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Cellular and Molecular Bioengineering, Vol. 3, No. 2, June 2010 ( 2010) pp. 91–105 DOI: 10.1007/s12195-010-0109-z

Molecular Biomechanics: The Molecular Basis of How Forces Regulate Cellular

1 2 3 4 5 GANG BAO, ROGER D. KAMM, WENDY THOMAS, WONMUK HWANG, DANIEL A. FLETCHER, 2 1 5 ALAN J. GRODZINSKY, CHENG ZHU, and MOHAMMAD R. K. MOFRAD 1Department of Biomedical , Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA; 2Departments of Mechanical and , MIT, Cambridge, MA 02139, USA; 3Department of Bioengineering, University of Washington, Seattle, WA 98195, USA; 4Department of , 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 of the structure, mechanical responses and biological molecular biomechanics as an essential element of modern functions of cells in different organs and tissues . These efforts focus on theoretical and experimental including, for example, lung, , , studies of the of and nucleic acids, and the understanding of the molecular mechanisms of stress vessels, and skeletal and cardiac muscles. These studies transmission, mechanosensing and in have led to a better understanding of how the biolog- living cells. In particular, single- biomechanics ical functions of a are regulated by mechanical studies of proteins and DNA, and mechanochemical cou- forces or deformation. They have also demonstrated pling in biomolecular motors have demonstrated the critical the central role that forces play in the initiation and importance of molecular mechanics as a new frontier in bioengineering and life sciences. To stimulate a more progression of numerous diseases such as atheroscle- systematic study of the basic issues in molecular biomechan- rosis, arthritis, asthma, to name a few. However, to ics, and attract a broader range of researchers to enter this decipher the fundamental mechanisms of force- emerging field, here we discuss its significance and relevance, induced control of cellular function, more systematic describe the important issues to be addressed and the most studies of deformation, structural and critical questions to be answered, summarize both experi- mental and theoretical/computational challenges, and iden- mechanochemical transduction in living cells and bio- tify some short-term and long-term goals for the field. The are needed. needs to train young researchers in molecular biomechanics As the basic unit of life, living cells perform an with a broader knowledge base, and to bridge and integrate enormous variety of functions through synthesis, molecular, subcellular and cellular level studies of biome- sorting, storage and transport of ; chanics are articulated. expression of genetic information; recognition, trans- mission and transduction of signals; and conversion Keywords—, Force, Cytoskeleton, Mecha- between different forms of . Many of these cel- notransduction, conformational change, Molecular lular processes involve mechanical force, or deforma- motors. tion, at the cellular, subcellular and molecular levels. For example, biomolecular motors and machines convert chemical energy into mechanical work in per- forming their diverse range of functions. During cell MOLECULAR BIOMECHANICS: migration, contractile forces are generated within the AN EMERGENT FIELD cell in order for the cell body to move forward. These Living cells are dynamic systems that perform contractile forces, in combination with the adhesion of integrated functions including , control, cells to extracellular matrix (ECM) through focal sensing, communication, growth, remodeling, repro- adhesion complexes enable cells to sense the stiffness of duction and apoptosis (programed cell death). During the surrounding substrate and respond to it. Many the past few decades, extensive studies have elucidated normal and pathological conditions are dependent upon or regulated by their mechanical environment. Some cells, such as osteoblasts and vascular cells, are Address correspondence to Gang Bao, Department of Biomedi- subjected to specific forces as part of their ‘native’ cal Engineering, Georgia Institute of Technology and Emory Uni- physiological environment. Others, such as muscle and versity, Atlanta, GA 30332, USA. Electronic mail: gang.bao@bme. 61 gatech.edu cochlear outer hair cells, perform their mechanical 91

1865-5025/10/0600-0091/0 2010 Biomedical Engineering Society 92 BAO et al.

FIGURE 1. Schematic of numerical of a mechanosensitive ion channel with (a) small and (b) large opening under the action of membrane tension. Left: side view showing the bilayer (white region with red phospholipid head groups) in an aqueous (blue) environment. Right: End view showing the membrane pore. Reproduced with permission from Yefimov et al.93 function either by converting an electrical or chemical mechanical signal, however little is known about how stimulus into mechanical motion or vice versa. these sensors work (see reviews in Kamm and Of particular importance is the ability of cells to Kaazempur-Mofrad46 and Vogel and Sheetz88). As sense mechanical force or deformation, and transduce illustrated in Fig. 1 using numerical simulation of a these mechanical signals into a biological response.96 mechanosensitive ion channel, one class of mem- For example, endothelial cells can recognize the mag- brane-localized mechanosensors is stretch-activated nitude, mode (steady or pulsatile), type (laminar or ion channels on the cell membrane that change their turbulent) and duration of applied shear flow, and conductance in response to forces.29 These ion chan- respond accordingly, maintaining healthy endothelium nels could sense increases in membrane tension as a or leading to vascular diseases including thrombosis result of applied mechanical load and open, thus and atherosclerosis.17,54 Vascular smooth muscle cells converting mechanical force exerted on the cell mem- in the arterial wall remodel when subjected to pressure- brane into electrical or biochemical signals.39,78 How- induced wall stress. Fibroblast cells ‘crawl’ like an ever, it is not yet clear if the force-induced activation of inchworm by pulling the cell body forward using ion channels is the dominant mechanism for mecha- contractile forces. Bone alters its structure to adapt to notransduction. Integrin molecules or other proteins changes in mechanical environment as occurs, for such as vinculin and talin in the focal adhesion com- example, in spaceflight. Stem cells sense the elasticity plex may serve as force sensors and transducers41,53,60 of the surrounding substrate and differentiate into as illustrated in Fig. 2 in which the molecular dynamics different phenotypes accordingly.22 These and other simulation of the force-induced activation of talin’s examples vividly demonstrate the ability of cells to vinculin binding site is shown. Talin, an essential sense and respond to their local mechanical environ- structural protein in the focal adhesion complex, con- ment. However, little is currently known about the tains the N-terminal five-helix bundle in the rod fundamental molecular mechanisms by which cells domain with a known cryptic vinculin binding site 1 sense mechanical force or deformation, and transduce (VBS1). The perturbation of this stable structure the mechanical signal into a biological response. through elevated temperature, destabilizing Ample evidence suggest that there are specialized and mechanical force activates vinculin binding.52 ‘force sensors’ on the cell membrane that detect the However the underlying molecular mechanism is only Molecular Biomechanics 93

FIGURE 2. Molecular dynamic simulation of the force-induced activation of talin’s vinculin binding site (VBS1) (red ribbon with 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), and some important polar residues (stick representation with color denoting the 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 the hydrophobic core. (b and d) At increased force, the hydrophobic residues rotate and become exposed to solvent. Hydrogen bonds between H5 and VBS1 are broken. The hydrophobic residues, or the vinculin binding residues, point into the page in (a) and point to left in (b). (c) Conformation at t = 0.86 ns viewed from top. The V-shaped VBS1 hydrophobic residues are packed within the hydrophobic core of TAL5 (cyan dotted lines). (d) Conformation at t = 9.24 ns showing VBS1 rotation. The hydrophobic residues H5 (white VDW) fit into the ‘V’ of the VBS1 hydrophobic residues (orange VDW). Reproduced with permission from Lee et al.52 beginning to be explored. Yet another possibility is coupling and interfacing issues. There is no doubt that that forces are transmitted via the cytoskeleton to progress in these areas will have a tremendous impact remote sites within the cell or nucleus where they elicit on the life sciences and our understanding of disease a response.35,42 There is a critical need to identify processes. definitively the molecular mechanisms of mechano- Studies of force sensing and mechanotransduction transduction in living cells that are common to most, if in cells are inevitably related to the mechanics of bio- not all, cell types. molecules (proteins and nucleic acids), and these As an emerging field, molecular biomechanics include the molecular analysis and constitutive modeling integrates mechanics, , , of single biomolecules and multi-molecular complexes. and biomolecular engineering, and Specifically, it is important to characterize how the encompasses three broadly defined and inter-related three-dimensional structural rigidity of DNA, RNA areas: (1) the molecular mechanisms of mechanosen- and proteins control their deformation under various sing and mechanotransduction; (2) the mechanics of loading conditions—stretching, twisting, bending and biomolecules, either individually or as part of a larger shear conditions—and how such deformation alters complex; (3) molecular motors or machines and their DNA condensation, gene replication and transcrip- use in bio/nano-devices, including mechanochemical tion, DNA–protein/RNA–protein interactions, protein 94 BAO et al. function, protein–protein interaction, and protein– ligand interaction.5–7,14,48,51,53,71 These research topics encompass bond formation, rates, and the and kinetics of biomolecular interac- tions. While there have been extensive single-molecule studies of the mechanics of DNA, to date only very limited theoretical and experimental studies of the mechanics of proteins have been conducted. A better understanding of the mechanical behavior of proteins, nucleic acids and other will provide the opportunity to decipher the fundamental mecha- nisms of mechanotransduction, and to predict mechanical function of protein complexes (including filaments, focal adhesion complexes, and subcellular structures) in living cells. With the recent advent of and nanomedicine, there is an increasing need to understand mechanochemical coupling in biomolecu- lar motors and , and to uncover the engi- neering design principles of proteins as nanomachines. Since DNA and proteins may be used as components of hybrid nanobiosystems, the optimal design of such systems inevitably requires an understanding of the mechanics of biomolecules, as well as how the indi- vidual components interact with each other. A specific example is the development of nanodevices utilizing, or powered by, biomolecular machines that can produce FIGURE 3. Two types of molecular motors. (a) Linear motor: linear, rotary, or even reciprocating motions such as Two linear molecular motors, kinesin and dynein, are shown. 25 kinesin, dynein and F1-ATPase (Fig. 3). The use of Both walk along a microtubule, but in opposite directions. In this schematic the motors are shown attached to vesicles for current technologies (e.g., batteries, electromagnetic intracellular transport along a neuronal process (reproduced motors or hydraulic energy sources) are often unable with permission from Vale85). (b) Rotary motor: Schematic of a to meet the needs of functionalized nanoscale inor- bacterial flagellar rotary motor. Rotational speed is over ganic machines with moving parts, especially when the 100,000 rpm and it measures just 50 nm across (Image pro- vided courtesy of A. Ishijima, Tohoku University, Japan). nanodevices are used in vivo. On the other hand, pro- gress in molecular biology has revealed intriguing features of the structures, mechanisms and functions of many biomolecular motors, including kinesin, myosin, deformation, and convert and combine such signals dynein, ATP synthase, DNA polymerase and RNA.37 with biochemical responses are not well understood. These biomotors directly convert chemical energy Living cells are dynamic and their structures can derived from ATPase activities into mechanical force change in response to mechanical load. This raises or motion with high efficiency. With their nanometer many fundamental questions essential to biomechan- size, they have the potential for use in multifunctional, ics: How do forces applied to a cell, either directly or self-powered nanosystems.76 How to integrate biomo- through cell–cell or cell–matrix adhesion sites, induce lecular motors with other nanodevices to perform reorganization of the cytoskeleton, thus changing its specific mechanical, biochemical or biological func- mechanical properties? How do the dynamics of tions in a controlled fashion remains a significant cytoskeleton affect cell spreading, rounding, crawling, challenge. and adhesion? How does the interaction between ECM and focal adhesion complexes transduce a mechanical signal (force or deformation) into cells? Answering MAJOR ISSUES AND NEW OPPORTUNITIES these and other questions will be crucial in understanding the structural basis of cellular function. It is now widely accepted that mechanical and bio- To date, the molecular mechanisms of force sensing and chemical functions of living cells are highly integrated mechanotransduction remain elusive. While numerous in controlling the phenotype of the cell in health mechanisms have been proposed, a strong candidate and disease. How cells sense mechanical forces and for the molecular mechanism of mechanosensing and Molecular Biomechanics 95 mechanotransduction is protein deformation, or protein TABLE 1. Specifications for three methods commonly used 41,51,53,87,95,96 to manipulate single molecules (adapted from Neuman conformational change under force. The et al.62). unique three-dimensional structure (i.e., conformation) of a protein largely determines its function. However, Specification AFM Magnetic trap Optical trap proteins in a cell are deformable and can assume dif- Bandwidth (Hz) 1000 10–1000 50–5000 ferent (altered) conformations under physical forces. Stiffness (pN nmÀ1) 1–105 10À6 0.005–1 Just as proteins can transform from a native or biolog- Spatial resolution (nm) 0.1–1 2–10 0.1–5 ically active state to a denatured or inactive state in Force range (pN) 5–103 10À3–104 0.1–100 response to small changes in temperature or pH in its environment, the application of mechanical forces can lead to protein domain conformational change and even how protein deformation affects protein–protein, unfolding, thus affecting protein–protein and protein– protein-DNA, and receptor–ligand interactions, and DNA recognition, binding/unbinding, enzymatic how such deformation is directly related to human activity, causing changes in downstream biochemical diseases. processes that trigger intracellular signaling pathways In the past 10 years, enormous strides have been and ultimately control cellular behavior. The underlying made in the field of molecular biomechanics, including reason is that the three-dimensional geometry and sur- most notably the development of methods to apply face chemistry local to the binding pocket of a receptor– forces in the range of 0.001 pN–10 nN to single mol- ligand pair or the protein–DNA binding site contributes ecules or small molecular complexes (Table 1). The significantly to the characteristics of their binding. Good magnitude of the forces under consideration in conformational matches usually lead to strong and long- molecular biomechanics studies is typically in the lasting bonds. However, the conformational match at range of 0.1–1000 pN. During this time, a small the binding site may change when the protein domains number of model systems have been extensively stud- are deformed or unfolded under mechanical forces. In ied, including elastic molecules such as titin56 and certain cases, an applied force can alter the affinity and fibronectin,64 receptor ligand pairs such as streptavi- lifetime of a receptor–ligand pair. In some other cases, din–biotin,94 selectin-PSGL-1 and FimH-mannose and protein deformation can expose (or bury) the binding motor proteins such as kinesin and various myosins.37 site, thus switching between the ‘on’ and ‘off’ states of These studies have pushed the envelope for methods protein, as illustrated by the extension and unfolding of development and for understanding the principles fibronectin.28 In vertebrate muscle, the giant elastic underlying molecular biomechanics. This creates the protein titin is involved in strain sensing via its C-ter- opportunity for the to apply these methods minal kinase domain (TK) at the sarcomeric M-band and approaches to current medical and technological and contributes to the of muscle in response problems. to changes in mechanical strain.69 To understand the essential roles of protein con- formational change in mechanosensing and mechano- Medical and Biological Applications transduction, theoretical and experimental studies need to be carried out, first to understand how forces are An opportunity exists to gain a better understanding distributed throughout the cell via its protein networks of diseases in which single molecules or molecular and molecular complexes. This information then needs complexes change function with the application of to be coupled with studies to analyze the constitutive mechanical force. Many diseases in which molecular behavior of proteins, including how proteins deform biomechanics plays a central role are diseases that under different mechanical loads, such as tension, involve in mechanotransduction. For exam- compression, shear, torsion, and their combinations, ple, it is well known that atherosclerotic plaques form and how such deformations are related to protein in regions of low and oscillatory wall shear stress.91 structural rigidities. It is necessary to study protein Even in patients who are biochemically susceptible to dynamics, including domain motion, the rate effect, atherosclerosis (e.g. due to LDL levels), most of their and to quantify how the dynamics of proteins, i.e., the arteries may be protected from the disease due to their modes and time scales of protein motions and defor- shear stress environment. Understanding this mecha- mations, are determined by the structural features of nism may enable alternative treatments for the disease proteins. It is necessary to demonstrate experimentally to be identified by providing targets for new drugs that that protein deformation (i.e., conformational change would mimic the protective effects of high steady shear under force) occurs in live cells under physiological stress. Mechanotransduction may also play a role in the conditions, and it is a key event of mechanosensing pathobiology of asthma. It has been shown that airway and mechanotransduction. It is also necessary to study epithelial cells sense compressive forces and respond by 96 BAO et al. activation of several intracellular signaling pathways including the epithelial growth factor (EGF) receptor pathway.82 Airway smooth muscle cells respond to both external mechanical stress and their own con- traction by remodeling and stiffening the cytoskeleton, which in turn increases stress. This suggests that a positive feedback loop might exist between stiffening and increased stress tending to exacerbate asthmatic conditions, and that bronchodilators function by decreasing contraction to break the cycle.20 Mechano- sensing by such as cilia allow detection of fluid flow in the inner ear, kidneys and other locations and malfunctions in the cilia cause a variety of dis- 11 FIGURE 4. AFM image of a single aggrecan monomer eases. For example, polycystic diseases involve extracted from human articular cartilage, consisting of a core in proteins that localize to the cilia,32 sug- protein substituted with almost 100 chondroitin sulfate and gesting that the detection of fluid flow by the cilia is 10–20 keratan sulfate glycosaminoglycan chains. Scale bar = 300 nm. The globular G1 domain at the left-most essential to proper renal function. However, the (N-terminal) end can bind to hyaluronic acid (HA), stabilized molecular mechanism(s) of how the mutations affect by co-binding of a 45 kDa link protein, thereby forming cilia mechanobiological function is not known. Perhaps supramolecular aggregates containing as many as 100 aggrecan monomers. Enzymatic cleavage of aggrecan by the most widely recognized example of mechanotrans- aggrecanase enzymes (e.g., ADAMTS-4, -5) at 5 or more sites duction in disease is that or connective along the core protein causes degradation and loss of these require mechanical forces to heal. Loss of forces due to monomers in diseases such as osteoarthritis (Image courtesy of H.-Y. Lee, A.J. Grodzinsky, and C. Ortiz). immobilization, bed rest or even low can cause disease or limit healing.73 Mechanosensing is also involved in cancer. First, matrix stiffness regulates the glycosaminoglycans (GAGs) attached to a core protein. ability of cancer cells to invade.2,66 Second, it has been Large proteoglycans containing 10–100 GAG chains hypothesized21 that cancer cells may metastasize in part are found in musculoskeletal and cardiovascular con- because they exhibit aberrant mechanosensing,30 which nective tissues where compressive or osmotic forces allows them to become adhesion independent and able act.43 Diseases involving these connective tissues often to travel around the body. A drug that interferes with involve the proteolytic degradation of constituent the tension-independent signaling by this pathway may proteoglycans, resulting in loss of tissue-level mechan- have fewer side effects than traditional chemotherapy ical function. An example is the ~3 MDa proteoglycan, drugs that kill all dividing cells.8 In each disease men- aggrecan, found in cartilaginous tissues and the inter- tioned here, the actual mechanotransducers—the pro- vertebral disc, shown in its monomeric form in Fig. 4.63 teins that sense mechanical force and transduce the The high negative charge density of the linear GAG mechanical signal to a biological response—are gener- chains results in molecular level electrostatic repulsion ally not known. What is known is that certain forces that significantly contribute to tissue compres- mechanical forces lead to the activation of many pro- sive stiffness and osmotic fluid retention. Glycosami- cesses including signaling pathways, transcription fac- noglycans such as hyaluronan are also found in tors and changes in gene expression. However, these synovial fluid and have been used for orthopedic ther- same processes are also involved in a wide variety of apy because of their role in joint lubrication.89 It is other regulatory responses and thus would make risky currently unknown how the mechanical properties of drug targets due to numerous side effects. In contrast, these molecules and their molecular complexes are identifying the actual mechanosensor and the mecha- changed in disease states such as osteoarthritis. An nism of force sensing and mechanotransduction might important step in addressing these issues is to image the provide for a drug target that is specific to the me- relevant molecules, using methods such as atomic force chanosensing process. microscopy (AFM) (Fig. 4), and to directly measure Other diseases involve a wide range of proteins that molecular level nanomechanical function, using tech- are subjected to mechanical forces, often outside the niques such as optical tweezers or AFM.18 cell, in various bodily compartments. In some cases, In other cases, proper mechanical functioning of the proper functioning of these proteins is needed to extracellular proteins is necessary to respond to maintain normal function in high-force situations. One abnormal force conditions to initiate certain processes. example is the involvement of proteoglycans in disease. For example, high shear stress induces platelets to bind These molecules contain high molecular weight to the blood serum protein von Willebrand factor negatively charged polysaccharide chains called (VWF) to mediate adhesion to the substratum and Molecular Biomechanics 97 thrombosis.15 This allows blood to clot upon injury to mechanical properties, or have specific functions arteries to prevent bleeding but can also cause activated by mechanical force.59 For example, they thrombotic occlusions leading to heart attack and might contract or expand with changes in pH, light, stroke in patients with cardiovascular disease. It has temperature, electrical current, or a chemical or bio- been proposed that shear stress changes the 3D struc- logical compound. The biocatalytic activity of nano- ture of the platelet glycoprotein Ib (GPIb), which is the assemblies consisting of polyelectrolyte multilayer receptor for VWF,55,72 or that VWF may form catch stratum loaded with enzymes can be switched on/off bonds with GPIb.50 The catch bonds between VWF reversibly by mechanical stretching.59 One challenge and GPIb have been demonstrated by AFM experi- over the next decade will be to design and incorpo- ments, their structural mechanism has been explained rate force-sensing or force-generating components using SMD simulation results, and their relationship to into larger devices in an oriented fashion. This will von Willebrand diseases (a bleeding disorder) has been require many of the concepts of self-assembly that proposed.92 Understanding the mechanism of shear- arise with the engineering and manufacture of all activation may allow novel therapies for both bleeding nanoscale devices. However, there will be the added and thrombotic disorders. Another example is that challenge of engineering linkages that have the right passive forces in muscle are elevated in some disease mechanical properties to transfer and properly dis- states. This may reflect changes in the elasticity of titin, tribute force without breaking. It is even possible that but while titin elasticity has been well-studied, changes the components can be made to reassemble into due to disease have not been addressed. alternate complexes as well as change their individual structure and function in response to mechanical force. Technological Applications Moving the field of molecular biomechanics toward Another broad area of application is the incorpo- solving biological, clinical and technological challenges ration of molecular biomechanics design principles will have many benefits. It will enhance the field of into nano- and micro-scale devices. For example, there research by attracting more funding and interest from are many potential technologies based on the force- industry and by raising new challenges that will inspire modulated adhesion of particles via biomolecules. novel research and methods. More importantly, the These include adhesion of nanoparticles in the blood novel solutions provided by this field may benefit stream, intestines, or lungs for either drug delivery or human health and the economy. molecular imaging.80 Another example is the capture of viruses, bacteria, or cells in microfluidic devices, all of which must bind when biological bonds are sub- COMPUTATIONAL AND EXPERIMENTAL jected to tensile force due to on the particle. This CHALLENGES tensile force may weaken adhesion for many bonds,10,23 but strengthens adhesion for a variety of The study of single-cell and molecular biomechanics bonds termed catch bonds.19,81,97 Another application has expanded enormously with the advent and of molecular mechanics in adhesion technology is the increasingly common usage of more precise experi- development of mechanically smart adhesives that mental tools and techniques, including embedded respond to mechanical force. For example, there has particle tracking, micropipette aspiration, atomic force been a great deal of interest in gecko adhesion4 as a microscopy (AFM) and optical tweezers. Although strong reversible adhesive that is mechanically regu- these experimental techniques have led to significant lated and thus appropriate for robotics. Can catch progress in single-molecule biomechanics, they still bonds be used to engineer smart nano-adhesives that lack atomic level resolution. Computational tech- respond to force rather than pH and other signals, for niques, including Molecular dynamics (MD), Monte use in medical microrobotics? A final example of smart Carlo methods, and Brownian dynamics calculations, adhesives is the need to develop imitation platelets and have the potential to complement the single-molecule their cofactors that will enable patients with bleeding experimental investigations, and provide insight into disorders to form thrombi in high shear conditions but the underlying mechanisms with atomic resolution. will not clot and block arteries at physiological levels For example, in the case of the motor protein kinesin, of shear stress. its force generation mechanism had been proposed by Molecular biomechanics may also be used to MD simulation40 which agreed well with subsequent design novel nanomechanical devices. For example, single-molecule .47 However, significant molecular machines can be used to move particles challenges exist in studying the conformational chan- instead of fluid in nanofluidic devices.33,79,86 Smart ges of biomolecules under force, including the non- polymers may be designed to have actively regulated equilibrium of conformational dynamics, and 98 BAO et al. the emergence of collective behavior while individual to the study of molecular biomechanics, there are proteins follow stochastic processes. several limitations. The greatest limitation is the Computational techniques have been used to study availability of protein structures solved by X-ray a variety of issues in molecular biomechanics. For crystallography and other experimental techniques. example, motivated by experimental studies using Further, the force fields used in are AFM and optical tweezers, MD and Monte Carlo empirically based and the accuracy of the simulations calculations have been used to explore the dynamic is limited by the accuracy of these force fields. The properties of nucleic acids that determine their treatment of solvent has also been a computational behavior during unfolding and refolding, and the challenge: implicit solvent representations are more structural basis underlying these properties, including efficient but less accurate, whereas explicit solvent the specific interaction among residues, and secondary representations, although they better capture the sol- structure rearrangements. Another example is the vent-induced conformational motion, are computa- computational simulation of the mechanical properties tionally very demanding. of ATP synthase and other molecular motors. In the A common question in MD simulations is how to case of ATP synthase, MD simulations have revealed mimic in vivo conditions. Specifically, how should the that the gamma subunit rotates as a consequence of mechanical signal be applied to the cell, constant dis- torsional forces generated by electron transport across placement (velocity) or constant force? What is the the membrane bound domain of the molecule.1 One of appropriate direction in which to apply the force? How the most important applications of computational do steric effects of protein complexes affect these molecular biomechanics is the study of mechano- studies? What boundary conditions are most appro- transduction, the process by which cells transduce priate in studying single protein molecules or multi- mechanical signal into biological processes. Numerous molecular complexes? How can one best bridge dif- experimental investigations have revealed that, when ferent length scales in molecular biomechanics simu- external forces are applied to the surface of a cell, lations? Since biomechanical phenomena span a large cytoskeletal filaments and associated molecules rear- range of time scales (from femtoseconds to days and range to form focal adhesion complexes, which act to even years) and length scales (from Angstroms to transmit mechanical signal into and out of cells meters), one important challenge is to develop novel through membrane-bound integrin molecules. MD algorithms and modeling approaches that can bridge simulations of locally applied forces to talin, one of the these disparate scales. Finally, while most of the bio- mechanically-sensitive molecules in a focal adhesion, logical processes in a living cell occur with a timescale have shown activation of its cryptic vinculin binding longer than a few milliseconds, available computing site, which is important to the linkage of actin fila- power prevents simulations over times longer than ments to integrin molecules41,53 (Fig. 2). Other MD microseconds. This severely limits the applicability of simulations have shown that the interaction between MD simulations. integrin and the arginine–glycine–aspartate (RGD) In addition to computational challenges, many peptide sequence in ECM filaments is due to a multi- experimental challenges exist in molecular biome- tude of stabilizing hydrogen bonds, which account for chanics studies of proteins and nucleic acids. Many of the strength of the interaction between the cell and the the experimental challenges in molecular biomechanics substrate. Still others have investigated the modulation involve the need to study molecular complexes in of transport though ion channels.31,93 conditions closer to their in vivo environment. Current Several computational tools have been developed methods allow researchers to apply forces to simple for molecular biomechanics studies. For example, molecular complexes in vitro with an AFM,26,70 optical steered molecular dynamics (SMD) has been used as a traps,12 biomembrane force probe (BFP),24 or other technique for simulating nonequilibrium conforma- single molecule force spectroscopy methods. Ideally, tional changes along a molecule’s free energy land- these methods require the complexes to be strongly scape by overcoming energy barriers with externally bound to two surfaces via two distinct sites. While applied forces. The umbrella sampling technique cal- many cross-linkers can achieve covalent binding, they culates the potential of mean force along the reaction recognize functional groups such as cysteines that are coordinates of the system. Normal mode analysis has increasingly common as the complexity of macromo- advanced as a technique for determining the natural lecular structures grow. Conversely, functional groups vibrational states of molecules. Together these com- such as gold binding polypeptide,67 His-tags, and putational techniques are used to determine mechani- streptavidin bind noncovalently and may detach under cal and physical properties of molecules that account the applied force before the molecular event to be for their functional roles. Although these computa- studied occurs. Therefore, a more specific method of tional techniques have demonstrated wide applicability linking proteins in an oriented fashion is needed Molecular Biomechanics 99 depending on the types of macromolecular complexes in a robust manner, have recently made major contri- that have a large number of components in vitro.Itis butions to many biomechanical and biological dis- also possible to apply forces to cells using beads that coveries, yet one must note that the GFP proteins are tethered to cell receptors and manipulated with a themselves are large (approximately 5 nm in size), magnet36 or optical tweezers,16 or by applying fluidic comparable in size to most proteins that are to be shear stress,84 or stretching the substratum.68 Finally, tagged. This caveat points out the need for less invasive we need to improve and apply methods to measure or approaches for marking proteins in vivo. Other new sense forces quantitatively on target molecules inside experimental techniques are required for mechanical living cells, so that we can determine how forces are regulation of proteins and other biomolecules. Similar applied to molecules of interest in vivo.58 It is possible to computational approaches, multiscale experimental to apply and sense forces using a single tool such as settings are also essential to the study of molecular optical trapping of liquid droplets to characterize basis of macroscale mechanical behavior of the cells. motor protein function in vivo.74 Alternatively, forces Taking into account the spatial organization of pro- that are applied by normal physiological processes may teins and cellular complexes, and how forces are dis- be sensed with a method such as a fluorescent force- tributed in molecular complexes proves critical here. sensitive probe58,75 or using cysteine labeling to iden- Many of these phenomena require breakthrough tify intracellular proteins that change their conforma- thinking as in many cases one needs to identify, devise, tion under force.45 Together, these methods will allow and build the appropriate building blocks for such researchers to apply and measure forces on compli- complexes and incorporate the suitable interactions cated molecular complexes to understand the force and force fields, similar to what is needed for the response of systems that bridge the length scale computational approaches. It is vitally important to between single molecules and cells. devise surrogate cell models in vitro where we can add one component at a time and study that by applying mechanical stimuli. Such cases rapidly become ROADMAP: SHORT-TERM AND LONG-TERM extremely complex, and will likely require a ‘‘systems GOALS biomechanics’’ approach. New computational modeling techniques are needed Numerous open questions and opportunities exist to model protein clusters and apply forces that realis- on the roadmap of molecular biomechanics. One most tically mimic biologically meaningful settings. How do relevant question is to specifically identify the we discover alternative protein states and identify the mechanosensing proteins, i.e. what proteins are likely effect of force on them? New computational methods to be deformed? What is the extent and importance of are necessary to show (slow) allosteric changes and we mechanosensing characteristics of different proteins in need to address the critical role of allosteric regulation the molecular machinery of the cell? How can we by force. Experimental methods are clearly essential. control molecular conformational changes and pro- We need experimental methods to better elucidate how tein’s modes of deformation to ascertain the biome- the structure is related to mechanical properties. In chanical phenomena under study? To demonstrate fact, it was revealed recently that integrin catch bonds experimentally that protein deformations actually provide a mechanism for mechanosensing and that occur in vivo and link them to certain biological force is required for outside-in signaling.49 Mutation functions, we need to distinguish with specificity the experiments represent a valuable tool to ascertain the distinct molecular conformations. We also need effect of localized changes in the molecular constructs to be able to alter the molecular conformation of of the protein machinery on their overall mechanical mechanosensing proteins or nucleic acids with thresh- behavior. Perhaps the most subtle step is to couple old specificity. Given that protein domain motions can these computational/theoretical and experimental often function as switches between distinct biological aspects. With the development of new techniques, functions, experimental techniques are necessary to theories and algorithms, it will become increasingly characterize and quantify how domain deformation feasible to understand the biological phenomena over a and unfolding alter interactions. In order to ascertain range of time and length scales. This will also offer a relevance of these localized molecular events to overall unique platform for probing the underlying mecha- biological phenomena, it is essential to identify how nisms involved in the initiation and development of conformational changes at one location may poten- many pathological conditions. Many disease examples tially change binding affinity at another location. It is remain to be explored, including atherosclerosis, can- also important to characterize allosteric effects. Opti- cer, infectious diseases, in which mechanics may play cal fluorescent techniques involving green fluorescent an important role. In atherosclerosis, for example, proteins (GFP), which can be genetically incorporated after more than 30 years of multifaceted investigations, 100 BAO et al.

bioengineering. Looking into the future, we can anticipate some of the advances that will be made, and the goals we might pose for future research. Many advances have occurred during the past decade in the visualization of molecular events within the cell (such as STED-4pi) or in vitro (see, Jares-Erijman and Jovin,44 O’Hare et al.,65 and Willig et al.90 for recent advances), which is likely to continue. In connection with protein mechanics, the potential exists now to probe large-scale conformational changes using Fluo- rescence-Resonance Energy Transfer (FRET) or its close cousin, Fluorescence Lifetime Imaging Micros- copy (FLIM) with the donor and acceptor fluoro- phores tethered to the same protein or to a pair of proteins. Using the theoretical dependence of FRET ratio on separation distance as a guide, it is possible to monitor changes in protein conformation. But many of the current fluorophores are quite large, and may influence protein conformation, and the number of probes is often limited to no more than one or two. New, small molecular weight fluorophores such as FLASH may prove more useful.34,57 In any event, new and more precise methods for measuring protein con- formational change are sorely needed. One especially promising new three-dimensional imaging technique, stochastic optical reconstruction microscopy, or STORM,38 provides a means of obtaining resolution on the order of 20–30 nm of intracellular structures. As a protein unfolds, the resisting force arises from a combination of entropic and enthalpic effects, where the latter involves rupture of internal non-covalent FIGURE 5. The force–extension relationship for two biolog- bonds. This can be seen in simulations and experiments ical molecules (DNA in (a) and protein in (b)) can be well on single proteins when rupture of an internal bond described by a worm-like chain (WLC) model. (a) Force vs. triggers complete unfolding, but more subtle transi- relative extension curves of single DNA molecules. The dots correspond to several experimental measurements performed tions also occur each time an internal bond is broken. over a wide range of forces. The full line curve is a best fit to While thermal fluctuation is another essential element the WLC model for forces smaller than 5 pN. The dashed that complicates the picture, analysis of the force– curve is the result of the freely jointed chain (FJC) model with the same persistence length. Above 70 pN, the length abruptly extension curves can still provide insight into the increases, corresponding to the appearance of S-DNA internal structure of a protein or other . 77 (reproduced with permission from Strick et al. ). (b) A force These force–extension relations could thus be viewed vs. extension curve of Filamin A protein in aqueous solution measured by AFM at room temperature. The WLC model fits as a ‘‘force signature’’ of a particular protein that the sawtooth pattern of the force vs. extension curve well arises from the internal structure and interactions. where the force gradually increased after the abrupt decrease Taking this one step further, the force signature of a in force (reproduced with permission from Furuike et al.27). protein should change whenever the protein confor- mation changes, as due to normal conformational we still do not know the molecular mechanisms fluctuation, or, more importantly, by activation or involved in the initiation and development of this binding to another protein. Thus, binding events disease. In cancer, it remains unclear whether physical between a single pair of proteins might be investigated factors including force play a role in metastasis. Thus, by observing changes in the force signature of a single there is a need to perform multiscale (tissue, cellular protein. Even single molecule stretching can display and molecular) studies of metastasis incorporating the diverse types of force–extension behavior (Fig. 5). key mechanical signaling pathways involved. Several methods are currently used to obtain force– Enormous opportunities exist for making progress extension data on single proteins, and these generally in molecular biomechanics and in its application employ either AFM or optical tweezers technologies more broadly to molecular and and (Table 1). Each has its advantages and disadvantages Molecular Biomechanics 101 in terms of force and distance resolution and the freedom of the system have been developed, but there complications of simultaneous force measurement and is no consensus as yet on the best approach to use for a optical (e.g., single molecule fluorescence) measure- given system, nor is there agreement in the research ments. Conventional AFM systems make it difficult to community regarding the accuracy of these methods. view the molecule under high magnification, but We will certainly see advances in computational methods are being developed to circumvent that methods during the next 5–10 years, and these should problem. Fluorescence measurements are compro- help to expand our capabilities to simulate the effects mised by the trapping laser, typically of high intensity of force on protein conformation. and likely to induce photobleaching, but new methods Coarse-graining or similar approaches might also that involve rapid switching between trapping and make possible true multi-scale simulations. At present, fluorescence measurement are being developed.13 time steps used in MD vs. Brownian Dynamics vs. Force resolution tends to be better at the low end in finite element methods can span over 15 orders of optical traps. AFM is capable of applying higher for- magnitude, so simultaneous calculations tend to be ces, but the range of applicability of both methods is unfeasible. Fortunately, they may be largely unneces- continually expanding. Still, room exists for new sary, as well, except in a few circumstances. At some approaches that facilitate simultaneous manipulation point in the future, methods will be developed to and visualization of single molecules so that we can simulate force transmission through the entire cell, sense force and observe conformational changes in a incorporating events down to molecular scale. But single . detailed knowledge of protein conformation may Clearly, to make significant progress on the com- only be necessary locally, at sites, for example, of putational front, more atomic structures are needed, mechanosensing. In such cases, network or even contin- and these are becoming available at an accelerating uum structural models may suffice for the bulk of the rate. X-ray crystallography has been the ‘‘gold stan- computational domain, with MD being applied to a dard’’ for structures, but cryoelectron microscopy, that single protein or even a subdomain of a larger protein. is capable of imaging proteins that are difficult to be Other situations, however, such as the simulation of crystallized, has made significant advances in resolu- multiple signaling molecules influenced by transmitted tion, and other methods, such as solution or solid-state stresses, in which the response of one is fundamentally nuclear magnetic resonance (NMR), and neutron or linked to conformational changes in another, may electron diffraction are also potentially useful tools.83 demand a true multi-scale, simultaneous simulation. Ultimately, we will need methods with Angstrom-level Progress will certainly be made in this direction, but it resolution that can determine the structure of single will likely be slow given the enormous magnitude of proteins, or even protein complexes. the problem. One approach that shows promise is the As we probe conformational change in force trans- use of coupled MD-FEM methods9 (Fig. 6). In a sense, mitting proteins, it will be important to gain the ability this is yet another example of coarse-graining, but gi- to map the effective intramolecular stress distribution. ven the maturity of finite element methods, effective This is somewhat analogous to the use of finite element and reliable coupling of these two approaches could programs to obtain stress distributions in macroscopic prove highly beneficial. continua, but on an atomistic length scale. Current Improvements are also inevitable in computational MD programs have the capability to calculate instan- speed. But in addition to the anticipated progression taneous or time-averaged force interactions, though, so along Moore’s Law, new computers are being designed taking the step of mapping these in 3D may be quite that specifically cater to the needs of MD simulation easily implemented. In a broader sense, however, new that could reap enormous benefits. These computers visualization schemes need to be developed that draw advantage from the use of a dedicated high- enhance the presentation of mechanical data. As with a performance processor specifically for non-bonded, macroscopic object with a complex geometry, regions long-range interactions that can often consume over of stress concentration in a protein may not be obvi- 99% of MD simulation times. Recent reports indicate ous, but knowledge of where these reside can help us to that speed increases of several hundred fold can be interpret force-induced changes in conformation. realized. (Blue Gene: A vision for protein science using One of the major limitations in using MD or SMD a petaflop supercomputer.3) to investigate single molecule mechanics has been the So what does the future hold in molecular biome- enormous computational demands, so that most sim- chanics? While we have no crystal ball, it can reason- ulations are limited to no more than about 50–100 ns. ably be predicted that we will develop new and greater Various improvements such as efficient implicit meth- capabilities to measure, monitor and model the ods for incorporating solvation effects, and coarse- response of single molecules to mechanical loads. At graining methods that reduce the number of degrees of the same time, working with our colleagues in biology 102 BAO et al.

FIGURE 6. Computational simulation of protein conformational changes using a coupled MD-FEM method. (a) Minimized energy molecular structure of the T4 lysozyme protein. (b) and (c) High and low resolution discretized meshes, respectively, based on the solvent-excluded surface of the structure in (a) and used for finite element simulations (reproduced with permission from Bathe9). and chemistry, we will begin to gain a better Finally, advances in these areas require individuals understanding of the interactions between intracellular with a unique knowledge and skill set, bridging biochemical signaling pathways, and what might be between chemistry, biology and bioengineering or termed, ‘‘mechanical signaling pathways’’, the trans- biophysics. To prepare for these challenges, we need to mission of signals through the network of linked pro- ensure that a new cadre of researchers is trained in teins by means of force transmission. Mechanical these fields. Cross-disciplinary teaching, which has signaling has already been widely appreciated, but its been discussed extensively, needs to be implemented. full impact on biological function is just now being Students of biomechanics need to understand not only recognized. And once its role is better understood, one continuum, but also , as well as might envision ‘‘mechanical therapies’’ in which dis- having a solid grasp of physical chemistry. Our pro- eases may be treated by altering the mechanical grams need to move to develop courses that span these properties of the relevant molecules, cells or tissues. disciplines and produce students who are truly multi- This is already happening to a limited extent and lingual, being comfortable working at this critical having significant impact in cancer treatment in the interface. context of molecular therapies that alter the ability of cells to generate new vascular networks to nourish a tumor, or the tendency of the tumor cells to migrate, CONCLUDING REMARKS extrasavate or intrasavate. Recognizing the pivotal role of mechanical signaling and mechanosensing opens In closure, we have presented a few of the challenges new opportunities for therapeutic control in a wide and milestones, both experimental and computational/ range of pathologies. theoretical, on the horizon for the advancement of Speculating further, one might envision ‘‘designer molecular biomechanics. Experimental techniques are proteins’’ with desired mechanical properties, that can required to capture intracellular forces and molecular turn intracellular processes on or off through the conformations. More sophisticated techniques are application of external force (e.g., with the introduc- essential to dissect changes in molecular conformation. tion of targeted magnetic particles). Since we know New computational modeling techniques are needed to that the forces experienced by a cell can influence a be able to model protein clusters and apply forces on multitude of cell functions, forces can conceivably be them to mimic biologically meaningful settings. These used to control cellular activity. Similarly, one might computational/theoretical and experimental aspects envision ‘‘mechanical beacons’’, that produce a signal, must be integrated to help understand the multiscale/ optical, for example, when a certain mechanical stim- multiphysics processes underlying biological phenom- ulus is sensed. This, or a similar approach, might be ena over a range of time and length scales. This rep- used to detect arterial blood flow conditions that resents both a challenge and an opportunity to contribute to atherogenesis as a means of training understand a disease or biological process in its individuals to avoid certain detrimental behaviors. entirety. This calls for a close coordination between Molecular Biomechanics 103 efforts in molecular biomechanics and the investiga- 14Brower-Toland, B. D., et al. 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