Dynamic Network Morphology and Tension Buildup in a 3D Model of Cytokinetic Ring Assembly

During fission yeast cytokinesis, actin filaments nucleated by cortical formin Cdc12 are captured by myosin motors bound to a band of cortical nodes and bundled by cross-linking proteins. The myosin motors exert forces on the actin filaments, resulting in a net pulling of the nodes into a contractile ring, while cross-linking interactions help align actin filaments and nodes into a single bundle. We used these mechanisms in a three-dimensional computational model of contractile ring assembly, with semiflexible actin filaments growing from formins at cortical nodes, capturing of filaments by neighboring nodes, and cross-linking among filaments through attractive interactions. The model was used to predict profiles of actin filament density at the cell cortex, morphologies of condensing node-filament networks, and regimes of cortical tension by varying the node pulling force and strength of cross-linking among actin filaments. Results show that cross-linking interactions can lead to confinement of actin filaments at the simulated cortical boundary. We show that the ring-formation region in parameter space lies close to regions leading to clumps, meshworks or double rings, and stars/cables. Since boundaries between regions are not sharp, transient structures that resemble clumps, stars, and meshworks can appear in the process of ring assembly. These results are consistent with prior experiments with mutations in actin-filament turnover regulators, myosin motor activity, and changes in the concentration of cross-linkers that alter the morphology of the condensing network. Transient star shapes appear in some simulations, and these morphologies offer an explanation for star structures observed in prior experimental images. Finally, we quantify tension along actin filaments and forces on nodes during ring assembly and show that the mechanisms describing ring assembly can also drive ring constriction once the ring is formed.     

A Highlights from MBoC Selection: Expansion and concatenation of nonmuscle myosin IIA filaments drive cellular contractile system formation during interphase and mitosis

Cell movement and cytokinesis are facilitated by contractile forces generated by the molecular motor, nonmuscle myosin II (NMII). NMII molecules form a filament (NMII-F) through interactions of their C-terminal rod domains, positioning groups of N-terminal motor domains on opposite sides. The NMII motors then bind and pull actin filaments toward the NMII-F, thus driving contraction. Inside of crawling cells, NMIIA-Fs form large macromolecular ensembles (i.e., NMIIA-F stacks), but how this occurs is unknown. Here we show NMIIA-F stacks are formed through two non–mutually exclusive mechanisms: expansion and concatenation. During expansion, NMIIA molecules within the NMIIA-F spread out concurrent with addition of new NMIIA molecules. Concatenation occurs when multiple NMIIA-Fs/NMIIA-F stacks move together and align. We found that NMIIA-F stack formation was regulated by both motor activity and the availability of surrounding actin filaments. Furthermore, our data showed expansion and concatenation also formed the contractile ring in dividing cells. Thus interphase and mitotic cells share similar mechanisms for creating large contractile units, and these are likely to underlie how other myosin II–based contractile systems are assembled.     

High Resolution Characterization of Myosin IIC Protein Tailpiece and Its Effect on Filament Assembly

The motor protein nonmuscle myosin II (NMII) must undergo dynamic oligomerization into filaments to perform its cellular functions. A small nonhelical region at the tail of the long coiled-coil region (tailpiece) is a common feature of all dynamically assembling myosin II proteins. This tailpiece is a key regulatory domain affecting NMII filament assembly properties and is subject to phosphorylation in vivo. We previously demonstrated that the positively charged region of the tailpiece binds to assembly-incompetent NMII-C fragments, inducing filament assembly. In the current study, we investigated the molecular mechanisms by which the tailpiece regulates NMII-C self-assembly. Using alanine scan, we found that specific positive and aromatic residues within the positively charged region of the tailpiece are important for inducing NMII-C filament assembly and for filament elongation. Combining peptide arrays with deletion studies allowed us to identify the tailpiece binding sites in the coiled-coil rod. Elucidation of the mechanism by which the tailpiece induces filament assembly permitted us further investigation into the role of tailpiece phosphorylation. Sedimentation and CD spectroscopy identified that phosphorylation of Thr¹⁹⁵⁷ or Thr¹⁹⁶⁰ inhibited the ability of the tailpiece to bind the coiled-coil rod and to induce NMII-C filament formation. This study provides molecular insight into the role of specific residues within the NMII-C tailpiece that are responsible for shifting the oligomeric equilibrium of NMII-C toward filament assembly and determining its morphology.     

Filament-dependent and -independent localization modes of Drosophila non-muscle myosin II

Myosin II assembles into force-generating filaments that drive cytokinesis and the organization of the cell cortex. Regulation of myosin II activity can occur through modulation of filament assembly and by targeting to appropriate cellular sites. Here we show, using salt-dependent solubility and a novel fluorescence resonance energy transfer assay, that assembly of the Drosophila non-muscle myosin II heavy chain, zipper, is mediated by a 90-residue region (1849-1940) of the coiled-coil tail domain. This filament assembly domain, transiently expressed in Drosophila S2 cells, does not localize to the interphase cortex or the cytokinetic cleavage furrow, whereas a 500-residue region (1350-1865) that overlaps the NH(2) terminus of the assembly domain localizes to the interphase cortex but not the cytokinetic cleavage furrow. Targeting to these two sites appears to utilize distinct localization mechanisms as the assembly domain is required for cleavage furrow recruitment of a truncated coiled-coil tail region but not targeting to the interphase cortex. These results delineate the requirements for zipper filament assembly and indicate that the ability to form filaments is necessary for targeting to the cleavage furrow but not to the interphase cortex.     

Coupling actin dynamics and membrane dynamics during endocytosis.

A convergence of cellular, genetic and biochemical studies supports the hypothesis that the actin cytoskeleton is coupled to endocytic processes, but the roles played by actin filaments during endocytosis are not yet clear. Recent studies have identified several proteins that may functionally link the endocytic machinery with actin filament dynamics. Three of these proteins, Abp1p, Pan1p and cortactin, are activators of actin assembly nucleated by the Arp2/3 complex, a key regulator of actin assembly in vivo. Two others, intersectin and syndapin, bind N-WASp, a potent activator of actin assembly via the Arp2/3 complex. All of these proteins also bind components of the endocytic machinery, and thus, could coordinately regulate actin assembly and trafficking events. Hip1R, an F-actin-binding protein that associates with clathrin-coated vesicles, may physically link endocytic vesicles to actin filaments. The GTPase dynamin is implicated in modulating actin filaments at specialized actin-rich structures of the cell cortex, suggesting that dynamin may regulate the organization of cortical actin filaments as well as regulate actin dynamics during endocytosis. Finally, myosin VI may generate actin-dependent forces for membrane invagination or vesicle movement during the early stages of endocytosis.     

Actin polymerization or myosin contraction: two ways to build up cortical tension for symmetry breaking

Cells use complex biochemical pathways to drive shape changes for polarization and movement. One of these pathways is the self-assembly of actin filaments and myosin motors that together produce the forces and tensions that drive cell shape changes. Whereas the role of actin and myosin motors in cell polarization is clear, the exact mechanism of how the cortex, a thin shell of actin that is underneath the plasma membrane, can drive cell shape changes is still an open question. Here, we address this issue using biomimetic systems: the actin cortex is reconstituted on liposome membranes, in an ‘outside geometry’. The actin shell is either grown from an activator of actin polymerization immobilized at the membrane by a biotin–streptavidin link, or built by simple adsorption of biotinylated actin filaments to the membrane, in the presence or absence of myosin motors. We show that tension in the actin network can be induced either by active actin polymerization on the membrane via the Arp2/3 complex or by myosin II filament pulling activity. Symmetry breaking and spontaneous polarization occur above a critical tension that opens up a crack in the actin shell. We show that this critical tension is reached by growing branched networks, nucleated by the Arp2/3 complex, in a concentration window of capping protein that limits actin filament growth and by a sufficient number of motors that pull on actin filaments. Our study provides the groundwork to understanding the physical mechanisms at work during polarization prior to cell shape modifications.     

The Positively Charged Region of the Myosin IIC Non-helical Tailpiece Promotes Filament Assembly

The motor protein, non-muscle myosin II (NMII), must undergo dynamic oligomerization into filaments to participate in cellular processes such as cell migration and cytokinesis. A small non-helical region at the tail of the long coiled-coil region (tailpiece) is a common feature of all dynamically assembling myosin II proteins. In this study, we investigated the role of the tailpiece in NMII-C self-assembly. We show that the tailpiece is natively unfolded, as seen by circular dichroism and NMR experiments, and is divided into two regions of opposite charge. The positively charged region (Tailpiece_1946–1967) starts at residue 1946 and is extended by seven amino acids at its N terminus from the traditional coiled-coil ending proline (Tailpiece_1953–1967). Pull-down and sedimentation assays showed that the positive Tailpiece_1946–1967 binds to assembly incompetent NMII-C fragments inducing filament assembly. The negative region, residues 1968–2000, is responsible for NMII paracrystal morphology as determined by chimeras in which the negative region was swapped between the NMII isoforms. Mixing the positive and negative peptides had no effect on the ability of the positive peptide to bind and induce filament assembly. This study provides molecular insight into the role of the structurally disordered tailpiece of NMII-C in shifting the oligomeric equilibrium of NMII-C toward filament assembly and determining its morphology.     

Non-muscle myosin IIB is essential for cytokinesis during male meiotic cell divisions

Cytokinesis, the final stage of cell division, bisects the cytoplasm into two daughter cells. In mitotic cells, this process depends on the activity of non-muscle myosin II (NMII), a family of actin-binding motor-proteins that participate in the formation of the cleavage furrow. The relevance of NMII for meiotic cell division, however, is poorly understood. The NMII family consists of three members, NMIIA, NMIIB, and NMIIC, containing different myosin heavy chains (MYH9, MYH10, and MYH14, respectively). We find that a single non-muscle myosin II, NMIIB, is required for meiotic cytokinesis in male but not female mice. Specifically, NMIIB-deficient spermatocytes exhibit cytokinetic failure in meiosis I, resulting in bi-nucleated secondary spermatocytes. Additionally, cytokinetic failure at meiosis II gives rise to bi-nucleated or even tetra-nucleated spermatids. These multi-nucleated spermatids fail to undergo normal differentiation, leading to male infertility. In spite of the presence of multiple non-muscle myosin II isoforms, we demonstrate that a single member, NMIIB, plays an essential and non-redundant role in cytokinesis during meiotic cell divisions of the male germline.     

Analysis of turnover dynamics of the submembranous actin cortex

The cell cortex is a thin network of actin, myosin motors, and associated proteins that underlies the plasma membrane in most eukaryotic cells. It enables cells to resist extracellular stresses, perform mechanical work, and change shape. Cortical structural and mechanical properties depend strongly on the relative turnover rates of its constituents, but quantitative data on these rates remain elusive. Using photobleaching experiments, we analyzed the dynamics of three classes of proteins within the cortex of living cells: a scaffold protein (actin), a cross-linker (α-actinin), and a motor (myosin). We found that two filament subpopulations with very different turnover rates composed the actin cortex: one with fast turnover dynamics and polymerization resulting from addition of monomers to free barbed ends, and one with slow turnover dynamics with polymerization resulting from formin-mediated filament growth. Our data suggest that filaments in the second subpopulation are on average longer than those in the first and that cofilin-mediated severing of formin-capped filaments contributes to replenishing the filament subpopulation with free barbed ends. Furthermore, α-actinin and myosin minifilaments turned over significantly faster than F-actin. Surprisingly, only one-fourth of α-actinin dimers were bound to two actin filaments. Taken together, our results provide a quantitative characterization of essential mechanisms under­lying actin cortex homeostasis.     

Dynamics of myosin,microtubules, and Kinesin-6 at the cortex during cytokinesis in Drosophila S2 cells

Signals from the mitotic spindle during anaphase specify the location of the actomyosin contractile ring during cytokinesis, but the detailed mechanism remains unresolved. Here, we have imaged the dynamics of green fluorescent protein–tagged myosin filaments, microtubules, and Kinesin-6 (which carries activators of Rho guanosine triphosphatase) at the cell cortex using total internal reflection fluorescence microscopy in flattened Drosophila S2 cells. At anaphase onset, Kinesin-6 relocalizes to microtubule plus ends that grow toward the cortex, but refines its localization over time so that it concentrates on a subset of stable microtubules and along a diffuse cortical band at the equator. The pattern of Kinesin-6 localization closely resembles where new myosin filaments appear at the cortex by de novo assembly. While accumulating at the equator, myosin filaments disappear from the poles of the cell, a process that also requires Kinesin-6 as well as possibly other signals that emanate from the elongating spindle. These results suggest models for how Kinesin-6 might define the position of cortical myosin during cytokinesis.     

Non-muscle myosin II regulates aortic stiffness through effects on specific focal adhesion proteins and the non-muscle cortical cytoskeleton

Non-muscle myosin II (NMII) plays a role in many fundamental cellular processes including cell adhesion, migration, and cytokinesis. However, its role in mammalian vascular function is not well understood. Here, we investigated the function of NMII in the biomechanical and signalling properties of mouse aorta. We found that blebbistatin, an inhibitor of NMII, decreases agonist-induced aortic stress and stiffness in a dose-dependent manner. We also specifically demonstrate that in freshly isolated, contractile, aortic smooth muscle cells, the non-muscle myosin IIA (NMIIA) isoform is associated with contractile filaments in the core of the cell as well as those in the non-muscle cell cortex. However, the non-muscle myosin IIB (NMIIB) isoform is excluded from the cell cortex and colocalizes only with contractile filaments. Furthermore, both siRNA knockdown of NMIIA and NMIIB isoforms in the differentiated A7r5 smooth muscle cell line and blebbistatin-mediated inhibition of NM myosin II suppress agonist-activated increases in phosphorylation of the focal adhesion proteins FAK Y925 and paxillin Y118. Thus, we show in the present study, for the first time that NMII regulates aortic stiffness and stress and that this regulation is mediated through the tension-dependent phosphorylation of the focal adhesion proteins FAK and paxillin.     

Sequential Assembly of Myosin II, an IQGAP-like Protein, and Filamentous Actin to a Ring Structure Involved in Budding Yeast Cytokinesis

We have identified a Saccharomyces cerevisiae protein, Cyk1p, that exhibits sequence similarity to the mammalian IQGAPs. Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle. Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother–bud junction after the initiation of anaphase. This ring contains filamentous actin and Myo1p, a myosin II homologue. In vivo observation with green fluorescent protein–tagged Myo1p showed that the ring decreases drastically in size during cell division and therefore may be contractile. These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring. The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis. The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments in the assembly of the actomyosin ring. The accumulation of actin in the cytokinetic ring was not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs.     

Functions of nonmuscle myosin II in assembly of the cellular contractile system

The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system's maturation heavily depends on nonmuscle myosin II (NMII). Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at early stages of this process. We show that inhibition of NMII by a specific inhibitor, blebbistatin, in addition to known effects, such as disassembly of stress fibers and mature focal adhesions, also causes transformation of lamellipodia into unattached ruffles, loss of immature focal complexes, loss of cytoskeleton-associated NMII filaments and peripheral accumulation of activated, but unpolymerized NMII. After blebbistatin washout, assembly of the contractile system begins with quick and coordinated recovery of lamellipodia and focal complexes that occurs before reappearance of NMII bipolar filaments. The initial formation of focal complexes and subsequent assembly of NMII filaments preferentially occurred in association with filopodial bundles and concave actin bundles formed by filopodial roots at the lamellipodial base. Over time, accumulating NMII filaments help to transform the precursor structures, focal complexes and associated thin bundles, into stress fibers and mature focal adhesions. However, semi-sarcomeric organization of stress fibers develops at much slower rate. Together, our data suggest that activation of NMII motor activity by light chain phosphorylation occurs at the cell edge and is uncoupled from NMII assembly into bipolar filaments. We propose that activated, but unpolymerized NMII initiates focal complexes, thus providing traction for lamellipodial protrusion. Subsequently, the mechanical resistance of focal complexes activates a load-dependent mechanism of NMII polymerization in association with attached bundles, leading to assembly of stress fibers and maturation of focal adhesions.     

A cytoskeletal demolition worker: myosin II acts as an actin depolymerization agent

Myosin II motors play several important roles in a variety of cellular processes, some of which involve active assembly/disassembly of cytoskeletal substructures. Myosin II motors have been shown to function in actin bundle turnover in neuronal growth cones and in the recycling of actin filaments during cytokinesis. Close examination had shown an intimate relationship between myosin II motor adenosine triphosphatase activity and actin turnover rate. However, the direct implication of myosin II in actin turnover is still not understood. Herein, we show, using high-resolution cryo-transmission electron microscopy, that myosin II motors control the turnover of actin bundles in a concentration-dependent manner in vitro. We demonstrate that disassembly of actin bundles occurs through two main stages: the first stage involves unbundling into individual filaments, and the second involves their subsequent depolymerization. These evidence suggest that, in addition to their “classical” contractile abilities, myosin II motors may be directly implicated in active actin depolymerization. We believe that myosin II motors may function similarly in vivo (e.g., in the disassembly of the contractile ring by fine tuning the local concentration/activity of myosin II motors).     

C-terminal anchoring of mid1p to membranes stabilizes cytokinetic ring position in early mitosis in fission yeast

mid1p is a key factor for the central positioning of the cytokinetic ring in Schizosaccharomyces pombe. In interphase and early mitosis, mid1p forms a medial cortical band overlying the nucleus, which may represent a landmark for cytokinetic ring assembly. It compacts before anaphase into a tight ring with other cytokinetic ring components. We show here that mid1p binds to the medial cortex by at least two independent means. First, mid1p C-terminus association with the cortex requires a putative amphipathic helix adjacent to mid1p nuclear localization sequence (NLS), which is predicted to insert directly into the lipid bilayer. This association is stabilized by the polybasic NLS. mid1p mutated within the helix and the NLS forms abnormal filaments in early mitosis that are not properly anchored to the medial cortex. Misplaced rings assemble in late mitosis, indicating that mid1p C-terminus binding to membranes stabilizes cytokinetic ring position. Second, the N terminus of mid1p has the ability to associate faintly with the medial cortex and is sufficient to form tight rings. In addition, we show that mid1p oligomerizes. We propose that membrane-bound oligomers of mid1p assemble recruitment "platforms" for cytokinetic ring components at the medial cortex and stabilize the ring position during its compaction.     

Actin filament bundling by fimbrin is important for endocytosis, cytokinesis, and polarization in fission yeast

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.     

FtsZ in Bacterial Cytokinesis: Cytoskeleton and Force Generator All in One

Summary: FtsZ, a bacterial homolog of tubulin, is well established as forming the cytoskeletal framework for the cytokinetic ring. Recent work has shown that purified FtsZ, in the absence of any other division proteins, can assemble Z rings when incorporated inside tubular liposomes. Moreover, these artificial Z rings can generate a constriction force, demonstrating that FtsZ is its own force generator. Here we review light microscope observations of how Z rings assemble in bacteria. Assembly begins with long-pitch helices that condense into the Z ring. Once formed, the Z ring can transition to short-pitch helices that are suggestive of its structure. FtsZ assembles in vitro into short protofilaments that are ∼30 subunits long. We present models for how these protofilaments might be further assembled into the Z ring. We discuss recent experiments on assembly dynamics of FtsZ in vitro, with particular attention to how two regulatory proteins, SulA and MinC, inhibit assembly. Recent efforts to develop antibacterial drugs that target FtsZ are reviewed. Finally, we discuss evidence of how FtsZ generates a constriction force: by protofilament bending into a curved conformation.     

UCS Protein Rng3p Is Essential for Myosin-II Motor Activity during Cytokinesis in Fission Yeast

UCS proteins have been proposed to operate as co-chaperones that work with Hsp90 in the de novo folding of myosin motors. The fission yeast UCS protein Rng3p is essential for actomyosin ring assembly and cytokinesis. Here we investigated the role of Rng3p in fission yeast myosin-II (Myo2p) motor activity. Myo2p isolated from an arrested rng3-65 mutant was capable of binding actin, yet lacked stability and activity based on its expression levels and inactivity in ATPase and actin filament gliding assays. Myo2p isolated from a myo2-E1 mutant (a mutant hyper-sensitive to perturbation of Rng3p function) showed similar behavior in the same assays and exhibited an altered motor conformation based on limited proteolysis experiments. We propose that Rng3p is not required for the folding of motors per se, but instead works to ensure the activity of intrinsically unstable myosin-II motors. Rng3p is specific to conventional myosin-II and the actomyosin ring, and is not required for unconventional myosin motor function at other actin structures. However, artificial destabilization of myosin-I motors at endocytic actin patches (using a myo1-E1 mutant) led to recruitment of Rng3p to patches. Thus, while Rng3p is specific to myosin-II, UCS proteins are adaptable and can respond to changes in the stability of other myosin motors.     

Calcium regulates scallop muscle by changing myosin flexibility

Muscle myosins are molecular motors that convert the chemical free energy available from ATP hydrolysis into mechanical displacement of actin filaments, bringing about muscle contraction. Myosin cross-bridges exert force on actin filaments during a cycle of attached and detached states that are coupled to each round of ATP hydrolysis. Contraction and ATPase activity of the striated adductor muscle of scallop is controlled by calcium ion binding to myosin. This mechanism of the so-called “thick filament regulation” is quite different to vertebrate striated muscle which is switched on and off via “thin filament regulation” whereby calcium ions bind to regulatory proteins associated with the actin filaments. We have used an optically based single molecule technique to measure the angular disposition adopted by the two myosin heads whilst bound to actin in the presence and absence of calcium ions. This has allowed us to directly observe the movement of individual myosin heads in aqueous solution at room temperature in real time. We address the issue of how scallop striated muscle myosin might be regulated by calcium and have interpreted our results in terms of the structures of smooth muscle myosin that also exhibit thick filament regulation. This paper is not being submitted elsewhere and the authors have no competing financial interests     

The UNC-45 chaperone mediates sarcomere assembly through myosin degradation in Caenorhabditis elegans

Myosin motors are central to diverse cellular processes in eukaryotes. Homologues of the myosin chaperone UNC-45 have been implicated in the assembly and function of myosin-containing structures in organisms from fungi to humans. In muscle, the assembly of sarcomeric myosin is regulated to produce stable, uniform thick filaments. Loss-of-function mutations in Caenorhabditis elegans UNC-45 lead to decreased muscle myosin accumulation and defective thick filament assembly, resulting in paralyzed animals. We report that transgenic worms overexpressing UNC-45 also display defects in myosin assembly, with decreased myosin content and a mild paralysis phenotype. We find that the reduced myosin accumulation is the result of degradation through the ubiquitin/proteasome system. Partial proteasome inhibition is able to restore myosin protein and worm motility to nearly wild-type levels. These findings suggest a mechanism in which UNC-45-related proteins may contribute to the degradation of myosin in conditions such as heart failure and muscle wasting.