Biophysical Perspective
A Perspective on the Role of Myosins as Mechanosensors ,2 Erkan Tu¨zel,2 and E. Michael Ostap3,* Michael J. Greenberg,1 Go¨ker Arpag 1 Biochemistry and Molecular Biophysics, Washington University, St. Louis, Missouri; 2Worcester Polytechnic Institute, Worcester, Massachusetts; and 3Pennsylvania Muscle Institute and Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
ABSTRACT Cells are dynamic systems that generate and respond to forces over a range of spatial and temporal scales, spanning from single molecules to tissues. Substantial progress has been made in recent years in identifying the molecules and pathways responsible for sensing and transducing mechanical signals to short-term cellular responses and longer-term changes in gene expression, cell identity, and tissue development. In this perspective article, we focus on myosin motors, as they not only function as the primary force generators in well-studied mechanobiological processes, but also act as key mechanosensors in diverse functions including intracellular transport, signaling, cell migration, muscle contraction, and sensory perception. We discuss how the biochemical and mechanical properties of different myosin isoforms are tuned to fulfill these roles in an array of cellular processes, and we highlight the underappreciated diversity of mechanosensing properties within the myosin superfamily. In particular, we use modeling and simulations to make predictions regarding how diversity in force sensing affects the lifetime of the actomyosin bond, the myosin power output, and the ability of myosin to respond to a perturbation in force for several nonprocessive myosin isoforms.
Exciting experiments in the field of mechanobiology have demonstrated that molecules, cells, and tissues are able to sense their environments and respond biologically to mechanical signals (1). Mechanical force is sensed by loadinduced changes in the conformation and/or activities of mechanosensitive macromolecules (i.e., mechanosensors), which in turn lead to a biological response. In mechanosensitive molecules such as p130Cas (2), von Willebrand factor (3), and titin (4), mechanical stress causes local protein unfolding that leads to the exposure of cryptic binding sites and/or sites of posttranslational modifications. In other proteins, such as Dam1 (5) and PSGL1 (6), force affects the interaction between a protein and its binding partner allosterically rather than through the exposure of cryptic sites. This allosteric tuning can either accelerate or slow the rate of bond dissociation. Forces can also directly alter enzymatic activities of mechanosensitive proteins such as myosin (below) or the opening probabilities of mechanosensitive ion channels (reviewed in (7)). The kinetics of the interaction between the molecular motor myosin and its cytoskeletal track, actin, can be allosterically tuned by force; however, in contrast to many
Submitted December 30, 2015, and accepted for publication May 16, 2016. *Correspondence: [email protected] Editor: Brian Salzberg. http://dx.doi.org/10.1016/j.bpj.2016.05.021 Ó 2016 Biophysical Society.
2568 Biophysical Journal 110, 2568–2576, June 21, 2016
other mechanosensitive proteins where the forces acting on the proteins are externally imposed, the forces affecting myosin can be externally imposed or internally generated by the myosin itself working against substrates of varied stiffnesses. It can be argued that skeletal muscle myosin, the motor that powers our locomotion, was one of the first recognized mechanosensors. In landmark experiments conducted in the 1920s, W. O. Fenn showed that a muscle contracting against a resisting load generates less heat (i.e., uses less ATP) than a muscle shortening in the absence of a load (8). These experiments were complemented by the work of A. V. Hill, which showed that the velocity of muscle shortening decreases nonlinearly with increasing force (9). Based on these observations, Hill proposed that force slows the enzymatic and motile activities of muscle myosin. In other words, besides acting as a force generator, muscle myosin also functions as a mechanosensor that changes its rate of motility in response to force. Subsequently, it has been shown that a muscle’s force sensitivity and rate of force development depend on its fiber type (10,11), suggesting that different myosin isoforms confer distinct mechanosensing properties to the muscle. We now know that the myosin gene family is large, and that the different isoforms have a range of force-generating properties that have evolved to function in a large array of physiological functions that extend beyond powering muscle contraction.
Tension Sensing by Myosin
Since the publication of these classic muscle physiology studies, single-molecule experiments have directly demonstrated that slowing of motility by force can be explained in part by changes in the attachment and detachment kinetics of actin-bound myosin (actomyosin). Depending on the magnitude of forces on myosin, actomyosin interactions can be described as catch- or slip-bonds, which have lifetimes that increase or decrease, respectively, with load. Notably, there are several features of the force dependence of the actomyosin interaction that distinguish its behavior from other catch- and slip-bonds, because these behaviors in myosin depend on its enzymatic activity. Moreover, different myosin isoforms can show distinct mechanosensing behaviors at a given force. Mechanosensing by myosin is regulated by chemical and mechanical forces To understand mechanosensing by myosin, knowledge of its mechanochemical pathway is required. All characterized myosins hydrolyze ATP via the same biochemical pathway and likely share similar mechanical intermediates (Fig. 1). Myosin detaches from actin upon ATP binding (Fig. 1, steps 1 and 2), myosin isomerizes to the prepowerstroke conformation, and ATP is hydrolyzed while myosin holds ADP and inorganic phosphate noncovalently in its active site (Fig. 1, step 3). Rebinding of myosin to actin induces the release of phosphate and the force-generating powerstroke (Fig. 1, steps 4 and 5), and ADP is released to complete the cycle (Fig. 1, step 6). The duty ratio, i.e., the fraction of time myosin spends attached to actin in its biochemical
cycle, correlates with the ability of myosin ensembles to generate force. It is crucial for the reader to note that 1) in many cases, the motility rate is limited by the detachment rate of myosin from actin (12); 2) the magnitude of force generated by an ensemble of myosins is proportional to the number of myosins bound strongly to actin; and 3) there are orders-of-magnitude variations among myosin isoforms in the rates and equilibrium constants that define their biochemical pathways, resulting in incredible diversity of sliding rates and force production (13). Critically, the rates and equilibrium constants of the biochemical and mechanical transitions shown in Fig. 1 can be modulated by force. To a first-order approximation, the effects of force on a kinetic rate (e.g., the rate of bond dissociation or ADP release) can be described by the Bell equation, which assumes an Arrhenius transition model (14): kðFÞ ¼ kf0 e
F , d kB T ;
(1)
where kf0 is the rate in the absence of force, F is the force on the molecule (where a positive F is a force that resists motion), kB is Boltzmann’s constant, T is the temperature, and d is the distance to the force-dependent transition state, also known as the distance parameter. Note, k(F) changes exponentially with d, which is a vector quantity that depends on the direction of the applied force. In this article, a positive d indicates a force that resists the forward motion of the myosin working stroke in the direction of the long-axis of the actin filament. At sufficiently high loads, all bonds become slip bonds, a behavior that is not captured in Eq. 1. It is important to note that the magnitude of d does not have to correspond to the
FIGURE 1 The myosin mechanochemical cycle. Although all characterized myosins follow this cycle, the individual rate and equilibrium constants can vary by several orders of magnitude for different myosin isoforms. Force can also affect the rates and equilibrium constants of the various transitions in an isoform-specific manner, leading to diversity of mechanosensing behaviors. Force can also promote actomyosin detachment through off-ATPase-pathway transitions (steps 7 and 8). To see this figure in color, go online.
Biophysical Journal 110, 2568–2576, June 21, 2016 2569
Greenberg et al.
size of the working stroke (15), and although d is proportional to the length of the lever arm in some myosin isoforms (16,17), the exact structural elements responsible for myosin mechanosensing are still not resolved. The effect of force on the actomyosin attachment lifetime depends on the magnitude of the force and whether the myosin is catalytically activated. In the case of rigor or ADP-bound skeletal muscle myosin (i.e., in the absence of ATP when the myosin is not enzymatically cycling), the actomyosin states from which myosin detaches directly from actin (Fig. 1, steps 7 and 8) can be described as catch bonds at forces 10 pN (18). Although noncycling actomyosin-V bound to ADP behaves as a catch bond, where force slows the rate of actomyosin-V dissociation, similar forces on ADP-bound actomyosin-VI cause the actomyosin bond to behave as a slip bond (19). However, it is important to note that these states dissociate very slowly from actin in the absence of force so that detachment from these states rarely occurs during active ATP cycling at physiological ATP concentrations. Rather, the pathway for dissociation that dominates under physiological conditions in cycling myosins is ATP-induced dissociation after ADP release (Fig. 1, step 2). In an active myosin, mechanical forces can theoretically affect any of the transitions among the actin-bound intermediates on the ATPase pathway. Moreover, the exact transition that limits turnover and is affected by force appears to be isoform specific, ranging from reversing the powerstroke to slowing the rates of ATP binding, ADP release, and/or an isomerization associated with ADP release (see below). Because these rates determine the attachment duration, sliding velocity, and power output, it is critical to consider exactly which transitions are affected by mechanical force. Force can affect the kinetic flux through different dissociation pathways (e.g., ATP-induced actomyosin dissociation, nucleotide-free actomyosin dissociation, and ADP-bound actomyosin dissociation), not just the probability of rupture of the actomyosin bond. As such, a simple two-state model, which is sometimes used to describe bond dissociation in the absence of enzymatic cycling, will often fail to capture the true behavior of the actomyosin interaction. Diversity of myosin mechanosensors There are 38 different myosin genes expressed in humans (20,21), and although many of these have been characterized biochemically, only a few have been studied mechanically. Here, we introduce to the reader four examples of nonprocessive myosins that demonstrate substantial diversity in biochemical and mechanosensing properties. Myosin-Ib (gene: MYO1B)
Myosin-Ib (Myo1b) is a single-headed, low-duty-ratio motor (in the absence of force) from the myosin-I family (22)
2570 Biophysical Journal 110, 2568–2576, June 21, 2016
that has been proposed to have roles in powering changes in Golgi morphology, endosomal movements, and signaling at the plasma membrane (23). Its motility rate is slow (120 nm/s at 37 C) and limited by the rate of ADP release (7 s1 at 37 C and 2.1 s1 at 20 C (17); Fig. 1, step 6). Single-molecule optical trapping experiments have shown that mechanical forces that resist the Myo1b powerstroke dramatically slow the rate of ADP release, which in turn slows the rate of motility (Fig. 1, step 6). Although ADP remains trapped in the active site for an extended period of time in the presence of force, at forces
A Perspective on the Role of Myosins as Mechanosensors ,2 Erkan Tu¨zel,2 and E. Michael Ostap3,* Michael J. Greenberg,1 Go¨ker Arpag 1 Biochemistry and Molecular Biophysics, Washington University, St. Louis, Missouri; 2Worcester Polytechnic Institute, Worcester, Massachusetts; and 3Pennsylvania Muscle Institute and Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
ABSTRACT Cells are dynamic systems that generate and respond to forces over a range of spatial and temporal scales, spanning from single molecules to tissues. Substantial progress has been made in recent years in identifying the molecules and pathways responsible for sensing and transducing mechanical signals to short-term cellular responses and longer-term changes in gene expression, cell identity, and tissue development. In this perspective article, we focus on myosin motors, as they not only function as the primary force generators in well-studied mechanobiological processes, but also act as key mechanosensors in diverse functions including intracellular transport, signaling, cell migration, muscle contraction, and sensory perception. We discuss how the biochemical and mechanical properties of different myosin isoforms are tuned to fulfill these roles in an array of cellular processes, and we highlight the underappreciated diversity of mechanosensing properties within the myosin superfamily. In particular, we use modeling and simulations to make predictions regarding how diversity in force sensing affects the lifetime of the actomyosin bond, the myosin power output, and the ability of myosin to respond to a perturbation in force for several nonprocessive myosin isoforms.
Exciting experiments in the field of mechanobiology have demonstrated that molecules, cells, and tissues are able to sense their environments and respond biologically to mechanical signals (1). Mechanical force is sensed by loadinduced changes in the conformation and/or activities of mechanosensitive macromolecules (i.e., mechanosensors), which in turn lead to a biological response. In mechanosensitive molecules such as p130Cas (2), von Willebrand factor (3), and titin (4), mechanical stress causes local protein unfolding that leads to the exposure of cryptic binding sites and/or sites of posttranslational modifications. In other proteins, such as Dam1 (5) and PSGL1 (6), force affects the interaction between a protein and its binding partner allosterically rather than through the exposure of cryptic sites. This allosteric tuning can either accelerate or slow the rate of bond dissociation. Forces can also directly alter enzymatic activities of mechanosensitive proteins such as myosin (below) or the opening probabilities of mechanosensitive ion channels (reviewed in (7)). The kinetics of the interaction between the molecular motor myosin and its cytoskeletal track, actin, can be allosterically tuned by force; however, in contrast to many
Submitted December 30, 2015, and accepted for publication May 16, 2016. *Correspondence: [email protected] Editor: Brian Salzberg. http://dx.doi.org/10.1016/j.bpj.2016.05.021 Ó 2016 Biophysical Society.
2568 Biophysical Journal 110, 2568–2576, June 21, 2016
other mechanosensitive proteins where the forces acting on the proteins are externally imposed, the forces affecting myosin can be externally imposed or internally generated by the myosin itself working against substrates of varied stiffnesses. It can be argued that skeletal muscle myosin, the motor that powers our locomotion, was one of the first recognized mechanosensors. In landmark experiments conducted in the 1920s, W. O. Fenn showed that a muscle contracting against a resisting load generates less heat (i.e., uses less ATP) than a muscle shortening in the absence of a load (8). These experiments were complemented by the work of A. V. Hill, which showed that the velocity of muscle shortening decreases nonlinearly with increasing force (9). Based on these observations, Hill proposed that force slows the enzymatic and motile activities of muscle myosin. In other words, besides acting as a force generator, muscle myosin also functions as a mechanosensor that changes its rate of motility in response to force. Subsequently, it has been shown that a muscle’s force sensitivity and rate of force development depend on its fiber type (10,11), suggesting that different myosin isoforms confer distinct mechanosensing properties to the muscle. We now know that the myosin gene family is large, and that the different isoforms have a range of force-generating properties that have evolved to function in a large array of physiological functions that extend beyond powering muscle contraction.
Tension Sensing by Myosin
Since the publication of these classic muscle physiology studies, single-molecule experiments have directly demonstrated that slowing of motility by force can be explained in part by changes in the attachment and detachment kinetics of actin-bound myosin (actomyosin). Depending on the magnitude of forces on myosin, actomyosin interactions can be described as catch- or slip-bonds, which have lifetimes that increase or decrease, respectively, with load. Notably, there are several features of the force dependence of the actomyosin interaction that distinguish its behavior from other catch- and slip-bonds, because these behaviors in myosin depend on its enzymatic activity. Moreover, different myosin isoforms can show distinct mechanosensing behaviors at a given force. Mechanosensing by myosin is regulated by chemical and mechanical forces To understand mechanosensing by myosin, knowledge of its mechanochemical pathway is required. All characterized myosins hydrolyze ATP via the same biochemical pathway and likely share similar mechanical intermediates (Fig. 1). Myosin detaches from actin upon ATP binding (Fig. 1, steps 1 and 2), myosin isomerizes to the prepowerstroke conformation, and ATP is hydrolyzed while myosin holds ADP and inorganic phosphate noncovalently in its active site (Fig. 1, step 3). Rebinding of myosin to actin induces the release of phosphate and the force-generating powerstroke (Fig. 1, steps 4 and 5), and ADP is released to complete the cycle (Fig. 1, step 6). The duty ratio, i.e., the fraction of time myosin spends attached to actin in its biochemical
cycle, correlates with the ability of myosin ensembles to generate force. It is crucial for the reader to note that 1) in many cases, the motility rate is limited by the detachment rate of myosin from actin (12); 2) the magnitude of force generated by an ensemble of myosins is proportional to the number of myosins bound strongly to actin; and 3) there are orders-of-magnitude variations among myosin isoforms in the rates and equilibrium constants that define their biochemical pathways, resulting in incredible diversity of sliding rates and force production (13). Critically, the rates and equilibrium constants of the biochemical and mechanical transitions shown in Fig. 1 can be modulated by force. To a first-order approximation, the effects of force on a kinetic rate (e.g., the rate of bond dissociation or ADP release) can be described by the Bell equation, which assumes an Arrhenius transition model (14): kðFÞ ¼ kf0 e
F , d kB T ;
(1)
where kf0 is the rate in the absence of force, F is the force on the molecule (where a positive F is a force that resists motion), kB is Boltzmann’s constant, T is the temperature, and d is the distance to the force-dependent transition state, also known as the distance parameter. Note, k(F) changes exponentially with d, which is a vector quantity that depends on the direction of the applied force. In this article, a positive d indicates a force that resists the forward motion of the myosin working stroke in the direction of the long-axis of the actin filament. At sufficiently high loads, all bonds become slip bonds, a behavior that is not captured in Eq. 1. It is important to note that the magnitude of d does not have to correspond to the
FIGURE 1 The myosin mechanochemical cycle. Although all characterized myosins follow this cycle, the individual rate and equilibrium constants can vary by several orders of magnitude for different myosin isoforms. Force can also affect the rates and equilibrium constants of the various transitions in an isoform-specific manner, leading to diversity of mechanosensing behaviors. Force can also promote actomyosin detachment through off-ATPase-pathway transitions (steps 7 and 8). To see this figure in color, go online.
Biophysical Journal 110, 2568–2576, June 21, 2016 2569
Greenberg et al.
size of the working stroke (15), and although d is proportional to the length of the lever arm in some myosin isoforms (16,17), the exact structural elements responsible for myosin mechanosensing are still not resolved. The effect of force on the actomyosin attachment lifetime depends on the magnitude of the force and whether the myosin is catalytically activated. In the case of rigor or ADP-bound skeletal muscle myosin (i.e., in the absence of ATP when the myosin is not enzymatically cycling), the actomyosin states from which myosin detaches directly from actin (Fig. 1, steps 7 and 8) can be described as catch bonds at forces 10 pN (18). Although noncycling actomyosin-V bound to ADP behaves as a catch bond, where force slows the rate of actomyosin-V dissociation, similar forces on ADP-bound actomyosin-VI cause the actomyosin bond to behave as a slip bond (19). However, it is important to note that these states dissociate very slowly from actin in the absence of force so that detachment from these states rarely occurs during active ATP cycling at physiological ATP concentrations. Rather, the pathway for dissociation that dominates under physiological conditions in cycling myosins is ATP-induced dissociation after ADP release (Fig. 1, step 2). In an active myosin, mechanical forces can theoretically affect any of the transitions among the actin-bound intermediates on the ATPase pathway. Moreover, the exact transition that limits turnover and is affected by force appears to be isoform specific, ranging from reversing the powerstroke to slowing the rates of ATP binding, ADP release, and/or an isomerization associated with ADP release (see below). Because these rates determine the attachment duration, sliding velocity, and power output, it is critical to consider exactly which transitions are affected by mechanical force. Force can affect the kinetic flux through different dissociation pathways (e.g., ATP-induced actomyosin dissociation, nucleotide-free actomyosin dissociation, and ADP-bound actomyosin dissociation), not just the probability of rupture of the actomyosin bond. As such, a simple two-state model, which is sometimes used to describe bond dissociation in the absence of enzymatic cycling, will often fail to capture the true behavior of the actomyosin interaction. Diversity of myosin mechanosensors There are 38 different myosin genes expressed in humans (20,21), and although many of these have been characterized biochemically, only a few have been studied mechanically. Here, we introduce to the reader four examples of nonprocessive myosins that demonstrate substantial diversity in biochemical and mechanosensing properties. Myosin-Ib (gene: MYO1B)
Myosin-Ib (Myo1b) is a single-headed, low-duty-ratio motor (in the absence of force) from the myosin-I family (22)
2570 Biophysical Journal 110, 2568–2576, June 21, 2016
that has been proposed to have roles in powering changes in Golgi morphology, endosomal movements, and signaling at the plasma membrane (23). Its motility rate is slow (120 nm/s at 37 C) and limited by the rate of ADP release (7 s1 at 37 C and 2.1 s1 at 20 C (17); Fig. 1, step 6). Single-molecule optical trapping experiments have shown that mechanical forces that resist the Myo1b powerstroke dramatically slow the rate of ADP release, which in turn slows the rate of motility (Fig. 1, step 6). Although ADP remains trapped in the active site for an extended period of time in the presence of force, at forces
