couplethese computational/theoretical and experimentalaspects. With the development of new techniques,theories and algorithms, it will become increasinglyfeasible to understand the biological phenomena over arange of time and length scales. This will also offer aunique platform for probing the underlying mecha-nisms involved in the initiation and development ofmany pathological conditions. Many disease examplesremain to be explored, including atherosclerosis, can-cer, infectious diseases, in which mechanics may playan important role. In atherosclerosis, for example,after more than 30 years of multifaceted investigations,


we still do not know the molecular mechanismsinvolved in the initiation and development of thisdisease. In cancer, it remains unclear whether physicalfactors including force play a role in metastasis. Thus,there is a need to perform multiscale (tissue, cellularand molecular) studies of metastasis incorporating thekey mechanical signaling pathways involved.

Enormous opportunities exist for making progressin molecular biomechanics and in its applicationmore broadly to molecular and cell biology and

bioengineering. Looking into the future, we cananticipate some of the advances that will be made, andthe goals we might pose for future research. Manyadvances have occurred during the past decade in thevisualization of molecular events within the cell (suchas STED-4pi) or in vitro (see, Jares-Erijman andJovin,44 O’Hare et al.,65 and Willig et al.90 for recentadvances), which is likely to continue. In connectionwith protein mechanics, the potential exists now toprobe large-scale conformational changes using Fluo-rescence-Resonance Energy Transfer (FRET) or itsclose cousin, Fluorescence Lifetime Imaging Micros-copy (FLIM) with the donor and acceptor fluoro-phores tethered to the same protein or to a pair ofproteins. Using the theoretical dependence of FRETratio on separation distance as a guide, it is possible tomonitor changes in protein conformation. But many ofthe current fluorophores are quite large, and mayinfluence protein conformation, and the number ofprobes is often limited to no more than one or two.New, small molecular weight fluorophores such asFLASH may prove more useful.34,57 In any event, newand more precise methods for measuring protein con-formational change are sorely needed. One especiallypromising new three-dimensional imaging technique,stochastic optical reconstruction microscopy, orSTORM,38 provides a means of obtaining resolutionon the order of 20–30 nm of intracellular structures.

As a protein unfolds, the resisting force arises froma combination of entropic and enthalpic effects, wherethe latter involves rupture of internal non-covalentbonds. This can be seen in simulations and experimentson single proteins when rupture of an internal bondtriggers complete unfolding, but more subtle transi-tions also occur each time an internal bond is broken.While thermal fluctuation is another essential elementthat complicates the picture, analysis of the force–extension curves can still provide insight into theinternal structure of a protein or other macromolecule.These force–extension relations could thus be viewedas a ‘‘force signature’’ of a particular protein thatarises from the internal structure and interactions.Taking this one step further, the force signature of aprotein should change whenever the protein confor-mation changes, as due to normal conformationalfluctuation, or, more importantly, by activation orbinding to another protein. Thus, binding eventsbetween a single pair of proteins might be investigatedby observing changes in the force signature of a singleprotein. Even single molecule stretching can displaydiverse types of force–extension behavior (Fig. 5).

Several methods are currently used to obtain force–extension data on single proteins, and these generallyemploy either AFM or optical tweezers technologies(Table 1). Each has its advantages and disadvantages

FIGURE 5. The force–extension relationship for two biolog-ical molecules (DNA in (a) and protein in (b)) can be welldescribed by a worm-like chain (WLC) model. (a) Force vs.relative extension curves of single DNA molecules. The dotscorrespond to several experimental measurements performedover a wide range of forces. The full line curve is a best fit tothe WLC model for forces smaller than 5 pN. The dashedcurve is the result of the freely jointed chain (FJC) model withthe same persistence length. Above 70 pN, the length abruptlyincreases, corresponding to the appearance of S-DNA(reproduced with permission from Strick et al.77). (b) A forcevs. extension curve of Filamin A protein in aqueous solutionmeasured by AFM at room temperature. The WLC model fitsthe sawtooth pattern of the force vs. extension curve wellwhere the force gradually increased after the abrupt decreasein force (reproduced with permission from Furuike et al.27).

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in terms of force and distance resolution and thecomplications of simultaneous force measurement andoptical (e.g., single molecule fluorescence) measure-ments. Conventional AFM systems make it difficult toview the molecule under high magnification, butmethods are being developed to circumvent thatproblem. Fluorescence measurements are compro-mised by the trapping laser, typically of high intensityand likely to induce photobleaching, but new methodsthat involve rapid switching between trapping andfluorescence measurement are being developed.13

Force resolution tends to be better at the low end inoptical traps. AFM is capable of applying higher for-ces, but the range of applicability of both methods iscontinually expanding. Still, room exists for newapproaches that facilitate simultaneous manipulationand visualization of single molecules so that we cansense force and observe conformational changes in asingle experiment.

Clearly, to make significant progress on the com-putational front, more atomic structures are needed,and these are becoming available at an acceleratingrate. X-ray crystallography has been the ‘‘gold stan-dard’’ for structures, but cryoelectron microscopy, thatis capable of imaging proteins that are difficult to becrystallized, has made significant advances in resolu-tion, and other methods, such as solution or solid-statenuclear magnetic resonance (NMR), and neutron orelectron diffraction are also potentially useful tools.83

Ultimately, we will need methods with Angstrom-levelresolution that can determine the structure of singleproteins, or even protein complexes.

As we probe conformational change in force trans-mitting proteins, it will be important to gain the abilityto map the effective intramolecular stress distribution.This is somewhat analogous to the use of finite elementprograms to obtain stress distributions in macroscopiccontinua, but on an atomistic length scale. CurrentMD programs have the capability to calculate instan-taneous or time-averaged force interactions, though, sotaking the step of mapping these in 3D may be quiteeasily implemented. In a broader sense, however, newvisualization schemes need to be developed thatenhance the presentation of mechanical data. As with amacroscopic object with a complex geometry, regionsof stress concentration in a protein may not be obvi-ous, but knowledge of where these reside can help us tointerpret force-induced changes in conformation.

One of the major limitations in using MD or SMDto investigate single molecule mechanics has been theenormous computational demands, so that most sim-ulations are limited to no more than about 50–100 ns.Various improvements such as efficient implicit meth-ods for incorporating solvation effects, and coarse-graining methods that reduce the number of degrees of

freedom of the system have been developed, but thereis no consensus as yet on the best approach to use for agiven system, nor is there agreement in the researchcommunity regarding the accuracy of these methods.We will certainly see advances in computationalmethods during the next 5–10 years, and these shouldhelp to expand our capabilities to simulate the effectsof force on protein conformation.

Coarse-graining or similar approaches might alsomake possible true multi-scale simulations. At present,time steps used in MD vs. Brownian Dynamics vs.finite element methods can span over 15 orders ofmagnitude, so simultaneous calculations tend to beunfeasible. Fortunately, they may be largely unneces-sary, as well, except in a few circumstances. At somepoint in the future, methods will be developed tosimulate force transmission through the entire cell,incorporating events down to molecular scale. Butdetailed knowledge of protein conformation mayonly be necessary locally, at sites, for example, ofmechanosensing. In such cases, network or even contin-uum structural models may suffice for the bulk of thecomputational domain, with MD being applied to asingle protein or even a subdomain of a larger protein.Other situations, however, such as the simulation ofmultiple signaling molecules influenced by transmittedstresses, in which the response of one is fundamentallylinked to conformational changes in another, maydemand a true multi-scale, simultaneous simulation.Progress will certainly be made in this direction, but itwill likely be slow given the enormous magnitude ofthe problem. One approach that shows promise is theuse of coupled MD-FEM methods9 (Fig. 6). In a sense,this is yet another example of coarse-graining, but gi-ven the maturity of finite element methods, effectiveand reliable coupling of these two approaches couldprove highly beneficial.

Improvements are also inevitable in computationalspeed. But in addition to the anticipated progressionalong Moore’s Law, new computers are being designedthat specifically cater to the needs of MD simulationthat could reap enormous benefits. These computersdraw advantage from the use of a dedicated high-performance processor specifically for non-bonded,long-range interactions that can often consume over99% of MD simulation times. Recent reports indicatethat speed increases of several hundred fold can berealized. (Blue Gene: A vision for protein science usinga petaflop supercomputer.3)

So what does the future hold in molecular biome-chanics? While we have no crystal ball, it can reason-ably be predicted that we will develop new and greatercapabilities to measure, monitor and model theresponse of single molecules to mechanical loads. Atthe same time, working with our colleagues in biology


and chemistry, we will begin to gain a betterunderstanding of the interactions between intracellularbiochemical signaling pathways, and what might betermed, ‘‘mechanical signaling pathways’’, the trans-mission of signals through the network of linked pro-teins by means of force transmission. Mechanicalsignaling has already been widely appreciated, but itsfull impact on biological function is just now beingrecognized. And once its role is better understood, onemight envision ‘‘mechanical therapies’’ in which dis-eases may be treated by altering the mechanicalproperties of the relevant molecules, cells or tissues.This is already happening to a limited extent andhaving significant impact in cancer treatment in thecontext of molecular therapies that alter the ability ofcells to generate new vascular networks to nourish atumor, or the tendency of the tumor cells to migrate,extrasavate or intrasavate. Recognizing the pivotal roleof mechanical signaling and mechanosensing opensnew opportunities for therapeutic control in a widerange of pathologies.

Speculating further, one might envision ‘‘designerproteins’’ with desired mechanical properties, that canturn intracellular processes on or off through theapplication of external force (e.g., with the introduc-tion of targeted magnetic particles). Since we knowthat the forces experienced by a cell can influence amultitude of cell functions, forces can conceivably beused to control cellular activity. Similarly, one mightenvision ‘‘mechanical beacons’’, that produce a signal,optical, for example, when a certain mechanical stim-ulus is sensed. This, or a similar approach, might beused to detect arterial blood flow conditions thatcontribute to atherogenesis as a means of trainingindividuals to avoid certain detrimental behaviors.

Finally, advances in these areas require individualswith a unique knowledge and skill set, bridgingbetween chemistry, biology and bioengineering orbiophysics. To prepare for these challenges, we need toensure that a new cadre of researchers is trained inthese fields. Cross-disciplinary teaching, which hasbeen discussed extensively, needs to be implemented.Students of biomechanics need to understand not onlycontinuum, but also statistical mechanics, as well ashaving a solid grasp of physical chemistry. Our pro-grams need to move to develop courses that span thesedisciplines and produce students who are truly multi-lingual, being comfortable working at this criticalinterface.

CONCLUDING REMARKS

In closure, we have presented a few of the challengesand milestones, both experimental and computational/theoretical, on the horizon for the advancement ofmolecular biomechanics. Experimental techniques arerequired to capture intracellular forces and molecularconformations. More sophisticated techniques areessential to dissect changes in molecular conformation.New computational modeling techniques are needed tobe able to model protein clusters and apply forces onthem to mimic biologically meaningful settings. Thesecomputational/theoretical and experimental aspectsmust be integrated to help understand the multiscale/multiphysics processes underlying biological phenom-ena over a range of time and length scales. This rep-resents both a challenge and an opportunity tounderstand a disease or biological process in itsentirety. This calls for a close coordination between

FIGURE 6. Computational simulation of protein conformational changes using a coupled MD-FEM method. (a) Minimized energymolecular structure of the T4 lysozyme protein. (b) and (c) High and low resolution discretized meshes, respectively, based on thesolvent-excluded surface of the structure in (a) and used for finite element simulations (reproduced with permission from Bathe9).

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efforts in molecular biomechanics and the investiga-tions focused on cell, tissue and organ biomechanics,necessitating developments of theoretical and compu-tational approaches to bridge the gap between thesedisparate scales.

ACKNOWLEDGMENTS

This work was supported by NIH as a Program ofExcellence in Nanotechnology (UO1HL80711-05 toGB), by NIH Grants R01GM076689-01 (RDK),R01AR033236-26 (AJG), R01GM087677-01A1 (WH),R01AI44902 (CZ) and R01AI38282 (CZ), and byNSF Grant CMMI-0645054 (WT), CBET-0829205(MRKM) and CAREER-0955291 (MRKM).

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