Double-headed binding of myosin II to F-actin shows the effect of strain on head structure.

Force production in muscle is achieved through the interaction of myosin and actin. Strong binding states in active muscle are associated with Mg·ADP bound to the active site; release of Mg·ADP allows rebinding of ATP and dissociation from actin. Thus, Mg·ADP binding is positioned for adaptation as a force sensor. Mechanical loads on the lever arm can affect the ability of myosin to release Mg·ADP but exactly how this is done is poorly defined. Here we use F-actin decorated with double-headed smooth muscle myosin fragments in the presence of Mg·ADP to visualize the effect of internally supplied tension on the paired lever arms using cryoEM. The interaction of the paired heads with two adjacent actin subunits is predicted to place one lever arm under positive and the other under negative strain. The converter domain is believed to be the most flexible domain within myosin head. Our results, instead, point to the segment of heavy chain between the essential and regulatory light chains as the location of the largest structural change. Moreover, our results suggest no large changes in the myosin coiled coil tail as the locus of strain relief when both heads bind F-actin. The method would be adaptable to double-headed members of the myosin family. We anticipate that the study of actin-myosin interaction using double-headed fragments enables visualization of domains that are typically noisy in decoration with single-headed fragments.

Mechanism of small molecule inhibition of Plasmodium falciparum myosin A informs antimalarial drug design.

Malaria results in more than 500,000 deaths per year and the causative Plasmodium parasites continue to develop resistance to all known agents, including different antimalarial combinations. The class XIV myosin motor PfMyoA is part of a core macromolecular complex called the glideosome, essential for Plasmodium parasite mobility and therefore an attractive drug target. Here, we characterize the interaction of a small molecule (KNX-002) with PfMyoA. KNX-002 inhibits PfMyoA ATPase activity in vitro and blocks asexual blood stage growth of merozoites, one of three motile Plasmodium life-cycle stages. Combining biochemical assays and X-ray crystallography, we demonstrate that KNX-002 inhibits PfMyoA using a previously undescribed binding mode, sequestering it in a post-rigor state detached from actin. KNX-002 binding prevents efficient ATP hydrolysis and priming of the lever arm, thus inhibiting motor activity. This small-molecule inhibitor of PfMyoA paves the way for the development of alternative antimalarial treatments.

Resistance of Acta2R149C/+ mice to aortic disease is associated with defective release of mutant smooth muscle α-actin from the chaperonin-containing TCP1 folding complex.

Pathogenic variants of the gene for smooth muscle α-actin (ACTA2), which encodes smooth muscle (SM) α-actin, predispose to heritable thoracic aortic disease. The ACTA2 variant p.Arg149Cys (R149C) is the most common alteration; however, only 60% of carriers have a dissection or undergo repair of an aneurysm by 70 years of age. A mouse model of ACTA2 p.Arg149Cys was generated using CRISPR/Cas9 technology to determine the etiology of reduced penetrance. Acta2R149C/+ mice had significantly decreased aortic contraction compared with WT mice but did not form aortic aneurysms or dissections when followed to 24 months, even when hypertension was induced. In vitro motility assays found decreased interaction of mutant SM α-actin filaments with SM myosin. Polymerization studies using total internal reflection fluorescence microscopy showed enhanced nucleation of mutant SM α-actin by formin, which correlated with disorganized and reduced SM α-actin filaments in Acta2R149C/+ smooth muscle cells (SMCs). However, the most prominent molecular defect was the increased retention of mutant SM α-actin in the chaperonin-containing t-complex polypeptide folding complex, which was associated with reduced levels of mutant compared with WT SM α-actin in Acta2R149C/+ SMCs. These data indicate that Acta2R149C/+ mice do not develop thoracic aortic disease despite decreased contraction of aortic segments and disrupted SM α-actin filament formation and function in Acta2R149C/+ SMCs. Enhanced binding of mutant SM α-actin to chaperonin-containing t-complex polypeptide decreases the mutant actin versus WT monomer levels in Acta2R149C/+ SMCs, thus minimizing the effect of the mutation on SMC function and potentially preventing aortic disease in the Acta2R149C/+ mice.

The actomyosin interface contains an evolutionary conserved core and an ancillary interface involved in specificity.

Plasmodium falciparum, the causative agent of malaria, moves by an atypical process called gliding motility. Actomyosin interactions are central to gliding motility. However, the details of these interactions remained elusive until now. Here, we report an atomic structure of the divergent Plasmodium falciparum actomyosin system determined by electron cryomicroscopy at the end of the powerstroke (Rigor state). The structure provides insights into the detailed interactions that are required for the parasite to produce the force and motion required for infectivity. Remarkably, the footprint of the myosin motor on filamentous actin is conserved with respect to higher eukaryotes, despite important variability in the Plasmodium falciparum myosin and actin elements that make up the interface. Comparison with other actomyosin complexes reveals a conserved core interface common to all actomyosin complexes, with an ancillary interface involved in defining the spatial positioning of the motor on actin filaments.

Unusual dynamics of the divergent malaria parasite PfAct1 actin filament.

Gliding motility and host cell invasion by the apicomplexan parasite Plasmodium falciparum (Pf), the causative agent of malaria, is powered by a macromolecular complex called the glideosome that lies between the parasite plasma membrane and the inner membrane complex. The glideosome core consists of a single-headed class XIV myosin PfMyoA and a divergent actin PfAct1. Here we use total internal reflection fluorescence microscopy to visualize growth of individual unstabilized PfAct1 filaments as a function of time, an approach not previously used with this actin isoform. Although PfAct1 was thought to be incapable of forming long filaments, filaments grew as long as 30 µm. Polymerization occurs via a nucleation-elongation mechanism, but with an ∼4 µM critical concentration, an order-of-magnitude higher than for skeletal actin. Protomers disassembled from both the barbed and pointed ends of the actin filament with similar fast kinetics of 10 to 15 subunits/s. Rapid treadmilling, where the barbed end of the filament grows and the pointed end shrinks while maintaining an approximately constant filament length, was visualized near the critical concentration. Once ATP has been hydrolyzed to ADP, the filament becomes very unstable, resulting in total dissolution in <40 min. Dynamics at the filament ends are suppressed in the presence of inorganic phosphate or more efficiently by BeFX A chimeric PfAct1 with a mammalian actin D-loop forms a more stable filament. These unusual dynamic properties distinguish PfAct1 from more canonical actins, and likely contribute to the difficultly in visualizing PfAct1 filaments in the parasite.

Tropomyosin Must Interact Weakly with Actin to Effectively Regulate Thin Filament Function.

Elongated tropomyosin, associated with actin-subunits along the surface of thin filaments, makes electrostatic interactions with clusters of conserved residues, K326, K328, and R147, on actin. The association is weak, permitting low-energy cost regulatory movement of tropomyosin across the filament during muscle activation. Interestingly, acidic D292 on actin, also evolutionarily conserved, lies adjacent to the three-residue cluster of basic amino acids and thus may moderate the combined local positive charge, diminishing tropomyosin-actin interaction and facilitating regulatory-switching. Indeed, charge neutralization of D292 is connected to muscle hypotonia in individuals with D292V actin mutations and linked to congenital fiber-type disproportion. Here, the D292V mutation may predispose tropomyosin-actin positioning to a myosin-blocking state, aberrantly favoring muscle relaxation, thus mimicking the low-Ca2+ effect of troponin even in activated muscles. To test this hypothesis, interaction energetics and in vitro function of wild-type and D292V filaments were measured. Energy landscapes based on F-actin-tropomyosin models show the mutation localizes tropomyosin in a blocked-state position on actin defined by a deeper energy minimum, consistent with augmented steric-interference of actin-myosin binding. In addition, whereas myosin-dependent motility of troponin/tropomyosin-free D292V F-actin is normal, motility is dramatically inhibited after addition of tropomyosin to the mutant actin. Thus, D292V-induced blocked-state stabilization appears to disrupt the delicately poised energy balance governing thin filament regulation. Our results validate the premise that stereospecific but necessarily weak binding of tropomyosin to F-actin is required for effective thin filament function.

Reconstitution of the core of the malaria parasite glideosome with recombinant Plasmodium class XIV myosin A and Plasmodium actin.

Motility of the apicomplexan malaria parasite Plasmodium falciparum is enabled by a multiprotein glideosome complex, whose core is the class XIV myosin motor, PfMyoA, and a divergent Plasmodium actin (PfAct1). Parasite motility is necessary for host-cell invasion and virulence, but studying its molecular basis has been hampered by unavailability of sufficient amounts of PfMyoA. Here, we expressed milligram quantities of functional full-length PfMyoA with the baculovirus/Sf9 cell expression system, which required a UCS (UNC-45/CRO1/She4p) family myosin chaperone from Plasmodium spp. In addition to the known light chain myosin tail interacting protein (MTIP), we identified an essential light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. The speed at which PfMyoA moved actin was fastest with both light chains bound, consistent with the light chain-binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfELC binding to the heavy chain required that MTIP also be bound to the heavy chain, unlike MTIP that bound the heavy chain independently of PfELC. Neither the presence of calcium nor deletion of the MTIP N-terminal extension changed the speed of actin movement. Of note, PfMyoA moved filaments formed from Sf9 cell-expressed PfAct1 at the same speed as skeletal muscle actin. Duty ratio estimates suggested that as few as nine motors can power actin movement at maximal speed, a feature that may be necessitated by the dynamic nature of Plasmodium actin filaments in the parasite. In summary, we have reconstituted the essential core of the glideosome, enabling drug targeting of both of its core components to inhibit parasite invasion.

Severe Molecular Defects Exhibited by the R179H Mutation in Human Vascular Smooth Muscle α-Actin.

Mutations in vascular smooth muscle α-actin (SM α-actin), encoded by ACTA2, are the most common cause of familial thoracic aortic aneurysms that lead to dissection (TAAD). The R179H mutation has a poor patient prognosis and is unique in causing multisystemic smooth muscle dysfunction (Milewicz, D. M., Østergaard, J. R., Ala-Kokko, L. M., Khan, N., Grange, D. K., Mendoza-Londono, R., Bradley, T. J., Olney, A. H., Ades, L., Maher, J. F., Guo, D., Buja, L. M., Kim, D., Hyland, J. C., and Regalado, E. S. (2010) Am. J. Med. Genet. A 152A, 2437-2443). Here, we characterize this mutation in expressed human SM α-actin. R179H actin shows severe polymerization defects, with a 40-fold higher critical concentration for assembly than WT SM α-actin, driven by a high disassembly rate. The mutant filaments are more readily severed by cofilin. Both defects are attenuated by copolymerization with WT. The R179H monomer binds more tightly to profilin, and formin binding suppresses nucleation and slows polymerization rates. Linear filaments will thus not be readily formed, and cells expressing R179H actin will likely have increased levels of monomeric G-actin. The cotranscription factor myocardin-related transcription factor-A, which affects cellular phenotype, binds R179H actin with less cooperativity than WT actin. Smooth muscle myosin moves R179H filaments more slowly than WT, even when copolymerized with equimolar amounts of WT. The marked decrease in the ability to form filaments may contribute to the poor patient prognosis and explain why R179H disrupts even visceral smooth muscle cell function where the SM α-actin isoform is present in low amounts. The R179H mutation has the potential to affect actin structure and function in both the contractile domain of the cell and the more dynamic cytoskeletal pool of actin, both of which are required for contraction.

Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin.

Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD.

Role of the essential light chain in the activation of smooth muscle myosin by regulatory light chain phosphorylation.

The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at serine 19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head-head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. Phosphorylation of serine 19 disrupts these head-head interactions by an unknown mechanism. Computational modeling (Tama et al., 2005. J. Mol. Biol. 345, 837-854) suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Therefore, altering relative motions between the ELC and the RLC at this locus might disrupt the inhibited state. Based on this hypothesis we have derived an atomic model for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). This model predicts a set of specific interactions between the N-terminal residues of the RLC with both the myosin HC and the ELC. Site directed mutagenesis was used to show that interactions between the phosphorylated N-terminus of the RLC and helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin.

A periodic pattern of evolutionarily conserved basic and acidic residues constitutes the binding interface of actin-tropomyosin.

Actin filament cytoskeletal and muscle functions are regulated by actin binding proteins using a variety of mechanisms. A universal actin filament regulator is the protein tropomyosin, which binds end-to-end along the length of the filament. The actin-tropomyosin filament structure is unknown, but there are atomic models in different regulatory states based on electron microscopy reconstructions, computational modeling of actin-tropomyosin, and docking of atomic resolution structures of tropomyosin to actin filament models. Here, we have tested models of the actin-tropomyosin interface in the "closed state" where tropomyosin binds to actin in the absence of myosin or troponin. Using mutagenesis coupled with functional analyses, we determined residues of actin and tropomyosin required for complex formation. The sites of mutations in tropomyosin were based on an evolutionary analysis and revealed a pattern of basic and acidic residues in the first halves of the periodic repeats (periods) in tropomyosin. In periods P1, P4, and P6, basic residues are most important for actin affinity, in contrast to periods P2, P3, P5, and P7, where both basic and acidic residues or predominantly acidic residues contribute to actin affinity. Hydrophobic interactions were found to be relatively less important for actin binding. We mutated actin residues in subdomains 1 and 3 (Asp(25)-Glu(334)-Lys(326)-Lys(328)) that are poised to make electrostatic interactions with the residues in the repeating motif on tropomyosin in the models. Tropomyosin failed to bind mutant actin filaments. Our mutagenesis studies provide the first experimental support for the atomic models of the actin-tropomyosin interface.

Tropomyosin is essential for processive movement of a class V myosin from budding yeast.

Myosin V is an actin-based motor protein involved in intracellular cargo transport [1]. Given this physiological role, it was widely assumed that all class V myosins are processive, able to take multiple steps along actin filaments without dissociating. This notion was challenged when several class V myosins were characterized as nonprocessive in vitro, including Myo2p, the essential class V myosin from S. cerevisiae [2-6]. Myo2p moves cargo including secretory vesicles and other organelles for several microns along actin cables in vivo. This demonstrated cargo transporter must therefore either operate in small ensembles or behave processively in the cellular context. Here we show that Myo2p moves processively in vitro as a single motor when it walks on an actin track that more closely resembles the actin cables found in vivo. The key to processivity is tropomyosin: Myo2p is not processive on bare actin but highly processive on actin-tropomyosin. The major yeast tropomyosin isoform, Tpm1p, supports the most robust processivity. Tropomyosin slows the rate of MgADP release, thus increasing the time the motor spends strongly attached to actin. This is the first example of tropomyosin switching a motor from nonprocessive to processive motion on actin.

Phosphorylated smooth muscle heavy meromyosin shows an open conformation linked to activation.

Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions between motor domains. The "free head" can bind to actin, but the actin binding interface of the "blocked head" is involved in interactions with the free head. We report here a three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional arrays. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the "blocked" head of dephosphorylated smHMM. We hypothesize that regulatory light chain phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.

Smooth muscle heavy meromyosin phosphorylated on one of its two heads supports force and motion.

Smooth muscle myosin is activated by regulatory light chain (RLC) phosphorylation. In the unphosphorylated state the activity of both heads is suppressed due to an asymmetric, intramolecular interaction between the heads. The properties of myosin with only one of its two RLCs phosphorylated, a state likely to be present both during the activation and the relaxation phase of smooth muscle, is less certain despite much investigation. Here we further characterize the mechanical properties of an expressed heavy meromyosin (HMM) construct with only one of its RLCs phosphorylated (HMM-1P). This construct was previously shown to have more than 50% of the ATPase activity of fully phosphorylated myosin (HMM-2P) and to move actin at the same speed in a motility assay as HMM-2P (Rovner, A. S., Fagnant, P. M., and Trybus, K. M. (2006) Biochemistry 45, 5280-5289). Here we show that the unitary step size and attachment time to actin of HMM-1P is indistinguishable from that of HMM-2P. Force-velocity measurements on small ensembles show that HMM-1P can generate approximately half the force of HMM-2P, which may relate to the observed duty ratio of HMM-1P being approximately half that of HMM-2P. Therefore, single-phosphorylated smooth muscle HMM molecules are active species, and the head associated with the unphosphorylated RLC is mechanically competent, allowing it to make a substantial contribution to both motion and force generation during smooth muscle contraction.

Mutation of a conserved glycine in the SH1-SH2 helix affects the load-dependent kinetics of myosin.

The ATP hydrolysis rate and shortening velocity of muscle are load-dependent. At the molecular level, myosin generates force and motion by coupling ATP hydrolysis to lever arm rotation. When a laser trap was used to apply load to single heads of expressed smooth muscle myosin (S1), the ADP release kinetics accelerated with an assistive load and slowed with a resistive load; however, ATP binding was mostly unaffected. To investigate how load is communicated within the motor, a glycine located at the putative fulcrum of the lever arm was mutated to valine (G709V). In the absence of load, stopped-flow and laser trap studies showed that the mutation significantly slowed the rates of ADP release and ATP binding, accounting for the approximately 270-fold decrease in actin sliding velocity. The load dependence of the mutant's ADP release rate was the same as that of wild-type S1 (WT) despite the slower rate. In contrast, load accelerated ATP binding by approximately 20-fold, irrespective of loading direction. Imparting mechanical energy to the mutant motor partially reversed the slowed ATP binding by overcoming the elevated activation energy barrier. These results imply that conformational changes near the conserved G709 are critical for the transmission of mechanochemical information between myosin's active site and lever arm.

Phosphorylation of a single head of smooth muscle myosin activates the whole molecule.

Regulatory light chain (RLC) phosphorylation activates smooth and non-muscle myosin II, but it has not been established if phosphorylation of one head turns on the whole molecule. Baculovirus expression and affinity chromatography were used to isolate heavy meromyosin (HMM) containing one phosphorylated and one dephosphorylated RLC (1-P HMM). Motility and steady-state ATPase assays indicated that 1-P HMM is nearly as active as HMM with two phosphorylated heads (2-P HMM). Single-turnover experiments further showed that both the dephosphorylated and phosphorylated heads of 1-P HMM can be activated by actin. Singly phosphorylated full-length myosin was also an active species with two cycling heads. Our results suggest that phosphorylation of one RLC abolishes the asymmetric inhibited state formed by dephosphorylated myosin [Liu, J., et al. (2003) J. Mol. Biol. 329, 963-972], allowing activation of both the phosphorylated and dephosphorylated heads. These findings help explain how smooth muscles are able to generate high levels of stress with low phosphorylation levels.

Expression of a nonpolymerizable actin mutant in Sf9 cells.

We have succeeded in expressing actin in the baculovirus/Sf9 cell system in high yield. The wild-type (WT) actin is functionally indistinguishable from tissue-purified actin in its ability to activate ATPase activity and to support movement in an in vitro motility assay. Having achieved this feat, we used a mutational strategy to express a monomeric actin that is incapable of polymerization. Native actin requires actin binding proteins or chemical modification to maintain it in a monomeric state. The mutant actin sediments in the analytical ultracentrifuge as a homogeneous monomeric species of 3.2 S in 100 mM KCl and 2 mM MgCl(2), conditions that cause WT actin to polymerize. The two point mutations that render actin nonpolymerizable are in subdomain 4 (A204E/P243K; "AP-actin"), distant from the myosin binding site. AP-actin binds to skeletal myosin subfragment 1 (S1) and forms a homogeneous complex as demonstrated by analytical ultracentrifugation. The ATPase activity of a cross-linked AP-actin.S1 complex is higher than that of S1 alone, although less than that supported by filamentous actin (F-actin). AP-Actin is an excellent candidate for structural studies of complexes of actin with motor proteins and other actin-binding proteins.

A mutant heterodimeric myosin with one inactive head generates maximal displacement.

Each of the heads of the motor protein myosin II is capable of supporting motion. A previous report showed that double-headed myosin generates twice the displacement of single-headed myosin (Tyska, M.J., D.E. Dupuis, W.H. Guilford, J.B. Patlak, G.S. Waller, K.M. Trybus, D.M. Warshaw, and S. Lowey. 1999. Proc. Natl. Acad. Sci. USA. 96:4402-4407). To determine the role of the second head, we expressed a smooth muscle heterodimeric heavy meromyosin (HMM) with one wild-type head, and the other locked in a weak actin-binding state by introducing a point mutation in switch II (E470A). Homodimeric E470A HMM did not support in vitro motility, and only slowly hydrolyzed MgATP. Optical trap measurements revealed that the heterodimer generated unitary displacements of 10.4 nm, strikingly similar to wild-type HMM (10.2 nm) and approximately twice that of single-headed subfragment-1 (4.4 nm). These data show that a double-headed molecule can achieve a working stroke of approximately 10 nm with only one active head and an inactive weak-binding partner. We propose that the second head optimizes the orientation and/or stabilizes the structure of the motion-generating head, thereby resulting in maximum displacement.

The two heads of smooth muscle myosin are enzymatically independent but mechanically interactive.

The interaction between the two heads of myosin II during motion and force production is poorly understood. To examine this issue, we developed an expression and purification strategy to isolate homogeneous populations of heterodimeric smooth muscle heavy meromyosins containing heads with different properties. As an extreme example, we characterized a heterodimer containing one native head and one head locked in a "weak binding" state by a point mutation in switch 2 (E470A). The in vitro actin filament motility of this heterodimer was the same as the homodimeric control with two cycling heads, suggesting that only one head of a pair actively interacts with actin to generate maximal velocity. A second naturally occurring heterodimer contained two cycling heads with 2-fold different activity, due to the presence or absence of a 7-amino acid insert near the active site. Enzymatically this (+/-) insert heterodimer was indistinguishable from a (50:50) mixture of the two homodimers, but its motility averaged 17% less than that of the mixture. These data suggest that one head of a heterodimer can disproportionately affect the mechanics of double-headed myosin, a finding relevant to our understanding of heterozygous mutant myosins found in disease states like familial hypertrophic cardiomyopathy.

The carboxyl-terminal isoforms of smooth muscle myosin heavy chain determine thick filament assembly properties.

The alternatively spliced SM1 and SM2 smooth muscle myosin heavy chains differ at their respective carboxyl termini by 43 versus 9 unique amino acids. To determine whether these tailpieces affect filament assembly, SM1 and SM2 myosins, the rod region of these myosin isoforms, and a rod with no tailpiece (tailless), were expressed in Sf 9 cells. Paracrystals formed from SM1 and SM2 rod fragments showed different modes of molecular packing, indicating that the tailpieces can influence filament structure. The SM2 rod was less able to assemble into stable filaments than either SM1 or the tailless rods. Expressed full-length SM1 and SM2 myosins showed solubility differences comparable to the rods, establishing the validity of the latter as a model for filament assembly. Formation of homodimers of SM1 and SM2 rods was favored over the heterodimer in cells coinfected with both viruses, compared with mixtures of the two heavy chains renatured in vitro. These results demonstrate for the first time that the smooth muscle myosin tailpieces differentially affect filament assembly, and suggest that homogeneous thick filaments containing SM1 or SM2 myosin could serve distinct functions within smooth muscle cells.