Smooth Muscle Myosin Filaments Research Articles

Effects of blebbistatin on force production, actin motility and the force-velocity relation produced by myosin II filaments and myofibrils.

Blebbistatin is a myosin II-specific inhibitor that is often used to study the relation between force generation and Pi release. The goal of this study was to investigate the effects of blebbistatin on force production and the actin motility while interacting with arrays of myosin motors. We used myosin filaments isolated from mammalian smooth muscles, rabbit psoas and the anterior byssus retractor (ABRM) muscles extracted from fresh Mytilus edulis in different experiments. We also used whole myofibrils isolated from the rabbit psoas. The force during myosin-actin interactions was measured with a system of micro-fabricated cantilevers, and the sliding velocity of actin was measured with a standard in-vitro motility assay. The force-velocity relation was measured in myofibrils with atomic force cantilevers. The force produced by the myosin filaments upon interaction with actin decreased with increasing concentrations of blebbistatin (5 μM-12.5 μM). The force produced by smooth and skeletal muscle myosin filaments were 95.93 ± 12.05 pN/μm before blebbistatin treatment, 71.45 ± 5.94 pN/μm with 5 μm blebbistatin, 62.05 ± 9.44 pN/μm with 7.5 μM blebbistatin, 38.28 ± 8.77 pN/μm with 10 μm blebbistatin and dropped to zero with 12.5 μM blebbistatin. All these differences were significantly different (p<0.0001). Sliding velocity of actin filaments also decreased consistently with increasing concentrations of blebbistatin. Before treatment, filaments slid over HMM at 3.68 ± 0.06 μm/s, decreasing to 2.30 ± 0.056 μm/s with 1 μM, 1.27 ± 0.043 μm/s with 5 μM, and 1.44 ± 0.032 μm/s with 30 μM. The maximal velocity decrease was observed at 5 μM of blebbistatin. These differences were significantly different (P<0.0001). There was a downward shift in the force-velocity relation in myofibrils treated with blebbistatin. These results suggest that blebbistatin regulates the mechanics of myosin motors working collectively in different experimental preparations.

Myosin assembly of smooth muscle: from ribbons and side polarity to a row polar helical model

After decades of debate over the structure of smooth muscle myosin filaments, it is still unclear whether they are helical, as in all other muscle types, or square in shape. In both cases bipolar building units are proposed, but the deduced cross-bridge arrangements are fundamentally different. The opposite polarity of the adjusting longitudinal rows is proposed for the helical structure, while in the case of square filaments, or myosin ribbons, only their two faces are appositively polarized. Analysis of our unpublished archival data on light meromyosin (LMM) paracrystals and myosin rod assemblies as well as the filaments themselves indicated that the rods were assembled with a 6°–7° tilt angle from the rods’ longitudinal axis, in contrast to the lack of tilt in LMM, both exhibiting a 14.3 nm myosin periodicity. Optical diffraction analysis of EM images of the rod assemblies and those of intact myosin confirmed their helical architecture characterized by 28 nm residue translations, 172 nm repeats and 516 nm pitch. A detailed helical model of these filaments was elucidated with bipolar tetramer building units made of two polar trimers. The filaments elongate at their two ends in a head-to-head manner, enabling targeted cross-bridge polarity of the adjacent rows, in the form of a unique Boerdijk–Coxeter type helix, similar to that of collagen or desmin fibers, with the covalent links replaced by a head-to-head clasp.

Self-assembly of smooth muscle myosin filaments: adaptation of filament length by telokin and Mg·ATP.

The contractile apparatus of smooth muscle is malleable to accommodate stress and strain exerted on the muscle cell and to maintain optimal contractility. Structural lability of smooth muscle myosin filaments is believed to play an important role in the cell's malleability. However, the mechanism and regulation of myosin filament formation is still poorly understood. In the present in vitro study, using a static light scattering method, length distributions were obtained from suspensions of short myosin filaments (SFs) formed by rapid dilution or long ones (LFs) formed by slow dialysis. The distributions indicated the presence of dynamic equilibriums between soluble myosin and the SFs; i.e.: trimers, hexamers and mini filaments, covering the range up to 0.75µm. The LFs were more stable, exhibiting favorable sizes at about 1.25, 2.4 and 4.5µm. More distinct distributions were obtained from filaments adsorbed to a glass surface, by evanescent wave scattering and local electric field enhancement. Addition of telokin (TL) to the suspensions of unphosphorylated SFs resulted in widening of the soluble range, while in the case of the LFs this shift was larger, and accompanied by reduced contribution of the soluble myosin species. Such changes were largely absent in the case of phosphorylated myosin. In contrast, the presence of Mg·ATP resulted in elongation of the filaments and clear separation of filaments from soluble myosin species. Thus, TL and Mg·ATP appeared to modify the distribution of myosin filament lengths, i.e., increasing the lengths in preparing for phosphorylation, or reducing it to aid dephosphorylation.

Evidence for S2 flexibility by direct visualization of quantum dot-labeled myosin heads and rods within smooth muscle myosin filaments moving on actin in vitro.

Myosins in muscle assemble into filaments by interactions between the C-terminal light meromyosin (LMM) subdomains of the coiled-coil rod domain. The two head domains are connected to LMM by the subfragment-2 (S2) subdomain of the rod. Our mixed kinetic model predicts that the flexibility and length of S2 that can be pulled away from the filament affects the maximum distance working heads can move a filament unimpeded by actin-attached heads. It also suggests that it should be possible to observe a head remain stationary relative to the filament backbone while bound to actin (dwell), followed immediately by a measurable jump upon detachment to regain the backbone trajectory. We tested these predictions by observing filaments moving along actin at varying ATP using TIRF microscopy. We simultaneously tracked two different color quantum dots (QDs), one attached to a regulatory light chain on the lever arm and the other attached to an LMM in the filament backbone. We identified events (dwells followed by jumps) by comparing the trajectories of the QDs. The average dwell times were consistent with known kinetics of the actomyosin system, and the distribution of the waiting time between observed events was consistent with a Poisson process and the expected ATPase rate. Geometric constraints suggest a maximum of ∼26 nm of S2 can be unzipped from the filament, presumably involving disruption in the coiled-coil S2, a result consistent with observations by others of S2 protruding from the filament in muscle. We propose that sufficient force is available from the working heads in the filament to overcome the stiffness imposed by filament-S2 interactions.

Muscle myosins form folded monomers, dimers, and tetramers during filament polymerization in vitro

Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail-head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.

The load dependence and the force-velocity relation in intact myosin filaments from skeletal and smooth muscles.

In the present study we evaluated the load dependence of force produced by isolated muscle myosin filaments interacting with fluorescently labeled actin filaments, using for the first time whole native myosin filaments. We used a newly developed approach that allowed the use of physiological levels of ATP. Single filaments composed of either skeletal or smooth muscle myosin and single filaments of actin were attached between pairs of nano-fabricated cantilevers of known stiffness. The filaments were brought into contact to produce force, which caused sliding of the actin filaments over the myosin filaments. We applied load to the system by either pushing or pulling the filaments during interactions and observed that increasing the load increased the force produced by myosin and decreasing the load decreased the force. We also performed additional experiments in which we clamped the filaments at predetermined levels of force, which caused the filaments to slide to adjust the different loads, allowing us to measure the velocity of length changes to construct a force-velocity relation. Force values were in the range observed previously with myosin filaments and molecules. The force-velocity curves for skeletal and smooth muscle myosins resembled the relations observed for muscle fibers. The technique can be used to investigate many issues of interest and debate in the field of muscle biophysics.

Abstract 107: Smooth Muscle α-actin Translocates to the Nucleus and Participates in Chromatin Remodeling at Smooth Muscle Contractile Gene Promoters

Smooth muscle cell specific α-actin (SMA, encoded by ACTA2 ), like other actins, polymerizes in the cytoplasm to form actin filaments that slide along smooth muscle myosin filaments to enact smooth muscle cell (SMC) contraction. Ubiquitously expressed β-actin polymerizes in filaments in the cytoplasm and also enters the nucleus and associates with various chromatin remodeling complexes. Nuclear β-actin has been shown to be important for the differentiation of a number of cell types, including neurons. We therefore asked whether SMA translocates to the nucleus and if nuclear SMA is critical for differentiation of smooth muscle cells. In explanted SMCs from human and mouse ascending aortas, cell fractionation and 2D gel electrophoresis identifies both SMA and β-actin in the nuclear isolates. Nuclear SMA but not β-actin accumulates with SMC differentiation driven by serum starvation and transforming growth factor β1 (TGFβ1) treatment. Chromatin immunoprecipitation with an SMA antibody reveals that the protein is bound to the promoters of SMC differentiation marker genes, including Acta2 , Cnn1, and Myh11 and that SMA is enriched over β-actin at these promoters with differentiation. Immunoprecipitation studies show that SMA binds specifically to the INO80 and the SWI/SNF chromatin remodeling complexes. In Acta2 -/- mouse SMCs, skeletal muscle α-actin (SKA) is ectopically expressed to compensate for the loss of SMA; SKA is in the nucleus and is enriched after TGFβ1 treatment, associating with both the INO80 and SWI/SNF complexes. Acta2 -/- SMCs are completely differentiated and respond normally to TGFβ1. However, in the mouse myoblast cell line C2C12, SKA is present in nuclear fractions but does not enrich with differentiation of these cells into myotubes, and is not associated with INO80 or SWI/SNF. We therefore propose that nuclear SMA is required for the remodeling of chromatin during the differentiation of SMCs, allowing contractile genes to become accessible for transcription, and that this remodeling is mediated by the involvement of SMA in the INO80 and SWI/SNF complexes These findings describe a novel role for SMA in the nucleus.

Using the SpyTag SpyCatcher system to label smooth muscle myosin II filaments with a quantum dot on the regulatory light chain

The regulatory light chain (RLC) of myosin is commonly tagged to monitor myosin behavior in vitro, in muscle fibers, and in cells. The goal of this study was to prepare smooth muscle myosin (SMM) filaments containing a single head labeled with a quantum dot (QD) on the RLC. We show that when the RLC is coupled to a QD at Cys-108 and exchanged into SMM, subsequent filament assembly is severely disrupted. To address this, we used a novel approach for myosin by implementing the SpyTag002 SpyCatcher002 system to prepare SMM incorporated with RLC constructs fused to SpyTag or SpyCatcher. We show that filament assembly, actin-activated steady-state ATPase activities, ability to be phosphorylated, and selected enzymatic and mechanical properties were essentially unaffected if either SpyTag or SpyCatcher were fused to the C-terminus of the RLC. Crucially for our application, we also show that a QD coupled to SpyCatcher can be covalently attached to a RLC-Spy incorporated into a SMM filament without disrupting the filament, and that the filaments can move along actin in vitro.

A Mixed-Kinetic Model Describes Unloaded Velocities of Smooth, Skeletal, and Cardiac Muscle Myosin Filaments in Vitro

In vitro motility assays, where purified myosin and actin move relative to one another, are used to better understand the mechanochemistry of the actomyosin adenosine triphosphatase (ATPase) cycle. We examined the relationship between the relative velocity (V) of actin and myosin and the number of available myosin heads (N) or [ATP] for smooth (SMM), skeletal (SKM), and cardiac (CMM) muscle myosin filaments moving over actin as well as V from actin filaments moving over a bed of monomeric SKM. These data do not fit well to a widely accepted model that predicts that V is limited by myosin detachment from actin (d/ton), where d equals step size and ton equals time a myosin head remains attached to actin. To account for these data, we have developed a mixed-kinetic model where V is influenced by both attachment and detachment kinetics. The relative contributions at a given V vary with the probability that a head will remain attached to actin long enough to reach the end of its flexible S2 tether. Detachment kinetics are affected by L/ton, where L is related to the tether length. We show that L is relatively long for SMM, SKM, and CMM filaments (59 ± 3 nm, 22 ± 9 nm, and 22 ± 2 nm, respectively). In contrast, L is shorter (8 ± 3 nm) when myosin monomers are attached to a surface. This suggests that the behavior of the S2 domain may be an important mechanical feature of myosin filaments that influences unloaded shortening velocities of muscle.

Force Produced by Smooth and Skeletal Muscle Myosin Filaments Measured with Micro-Fabricated Cantilevers

Force Produced by Smooth and Skeletal Muscle Myosin Filaments Measured with Micro-Fabricated Cantilevers

A mixed-kinetic model describes unloaded velocities of smooth, skeletal, and cardiac muscle myosin filaments in vitro.

In vitro motility assays, where purified myosin and actin move relative to one another, are used to better understand the mechanochemistry of the actomyosin adenosine triphosphatase (ATPase) cycle. We examined the relationship between the relative velocity (V) of actin and myosin and the number of available myosin heads (N) or [ATP] for smooth (SMM), skeletal (SKM), and cardiac (CMM) muscle myosin filaments moving over actin as well as V from actin filaments moving over a bed of monomeric SKM. These data do not fit well to a widely accepted model that predicts that V is limited by myosin detachment from actin (d/ton), where d equals step size and ton equals time a myosin head remains attached to actin. To account for these data, we have developed a mixed-kinetic model where V is influenced by both attachment and detachment kinetics. The relative contributions at a given V vary with the probability that a head will remain attached to actin long enough to reach the end of its flexible S2 tether. Detachment kinetics are affected by L/ton, where L is related to the tether length. We show that L is relatively long for SMM, SKM, and CMM filaments (59 ± 3 nm, 22 ± 9 nm, and 22 ± 2 nm, respectively). In contrast, L is shorter (8 ± 3 nm) when myosin monomers are attached to a surface. This suggests that the behavior of the S2 domain may be an important mechanical feature of myosin filaments that influences unloaded shortening velocities of muscle.

Helical model of smooth muscle myosin filament and the ribbons made of caldesmon: history revisited.

In early studies on smooth muscle, I described a crude myosin fraction (CMF) in which self-assembly of myosin filaments was observed. For the first time, the 14-nm periodicity stemming from regular arrangement of myosin heads on the filament surface was observed (Sobieszek in J Mol Biol 70:741-744, 1972). In this fraction, we also observed formation of long ribbon-shaped aggregates exhibiting a 5.6-nm periodicity, characteristic of tropomyosin (TM) paracrystals (Sobieszek and Small in Phil Trans R Soc Lond B 265:203-212, 1973). We therefore concluded that these ribbons were made of TM and they might be related to the myosin ribbons observed in electron micrographs (EM) of intact smooth muscle (Lowy and Small in Nature 227:46-51, 1970; Small and Squire in Mol Biol 67:117-149, 1972). Subsequently, Small (J Cell Sci 24:327-349, 1977) concluded that the ribbons observed in the EM sections were an artifact, but their observation in the CMF was not addressed. I have now revisited two aspects of the above studies. Firstly, based on my new multi-angle laser-scattering data and considering the length and stability of the building unit for the filament, a myosin trimer fit better to the previously proposed helical structure. Secondly, after two decades of systematic examinations of protein compositions in multiple smooth muscle extracts and isolated filaments, I concluded that the ribbons were made of caldesmon and not TM. Thirdly, actin-activated ATPase activity measurements indicated that modulation of this activity (by CaD and TM) was synergistic, cooperative and depended on myosin to actin ratio.

Velocities of unloaded muscle filaments are not limited by drag forces imposed by myosin cross-bridges

It is not known which kinetic step in the acto-myosin ATPase cycle limits contraction speed in unloaded muscles (V0). Huxley's 1957 model [Huxley AF (1957) Prog Biophys Biophys Chem 7:255-318] predicts that V0 is limited by the rate that myosin detaches from actin. However, this does not explain why, as observed by Bárány [Bárány M (1967) J Gen Physiol 50(6, Suppl):197-218], V0 is linearly correlated with the maximal actin-activated ATPase rate (vmax), which is limited by the rate that myosin attaches strongly to actin. We have observed smooth muscle myosin filaments of different length and head number (N) moving over surface-attached F-actin in vitro. Fitting filament velocities (V) vs. N to a detachment-limited model using the myosin step size d=8 nm gave an ADP release rate 8.5-fold faster and ton (myosin's attached time) and r (duty ratio) ∼10-fold lower than previously reported. In contrast, these data were accurately fit to an attachment-limited model, V=N·v·d, over the range of N found in all muscle types. At nonphysiologically high N, V=L/ton rather than d/ton, where L is related to the length of myosin's subfragment 2. The attachment-limited model also fit well to the [ATP] dependence of V for myosin-rod cofilaments at three fixed N. Previously published V0 vs. vmax values for 24 different muscles were accurately fit to the attachment-limited model using widely accepted values for r and N, giving d=11.1 nm. Therefore, in contrast with Huxley's model, we conclude that V0 is limited by the actin-myosin attachment rate.

Phosphate and ADP Differently Inhibit Coordinated Smooth Muscle Myosin Groups

Actin filaments propelled in vitro by groups of skeletal muscle myosin motors exhibit distinct phases of active sliding or arrest, whose occurrence depends on actin length (L) within a range of up to 1.0 μm. Smooth muscle myosin filaments are exponentially distributed with ≈150 nm average length in vivo—suggesting relevance of the L-dependence of myosin group kinetics. Here, we found L-dependent actin arrest and sliding in in vitro motility assays of smooth muscle myosin. We perturbed individual myosin kinetics with varying, physiological concentrations of phosphate (Pi, release associated with main power stroke) and adenosine diphosphate (ADP, release associated with minor mechanical step). Adenosine triphosphate was kept constant at physiological concentration. Increasing [Pi] lowered the fraction of time for which actin was actively sliding, reflected in reduced average sliding velocity (ν) and motile fraction (fmot, fraction of time that filaments are moving); increasing [ADP] increased the fraction of time actively sliding and reduced the velocity while sliding, reflected in reduced ν and increased fmot. We introduced specific Pi and ADP effects on individual myosin kinetics into our recently developed mathematical model of actin propulsion by myosin groups. Simulations matched our experimental observations and described the inhibition of myosin group kinetics. At low [Pi] and [ADP], actin arrest and sliding were reflected by two distinct chemical states of the myosin group. Upon [Pi] increase, the probability of the active state decreased; upon [ADP] increase, the probability of the active state increased, but the active state became increasingly similar to the arrested state.

Varying the Number of Heads in Phosphorylated Smooth Muscle Myosin Filaments Provides Evidence for Attachment Limited Kinetics of in vitro Actin-Sliding Velocities

Previously we have shown that the ATP-dependence of EDC-stabilized fluorescent smooth muscle myosin (SMM) filament motility is very similar to solution ATPase values (KATP = 8.5 and 9.2 µM, respectively). This suggests both processes are influenced by the same kinetic step, acto-myosin attachment not detachment, presumably linked to Pi release. This mechanism predicts that velocities will be linearly related to the number of working heads. Therefore, we prepared SMM- rod co-filaments that contain fewer molecules per filament length as well as longer SMM filaments that contain more molecules per filament to determine the relationship between the number of heads and velocity. SMM:rod co-filaments (at 50:50, 25:75, and 15:25 ratios) were approximately the same length as normal SMM filaments (∼0.6 µm), and the number of molecules incorporated was directly related to the ratio of SMM to rods. As the number of heads decreased, the maximal velocity at saturating ATP also decreased, and the KATP was lower for all of the co-filaments compared to the normal filaments. These findings provide evidence that the velocity of SMM filaments is influenced by attachment limited kinetics. The longer SMM filaments (average length of 2.3 ± 0.81 µm) will allow us to visualize the geometry of acto-myosin interactions during motion. Also, the relationship between velocity and filament length will be determined at various [ATP]. The long filaments also better demonstrate our previously described “parking” behavior where filaments move to the end of actin filaments but remain attached for long periods even at saturating [ATP] (Haldeman et al, JBC, 2014). Interestingly, most of the SMM filament hangs off the end of the actin suggesting the parking is mediated by a small fraction of the heads.

Kinetic Characterization of Stabilized Smooth Muscle Myosin Filaments

We have measured kinetic parameters of smooth muscle myosin (SMM) filament interactions with actin, ADP, and ATP using stopped-flow fluorimetry. Our objective was to determine which parameters, if any, are different from those of monomeric myosins, S1 and HMM, using buffers at physiologic ionic strength. Previously we measured the ATP-dependence of SMM filament velocities (using EDC-cross-linked filaments that are stable to ATP-induced depolymerization) in an inverted in vitro motility assay with myosin filaments moving over surface-attached actin. We compared this to the ATP-dependence of solution actin-activated ATPase measurements. The two relationships are very similar, suggesting that they are both attachment-limited and regulated by the same kinetic step, presumably phosphate release (Haldeman et al JBC 2014). This is in contrast to the kinetics of actin moving over a surface of monomeric SMM (standard vitro motility assay), known to be predominately detachment-limited, limited by ADP release. We hypothesized that heads within moving filaments are attached to unhindered and thus flexible S2 regions putting minimal strain on heads put in the drag position. In contrast, the S2 region of monomeric myosin is bound to the surface and not as flexible allowing the relative stiff head domains to become strained, which affects the underlying kinetics of acto-SMM interactions. All kinetic parameters measured so far in solution for myosin or acto-myosin, ADP association, ADP dissociation, ATP binding, ATP-induced dissociation, and acto-myosin affinity, are very similar to those for S1 and HMM. Measurements of Pi release kinetics by limited turnover are planned. If Pi release kinetics is also similar, this will suggest that the attachment-limited kinetics that is unique to filaments is not due to alterations in any fundamental kinetic parameters, but instead is due to differences in the effective head or head/S2 stiffness during the assays.

The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in vitro

The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in vitro

The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in Vitro

Actin-myosin interactions are well studied using soluble myosin fragments, but little is known about effects of myosin filament structure on mechanochemistry. We stabilized unphosphorylated smooth muscle myosin (SMM) and phosphorylated smooth muscle myosin (pSMM) filaments against ATP-induced depolymerization using a cross-linker and attached fluorescent rhodamine (XL-Rh-SMM). Electron micrographs showed that these side polar filaments are very similar to unmodified filaments. They are ~0.63 μm long and contain ~176 molecules. Rate constants for ATP-induced dissociation and ADP release from acto-myosin for filaments and S1 heads were similar. Actin-activated ATPases of SMM and XL-Rh-SMM were similarly regulated. XL-Rh-pSMM filaments moved processively on F-actin that was bound to a PEG brush surface. ATP dependence of filament velocities was similar to that for solution ATPases at high [actin], suggesting that both processes are limited by the same kinetic step (weak to strong transition) and therefore are attachment- limited. This differs from actin sliding over myosin monomers, which is primarily detachment-limited. Fitting filament data to an attachment-limited model showed that approximately half of the heads are available to move the filament, consistent with a side polar structure. We suggest the low stiffness subfragment 2 (S2) domain remains unhindered during filament motion in our assay. Actin-bound negatively displaced heads will impart minimal drag force because of S2 buckling. Given the ADP release rate, the velocity, and the length of S2, these heads will detach from actin before slack is taken up into a backwardly displaced high stiffness position. This mechanism explains the lack of detachment- limited kinetics at physiological [ATP]. These findings address how nonlinear elasticity in assemblies of motors leads to efficient collective force generation.

Kinetic Characterization of Interactions between Stabilized Smooth Muscle Myosin Filaments and Actin

Using our previously described EDC-cross-linked smooth muscle myosin (SMM) filaments that are stable to ATP-induced depolymerization, we measured kinetic parameters of the SMM filament interactions with actin comparing the filamentous physiological state with monomeric forms of SMM. We use stopped-flow spectrometry, actin-activated steady state Mg-ATPase, and in vitro motility assays to measure the fundamental kinetic differences between SMM monomers and filaments. Using stopped-flow, we show that the apparent second order rate constant for ATP binding to the complex of pyrene-actin with SMM filaments is 0.21 µM-1s-1, similar to HMM monomers with two heads, at 0.46 µM-1s-1, and single-headed S1 at 0.47 µM-1s-1. Using TIRF microscopy of filaments bound to surface-attached F-actin, we show ATP decreases the number of unphosphorylated SMM filaments bound per µm actin, giving an apparent KATP of 4.8 µM. We show that the ATP concentration that gives half-maximal velocities of actin moving over surface-attached phosphorylated SMM monomers is 21.2 µM (KATP). Using inverted-geometry to measure the velocities of phosphorylated SMM filaments moving over surface-attached actin gave a much lower KATP = 8.5 µM. Actin-activated steady-state Mg-ATPase of phosphorylated filaments gave a KATP = 9.2 µM. The similarities in the two KATPs for filaments suggest that filament velocities and ATPase are limited by the same kinetic step, which best fits an attachment-limited over a detachment-limited model. Using TIRF, we show that phosphorylated SMM filaments move over surface-attached actin filaments until they reach the ends from which they do not detach, even upon dephosphorylation of the SMM. This behavior is specific to SMM filaments since skeletal filaments readily detach from actin filament ends. These observations suggest that phosphorylated SMM filaments that have been dephosphorylated are in a different state than unphosphorylated filaments in the presence of ATP.

Myosin filaments in smooth muscle cells do not have a constant length

Myosin molecules from smooth muscle and non-muscle cells are known to self-assemble into side-polar filaments in vitro. However, the in situ mechanism of filament assembly is not clear and the question of whether there is a unique length for myosin filaments in smooth muscle is still under debate. In this study we measured the lengths of 16,587 myosin filaments in three types of smooth muscle cells using serial electron microscopy (EM). Sheep airway and pulmonary arterial smooth muscle as well as rabbit carotid arterial smooth muscle were fixed for EM and serial ultra-thin (50-60 nm) sections were obtained. Myosin filaments were traced in consecutive sections to determine their lengths. The results indicate that there is not a single length for the myosin filaments; instead there is a wide variation in lengths. The plots of observation frequency versus myosin filament length follow an exponential decay pattern. Analysis suggests that in situ assembly of myosin filaments in smooth muscle is governed by random processes of linear polymerization and de-polymerization, and that the dynamic equilibrium of these processes determines the observed length distribution.