Myosin Assembly Research Articles

Myosin Assembly Regulation by 14-3-3

The regulation of non-muscle myosin II is key to a number of processes, including cell migration, tissue morphogenesis, and cytokinesis. We have been using the social amoeba Dictyostelium discoideum as a model organism to study how myosin II is regulated in cytokinesis. One major pathway that we have discovered is the racE–14-3-3 pathway, which maintains the uniformity of myosin II at the cortex and increases myosin II turnover, as shown by FRAP analysis and in vitro studies. We have demonstrated that 14-3-3 associates directly with the myosin II assembly domain to inhibit bipolar thick filament (BTF) assembly. This effect is observed in wild-type as well as in constitutively assembled or unassembled mutant forms of myosin II, and the exact 14-3-3 binding site is being mapped by mutational analysis. Furthermore, a bioinformatics analysis has predicted a similar 14-3-3 binding site in the mammalian non-muscle myosin IIA and IIB assembly domains. We are investigating if the 14-3-3–myosin II interactions that we have observed in Dictyostelium are also present in the mammalian system. We are thus pursuing a possible conserved mechanism for phosphorylation-independent regulation of myosin assembly by 14-3-3 proteins.

Dynamics of Non-Muscle Myosin II Organization into Stress Fibers and Contractile Networks

The cellular morphology of adhered cells critically depends on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of non-muscle myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin which disrupts actomyosin stress fibers. Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared them to studies by other groups. This analysis suggested that the following processes contribute to the assembly of cortical actomyosin and stress fibers: random myosin mini-filament assembly and disassembly along the cortex; myosin mini-filament aligning and contraction; stabilization of cortical myosin upon increasing contractile tension. We developed simple numerical simulations that include those processes. The results of simulations of cells at steady state and in response to blebbistatin capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness.

‘Marker of Self’, CD47, Modulates Mechanical Forces Imposed by Macrophages during Phagocytosis

Macrophages are innate immune cells, which engulf and digest pathogens and opsonized cells. This uptake process, known as phagocytosis, is actin-driven and becomes more efficient in the presence of phospho-activated non-muscle myosin IIA (NMMIIA). CD47 is a transmembrane membrane protein which acts as a marker of self by inhibiting phagocytosis by macrophages. An IgG-opsonized target cell or particle lacking CD47 binds macrophage FcR which then activates the assembly of paxillin, F-actin, and nonmuscle myosin IIA at the phagocytic synapse. In contrast, parallel interactions with CD47 signal through SIRP to inhibit myosin assembly and contractile contributions to efficient phagocytosis. We have studied the force and kinetics of phagocytosis by human macrophages. To this aim, we studied phagocytosis of opsonized red blood cells. The force imparted during phagocytosis was calculated based on blood cells' shape deformation and elastic properties. Our analysis shows that the range of the resultant force imposed by Macrophages is up to 100pN and in 10 minutes after initial contact. Further we show that CD47 modulates the force generated by macrophages on phagocytic targets.

Anisotropy of Crumbs and aPKC Drives Myosin Cable Assembly during Tube Formation

SummaryThe formation of tubular structures from epithelial sheets is a key process of organ formation in all animals, but the cytoskeletal rearrangements that cause the cell shape changes that drive tubulogenesis are not well understood. Using live imaging and super-resolution microscopy to analyze the tubulogenesis of the Drosophila salivary glands, I find that an anisotropic plasma membrane distribution of the protein Crumbs, mediated by its large extracellular domain, determines the subcellular localization of a supracellular actomyosin cable in the cells at the placode border, with myosin II accumulating at edges where Crumbs is lowest. Laser ablation shows that the cable is under increased tension, implying an active involvement in the invagination process. Crumbs anisotropy leads to anisotropic distribution of aPKC, which in turn can negatively regulate Rok, thus preventing the formation of a cable where Crumbs and aPKC are localized.

Self-Organization of Myosin II in Reconstituted Actomyosin Bundles

Cells assemble a variety of bundled actomyosin structures in the cytoskeleton for activities such as cell-shape regulation, force production, and cytokinesis. Although these linear structures exhibit varied architecture, two common organizational themes are a punctate distribution of myosin II and distinct patterns of actin polarity. The mechanisms that cells use to assemble and maintain these organizational features are poorly understood. To study these, we reconstituted actomyosin bundles in vitro that contained only actin filaments and myosin II. Upon addition of ATP, the bundles contracted and the uniformly distributed myosin spontaneously reorganized into discrete clusters. We developed a mathematical model in which the motion of myosin II filaments is governed by the polarities of the actin filaments with which they interact. The model showed that the assembly of myosins into clusters is driven by their tendency to migrate to locations with zero net actin filament polarity. With no fitting parameters, the predicted distribution of myosin cluster separations was in close agreement with our experiments, including a −3/2 power law decay for intermediate length scales. Thus, without an organizing template or accessory proteins, a minimal bundle of actin and myosin has the inherent capacity to self-organize into a heterogeneous banded structure.

Tracking UNC-45 Chaperone-Myosin Interaction with a Titin Mechanical Reporter

Myosins are molecular motors that convert chemical energy into mechanical work. Allosterically coupling ATP-binding, hydrolysis, and binding/dissociation to actin filaments requires precise and coordinated structural changes that are achieved by the structurally complex myosin motor domain. UNC-45, a member of the UNC-45/Cro1/She4p family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly of myosin into muscle thick filaments in vivo. The molecular mechanisms by which UNC-45 interacts with myosin to promote proper folding of the myosin head domain are not known. We have devised a novel approach, to our knowledge, to analyze the interaction of UNC-45 with the myosin motor domain at the single molecule level using atomic force microscopy. By chemically coupling a titin I27 polyprotein to the motor domain of myosin, we introduced a mechanical reporter. In addition, the polyprotein provided a specific attachment point and an unambiguous mechanical fingerprint, facilitating our atomic force microscopy measurements. This approach enabled us to study UNC-45–motor domain interactions. After mechanical unfolding, the motor domain interfered with refolding of the otherwise robust I27 modules, presumably by recruiting them into a misfolded state. In the presence of UNC-45, I27 folding was restored. Our single molecule approach enables the study of UNC-45 chaperone interactions with myosin and their consequences for motor domain folding and misfolding in mechanistic detail.

ADF/Cofilin Regulates Actomyosin Assembly through Competitive Inhibition of Myosin II Binding to F-Actin

The contractile actin cortex is important for diverse fundamental cell processes, but little is known about how the assembly of F-actin and myosin II motors is regulated. We report that depletion of actin depolymerizing factor (ADF)/cofilin proteins in human cells causes increased contractile cortical actomyosin assembly. Remarkably, our data reveal that the major cellular defects resulting from ADF/cofilin depletion, including cortical F-actin accumulation, were largely due to excessive myosin II activity. We identify that ADF/cofilins from unicellular organisms to humans share a conserved activity to inhibit myosin II binding to F-actin, indicating a mechanistic rationale for our cellular results. Our study establishes an essential requirement for ADF/cofilin proteins in the control of normal cortical contractility and in processes such as mitotic karyokinesis. We propose that ADF/cofilin proteins are necessary for controlling actomyosin assembly and intracellular contractile force generation, a function of equal physiological importance to their established roles in mediating F-actin turnover.

Effects of Pathogenic Proline Mutations on Myosin Assembly

Laing distal myopathy (MPD1) is a genetically dominant myopathy characterized by early and selective weakness of the distal muscles. Mutations in the MYH7 gene encoding for the β-myosin heavy chain are the underlying genetic cause of MPD1. However, their pathogenic mechanisms are currently unknown. Here, we measure the biological effects of the R1500P and L1706P MPD1 mutations in different cellular systems. We show that, while the two mutations inhibit myosin self-assembly in non-muscle cells, they do not prevent incorporation of the mutant myosin into sarcomeres. Nevertheless, we find that the L1706P mutation affects proper antiparallel myosin association by accumulating in the bare zone of the sarcomere. Furthermore, bimolecular fluorescence complementation assay shows that the α-helix containing the R1500P mutation folds into homodimeric (mutant/mutant) and heterodimeric [mutant/wild type (WT)] myosin molecules that are competent for sarcomere incorporation. Both mutations also form aggregates consisting of cytoplasmic vacuoles surrounding paracrystalline arrays and amorphous rod-like inclusions that sequester WT myosin. Myosin aggregates were also detected in transgenic nematodes expressing the R1500P mutation. By showing that the two MPD1 mutations can have dominant effects on distinct components of the contractile apparatus, our data provide the first insights into the pathogenesis of the disease.

Analysis of the role of Ser1/Ser2/Thr9 phosphorylation on myosin II assembly and function in live cells

BackgroundPhosphorylation of non-muscle myosin II regulatory light chain (RLC) at Thr18/Ser19 is well established as a key regulatory event that controls myosin II assembly and activation, both in vitro and in living cells. RLC can also be phosphorylated at Ser1/Ser2/Thr9 by protein kinase C (PKC). Biophysical studies show that phosphorylation at these sites leads to an increase in the Km of myosin light chain kinase (MLCK) for RLC, thereby indirectly inhibiting myosin II activity. Despite unequivocal evidence that PKC phosphorylation at Ser1/Ser2/Thr9 can regulate myosin II function in vitro, there is little evidence that this mechanism regulates myosin II function in live cells.ResultsThe purpose of these studies was to investigate the role of Ser1/Ser2/Thr9 phosphorylation in live cells. To do this we utilized phospho-specific antibodies and created GFP-tagged RLC reporters with phosphomimetic aspartic acid substitutions or unphosphorylatable alanine substitutions at the putative inhibitory sites or the previously characterized activation sites. Cell lines stably expressing the RLC-GFP constructs were assayed for myosin recruitment during cell division, the ability to complete cell division, and myosin assembly levels under resting or spreading conditions. Our data shows that manipulation of the activation sites (Thr18/Ser19) significantly alters myosin II function in a number of these assays while manipulation of the putative inhibitory sites (Ser1/Ser2/Thr9) does not.ConclusionsThese studies suggest that inhibitory phosphorylation of RLC is not a substantial regulatory mechanism, although we cannot rule out its role in other cellular processes or perhaps other types of cells or tissues in vivo.

Actin cross-linking proteins cortexillin I and II are required for cAMP signaling duringDictyosteliumchemotaxis and development

Starvation induces Dictyostelium amoebae to secrete cAMP, toward which other amoebae stream, forming multicellular mounds that differentiate and develop into fruiting bodies containing spores. We find that the double deletion of cortexillin (ctx) I and II alters the actin cytoskeleton and substantially inhibits all molecular responses to extracellular cAMP. Synthesis of cAMP receptor and adenylyl cyclase A (ACA) is inhibited, and activation of ACA, RasC, and RasG, phosphorylation of extracellular signal regulated kinase 2, activation of TORC2, and stimulation of actin polymerization and myosin assembly are greatly reduced. As a consequence, cell streaming and development are completely blocked. Expression of ACA-yellow fluorescent protein in the ctxI/ctxII-null cells significantly rescues the wild-type phenotype, indicating that the primary chemotaxis and development defect is the inhibition of ACA synthesis and cAMP production. These results demonstrate the critical importance of a properly organized actin cytoskeleton for cAMP-signaling pathways, chemotaxis, and development in Dictyostelium.

Light‐Triggered Myosin Activation for Probing Dynamic Cellular Processes

Myosin II is an ATPase motor protein essential for many cellular functions including cell migration[1] and division.[2] In nonmuscle cells, myosin modulates protrusions at the leading edge and promotes retraction at the trailing edge during migration,[3] while during cytokinesis, myosin is required for contraction of the cleavage furrow.[4] For nonmuscle myosin, these varied functions are regulated by phosphorylation of the associated myosin regulatory light chain (mRLC) protein at Ser19, which activates the myosin complex to promote myosin assembly, contractility, and stress fiber formation.[5] Upon phosphorylation of the mRLC at both Thr18 and Ser19, these activities are further enhanced.[6 ] The dramatic effects of phosphorylation can also be recapitulated in vitro. Specifically, myosin and the proteolytic derivative heavy meromyosin (HMM),[7] which contains only one-third of the C-terminal myosin tail, exhibit low in vitro activities when associated with the nonphosphorylated mRLC. Phosphorylation of Ser19 amplifies actin-activated ATPase activities 10 – 1000-fold[8] and leads to myosin-mediated actin translocation.[9]

Role of B Regulatory Subunits of Protein Phosphatase Type 2A in Myosin II Assembly Control in Dictyostelium discoideum

In Dictyostelium discoideum, myosin II resides predominantly in a soluble pool as the result of phosphorylation of the myosin heavy chain (MHC), and dephosphorylation of the MHC is required for myosin II filament assembly, recruitment to the cytoskeleton, and force production. Protein phosphatase type 2A (PP2A) was identified in earlier studies in Dictyostelium as a key biochemical activity that can drive MHC dephosphorylation. We report here gene targeting and cell biological studies addressing the roles of candidate PP2A B regulatory subunits (phr2aBα and phr2aBβ) in myosin II assembly control in vivo. Dictyostelium phr2aBα- and phr2aBβ-null cells show delayed development, reduction in the assembly of myosin II in cytoskeletal ghost assays, and defects in cytokinesis when grown in suspension compared to parental cell lines. These results demonstrate that the PP2A B subunits phr2aBα and phr2aBβ contribute to myosin II assembly control in vivo, with phr2aBα having the predominant role facilitating MHC dephosphorylation to facilitate filament assembly.

Rho GTPases and their role in organizing the actin cytoskeleton

Cells receive extracellular stimuli in various ways: in the form of soluble molecules (growth factors, cytokines and hormones) that interact with cell-surface receptors; from adhesive interactions with the extracellular matrix; and from cell–cell adhesions. These stimuli act to generate changes in

A Proteomic Study of Myosin II Motor Proteins during Tumor Cell Migration

Myosin II motor proteins play important roles in cell migration. Although myosin II filament assembly plays a key role in the stabilization of focal contacts at the leading edge of migrating cells, the mechanisms and signaling pathways regulating the localized assembly of lamellipodial myosin II filaments are poorly understood. We performed a proteomic analysis of myosin heavy chain (MHC) phosphorylation sites in MDA-MB 231 breast cancer cells to identify MHC phosphorylation sites that are activated during integrin engagement and lamellar extension on fibronectin. Fibronectin-activated MHC phosphorylation was identified on novel and previously recognized consensus sites for phosphorylation by protein kinase C and casein kinase II (CK-II). S1943, a CK-II consensus site, was highly phosphorylated in response to matrix engagement, and phosphoantibody staining revealed phosphorylation on myosin II assembled into leading-edge lamellae. Surprisingly, neither pharmacological reduction nor small inhibitory RNA reduction in CK-II activity reduced this stimulated S1943 phosphorylation. Our data demonstrate that S1943 phosphorylation is upregulated during lamellar protrusion, and that CK-II does not appear to be the kinase responsible for this matrix-induced phosphorylation event.

Myosin Cortical Assembly and Spindle Oscillations During Hela Cell Division

The cortical actomyosin layer below the membrane of dividing cells and the microtubules of the mitotic spindle are coupled through internal feedback mechanisms. Identifying the factors that control molecular motor distribution is essential for understanding cell division. The equatorial contractile ring forms by myosin assembly in the middle and disassembly in the flanking regions. To better understand the mechanical and biochemical mechanisms of contractile ring assembly, we imaged HeLa cells expressing MRLC-GFP by confocal microscopy at high spatial and temporal resolution. MRLC-GFP also marks spindle poles; this allowed us to study the correlation between mitotic spindle movements and cortical myosin distribution. We confirmed that the onset of myosin assembly in the contractile ring occurs in anaphase. We observed that the mitotic spindle exhibited oscillations upon entry into anaphase, similar to oscillations observed during the asymmetric division of C. elegans embryos by other groups. We used particle tracking software to follow spindle position and JFilament to track cortical myosin intensity distribution during these oscillations. We further exerted controlled and localized forces while following the cellular response using fluorescence imaging. Anti-integrin coated micro-beads were attached to HeLa cells expressing MRLC-GFP. We applied external forces to a bead by an optical tweezers and imaged the dynamics of myosin and mitotic spindle during cell division. Experimental results showed that the spindle appeared to move to an asymmetric post perturbation position. This observation is consistent with the expected coupling between force-dependent cortical flow and spindle position and will be useful in more detailed studies of the response of cytoskeleton to external or internal forces.

Determinants of Myosin II Cortical Localization during Cytokinesis

Myosin II is an essential component of the contractile ring that divides the cell during cytokinesis. Previous work showed that regulatory light chain (RLC) phosphorylation is required for localization of myosin at the cellular equator. However, the molecular mechanisms that concentrate myosin at the site of furrow formation remain unclear. By analyzing the spatiotemporal dynamics of mutant myosin subunits in Drosophila S2 cells, we show that myosin accumulates at the equator through stabilization of interactions between the cortex and myosin filaments and that the motor domain is dispensable for localization. Filament stabilization is tightly controlled by RLC phosphorylation. However, we show that regulatory mechanisms other than RLC phosphorylation contribute to myosin accumulation at three different stages: (1) turnover of thick filaments throughout the cell cycle, (2) myosin heavy chain-based control of myosin assembly at the metaphase-anaphase transition, and (3) redistribution and/or activation of myosin binding sites at the equator during anaphase. Surprisingly, the third event can occur to a degree in a Rho-independent fashion, gathering preassembled filaments to the equatorial zone via cortical flow. We conclude that multiple regulatory pathways cooperate to control myosin localization during mitosis and cytokinesis to ensure that this essential biological process is as robust as possible.

UNC45A Confers Resistance to Histone Deacetylase Inhibitors and Retinoic Acid

To identify potential biomarkers of therapy response, we have previously done a large-scale gain-of-function genetic screen to identify genes whose expression confers resistance to histone deacetylase inhibitors (HDACI). This genetic screen identified two genes with a role in retinoic acid signaling, suggesting that HDACIs target retinoic acid signaling as part of their anticancer effect. We study here a third gene identified in this genetic screen, UNC45A, and assess its role in retinoic acid signaling and responses to HDACIs using cell-based proliferation and differentiation assays and transcriptional reporter gene assays. The vertebrate Unc45 genes are known for their roles in muscle development and the assembly and cochaperoning of the muscle motor protein myosin. Here, we report that human UNC45A (GCUNC45) can render transformed cells resistant to treatment with HDACIs. We show that UNC45A also inhibits signaling through the retinoic acid receptor alpha. Expression of UNC45A inhibits retinoic acid-induced proliferation arrest and differentiation of human neuroblastoma cells and inhibits the induction of endogenous retinoic acid receptor target genes. These data establish an unexpected role for UNC45A in causing resistance to both HDACI drugs and retinoic acid. Moreover, our data lend further support to the notion that HDACIs exert their anticancer effect, at least in part, through an effect on retinoic acid signaling.

Myosin II regulation duringC. elegansembryonic elongation:LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles

Myosin II plays a central role in epithelial morphogenesis; however, its role has mainly been examined in processes involving a single cell type. Here we analyze the structure, spatial requirement and regulation of myosin II during C. elegans embryonic elongation, a process that involves distinct epidermal cells and muscles. We developed novel GFP probes to visualize the dynamics of actomyosin remodeling, and found that the assembly of myosin II filaments, but not actin microfilaments, depends on the myosin regulatory light chain (MLC-4) and essential light chain (MLC-5, which we identified herein). To determine how myosin II regulates embryonic elongation, we rescued mlc-4 mutants with various constructs and found that MLC-4 is essential in a subset of epidermal cells. We show that phosphorylation of two evolutionary conserved MLC-4 serine and threonine residues is important for myosin II activity and organization. Finally, in an RNAi screen for potential myosin regulatory light chain kinases, we found that the ROCK, PAK and MRCK homologs act redundantly. The combined loss of ROCK and PAK, or ROCK and MRCK, completely prevented embryonic elongation, but a constitutively active form of MLC-4 could only rescue a lack of MRCK. This result, together with systematic genetic epistasis tests with a myosin phosphatase mutation, suggests that ROCK and MRCK regulate MLC-4 and the myosin phosphatase. Moreover, we suggest that ROCK and PAK regulate at least one other target essential for elongation, in addition to MLC-4.

Elastic Matrices that mimic normal heart are best for beating Cardiomyocytes - Beating stops on mechanical mimics of Scars

Fibrotic rigidification following a myocardial infarct is known to impair cardiac output, and it is also known that cardiomyocytes on rigid culture substrates show a progressive loss of rhythmic beating. Here, isolated embryonic cardiomyocytes cultured on a series of flexible substrates show that matrices which mimic the elasticity of the developing myocardial microenvironment are optimal for transmitting contractile work to the matrix and for promoting actomyosin striation and 1 Hz beating. On hard matrices that mechanically mimic a post-infarct fibrotic scar, cells over-strain themselves, lack striated myofibrils and stop beating; on very soft matrices, cells preserve contractile beating for days in culture but do very little work. Optimal matrix leads to a strain match between cell and matrix and suggests dynamic differences in intracellular protein structures. A “Cysteine Shotgun” method of labeling the in situ proteome reveals differences in assembly or conformation of several abundant cytoskeletal proteins, including vimentin, filamin, and myosin. Combined with recent results that show stem cell differentiation is also highly sensitive to matrix elasticity, the results here highlight the need for greater attention to fibrosis and mechanical microenvironments in cell therapy.

Multiple Regulatory Steps Control Mammalian Nonmuscle Myosin II Assembly in Live Cells

To better understand the mechanism controlling nonmuscle myosin II (NM-II) assembly in mammalian cells, mutant NM-IIA constructs were created to allow tests in live cells of two widely studied models for filament assembly control. A GFP-NM-IIA construct lacking the RLC binding domain (DeltaIQ2) destabilizes the 10S sequestered monomer state and results in a severe defect in recycling monomers during spreading, and from the posterior to the leading edge during polarized migration. A GFP-NM-IIA construct lacking the nonhelical tailpiece (Deltatailpiece) is competent for leading edge assembly, but overassembles, suggesting defects in disassembly from lamellae subsequent to initial recruitment. The Deltatailpiece phenotype was recapitulated by a GFP-NM-IIA construct carrying a mutation in a mapped tailpiece phosphorylation site (S1943A), validating the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6S/10S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with R1933-stop alleles of patients afflicted with human MYH9-related disorder.