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The elongation of Caenorhabditis elegans embryos allows examination of mechanical interactions between adjacent tissues. Muscle contractions during late elongation induce the remodeling of epidermal circumferential actin filaments through mechanotransduction. Force inputs from the muscles deform circumferential epidermal actin filament, which causes them to be severed, eventually reformed, and shortened. This squeezing force drives embryonic elongation. We investigated the possible role of the nonmuscle myosins NMY-1 and NMY-2 in this process using nmy-1 and nmy-2 thermosensitive alleles. Our findings show these myosins act redundantly in late elongation, since double nmy-2(ts); nmy-1(ts) mutants immediately stop elongation when raised to 25°C. Their inactivation does not reduce muscle activity, as measured from epidermis deformation, suggesting that they are directly involved in the multistep process of epidermal remodeling. Furthermore, NMY-1 and NMY-2 inactivation is reversible when embryos are kept at the nonpermissive temperature for a few hours. However, after longer exposure to 25°C double mutant embryos fail to resume elongation, presumably because NMY-1 was seen to form protein aggregates. We propose that the two C. elegans nonmuscle myosin II act during actin remodeling either to bring severed ends or hold them. [ABSTRACT FROM AUTHOR]

PURPOSE: Nonmuscle myosin II complexes are master regulators of actin dynamics that play essential roles during embryogenesis with vertebrates possessing 3 nonmuscle myosin II heavy chain genes, MYH9, MYH10, and MYH14. As opposed to MYH9 and MYH14, no recognizable disorder has been associated with MYH10. We sought to define the clinical characteristics and molecular mechanism of a novel autosomal dominant disorder related to MYH10. METHODS: An international collaboration identified the patient cohort. CAS9-mediated knockout cell models were used to explore the mechanism of disease pathogenesis. RESULTS: We identified a cohort of 16 individuals with heterozygous MYH10 variants presenting with a broad spectrum of neurodevelopmental disorders and variable congenital anomalies that affect most organ systems and were recapitulated in animal models of altered MYH10 activity. Variants were typically de novo missense changes with clustering observed in the motor domain. MYH10 knockout cells showed defects in primary ciliogenesis and reduced ciliary length with impaired Hedgehog signaling. MYH10 variant overexpression produced a dominant-negative effect on ciliary length. CONCLUSION: These data presented a novel genetic cause of isolated and syndromic neurodevelopmental disorders related to heterozygous variants in the MYH10 gene with implications for disrupted primary cilia length control and altered Hedgehog signaling in disease pathogenesis.

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state results in the loss of regenerative potential of the mammalian heart shortly after birth. Nonmuscle myosin IIB (NM IIB)-mediated actomyosin contractility regulates cardiomyocyte cytokinesis in the embryonic heart, and NM IIB levels decline after birth, suggesting a role for cellular tension in the regulation of cardiomyocyte cell cycle activity in the postnatal heart. To investigate the role of actomyosin contractility in cardiomyocyte cell cycle arrest, we conditionally activated ROCK2 kinase domain (ROCK2:ER) in the murine postnatal heart. Here, we show that α5/β1 integrin and fibronectin matrix increase in response to actomyosinmediated tension. Moreover, activation of ROCK2:ER promotes nuclear translocation of Yap, a mechanosensitive transcriptional co-activator, and enhances cardiomyocyte proliferation. Finally, we show that reduction of myocardial α5 integrin rescues the myocardial proliferation phenotype in ROCK2:ER hearts. These data demonstrate that cardiomyocytes respond to increased intracellular tension by altering their intercellular contacts in favor of cell-matrix interactions, leading to Yap nuclear translocation, thus uncovering a function for nonmuscle myosin contractility in promoting cardiomyocyte proliferation in the postnatal heart. [ABSTRACT FROM AUTHOR]

Background: There is no clear information on the regulation of liver blood flow by the autonomic nervous system. We conducted this study to investigate whether quiescent hepatic stellate cells (qHSCs) regulate liver blood flow in response to the sympathetic neurotransmitter norepinephrine (NE)., Methods: qHSCs isolated from mice were cultured in Dulbecco's modified Eagle medium without fetal bovine serum for 1 day on collagen gel. NE-induced qHSC contraction was evaluated using the quantitative single-cell contraction measurement method that we had developed previously. For the measurement of liver perfusion pressure in situ, a buffer solution was perfused from the portal vein in mice., Results: NE-induced a reversible contraction of qHSCs. This contraction was suppressed by the nonmuscle myosin II inhibitor blebbistatin, the myosin light chain kinase inhibitor ML-9, the Rho kinase inhibitor H-1152, the calmodulin inhibitor W-7, the store-operated calcium channel inhibitor YM-58483, and the IP 3 receptor inhibitor xestospongin C. In contrast, the transient receptor potential C channel inhibitor SKF96365 did not affect the NE-induced contraction., Conclusion: These results suggest that qHSCs contract in response to NE., New & Noteworthy: The present study provides direct evidence for the first time that norepinephrine (NE) induces a reversible contraction of isolated single quiescent hepatic stellate cells (qHSCs) and further suggests that the NE-mediated qHSC contraction participates in the regulation of liver blood flow in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cingulin (CGN) tethers nonmuscle myosin 2B (NM2B; heavy chain encoded by MYH10) to tight junctions (TJs) to modulate junctional and apical cortex mechanics. Here, we studied the role of the CGN-nonmuscle myosin 2 (NM2) interaction in epithelial morphogenesis and nanoscale organization of CGN by expressing wild-type and mutant CGN constructs in CGN-knockout Madin-Darby canine kidney (MDCK) epithelial cells. We show that the NM2-binding region of CGN is required to promote normal cyst morphogenesis of MDCK cells grown in three dimensions and to maintain the C-terminus of CGN in a distal position with respect to the ZO-2 (or TJP2)-containing TJ submembrane region, whereas the N-terminus of CGN is localized more proximal to the TJ membrane. We also show that the CGN mutant protein that causes deafness in human and mouse models is localized at TJs but does not bind to NM2B, resulting in decreased TJ membrane tortuosity. These results indicate that the interaction between CGN and NM2B regulates epithelial tissue morphogenesis and nanoscale organization of CGN and suggest that CGN regulates the auditory function of hair cells by organizing the actomyosin cytoskeleton to modulate the mechanics of the apical and junctional cortex., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The auditory papilla of geckos contains two zones of sensory hair cells, one covered by a continuous tectorial membrane affixed to the hair bundles and the other by discrete tectorial sallets each surmounting a transverse row of bundles. Gecko papillae are thought to encode sound frequencies up to 5 kHz, but little is known about the hair cell electrical properties or their role in frequency tuning. We recorded from hair cells in the isolated auditory papilla of the crested gecko, Correlophus ciliatus, and found that in both the nonsalletal region and part of the salletal region, the cells displayed electrical tuning organized tonotopically. Along the salletal zone, occupying the apical two-thirds of the papilla, hair bundle length decreased threefold and stereociliary complement increased 1.5-fold. The two morphological variations predict a 13-fold gradient in bundle stiffness, confirmed experimentally, which, when coupled with salletal mass, could provide passive mechanical resonances from 1 to 6 kHz. Sinusoidal electrical currents injected across the papilla evoked hair bundle oscillations at twice the stimulation frequency, consistent with fast electromechanical responses from hair bundles of two opposing orientations across the papilla. Evoked bundle oscillations were diminished by reducing Ca2+ influx, but not by blocking the mechanotransduction channels or inhibiting prestin action, thereby distinguishing them from known electromechanical mechanisms in hair cells. We suggest the phenomenon may be a manifestation of an electromechanical amplification that augments the passive mechanical tuning of the sallets over the high-frequency region. [ABSTRACT FROM AUTHOR]

During asymmetrical division of the endomesodermal precursor cell EMS, a cortical flow arises, and the daughter cells, endodermal precursor E and mesodermal precursor MS, have an enduring difference in the levels of F-actin and non-muscular myosin. Ablation of the cell cortex suggests that these observed differences lead to differences in cortical tension. The higher F-actin and myosin levels in the MS daughter coincide with cell shape changes and relatively lower tension, indicating a soft, actively moving cell, whereas the lower signal in the E daughter cell is associated with higher tension and a more rigid, spherical shape. The cortical flow is under control of the Wnt signaling pathway. Perturbing the pathway removes the asymmetry arising during EMS division and induces subtle defects in the cellular movements at the eight-cell stage. The perturbed cellular movement appears to be associated with an asymmetric distribution of E-cadherin across the EMS cytokinesis groove. ABpl forms a lamellipodium which preferentially adheres to MS by the E-cadherin HMR-1. The HMR-1 asymmetry across the groove is complete just at the moment cytokinesis completes. Perturbing Wnt signaling equalizes the HMR-1 distribution across the lamellipodium. We conclude that Wnt signaling induces a cortical flow during EMS division, which results in a transition in the cortical contractile network for the daughter cells, as well as an asymmetric distribution of E-cadherin.

Embryonic morphogenesis relies on the intrinsic ability of cells, often through remodeling the cytoskeleton, to shape epithelial tissues during development. Epithelial invagination is an example of morphogenesis that depends on this remodeling but the cellular mechanisms driving arrangement of cytoskeletal elements needed for tissue deformation remain incompletely characterized. To elucidate these mechanisms, live fluorescent microscopy and immunohistochemistry on fixed specimens were performed on chick and mouse lens placodes. This analysis revealed the formation of peripherally localized, circumferentially orientated and aligned junctions enriched in F-actin and MyoIIB. Once formed, the aligned junctions contract in a Rho-kinase and non-muscle myosin dependent manner. Further molecular characterization of these junctions revealed a Rho-kinase dependent accumulation of Arhgef11, a RhoA-specific guanine exchange factor known to regulate the formation of actomyosin cables and junctional contraction. In contrast, the localization of the Par-complex protein Par3, was reduced in these circumferentially orientated junctions. In an effort to determine if Par3 plays a negative role in MyoIIB accumulation, Par3-deficient mouse embryos were analyzed which not only revealed an increase in bicellular junctional accumulation of MyoIIB, but also a reduction of Arhgef11. Together, these results highlight the importance of the formation of the multicellular actomyosin cables that appear essential to the initiation of epithelial invagination and implicate the potential role of Arhgef11 and Par3 in their contraction and formation. • Chick and mouse lens placode invagination involves the contraction of encircling multicellular actomyosin cables. • Contraction of these cables are Rho-kinase and non-muscle myosin dependent and are enriched with Arhgef11. • Pseudo-sarcomeric repeat like-structures effectively mark the contraction state of bicellular junctions in the lens placode. • Reduction of Par3 facilitates bicellular myosin localization and actomyosin cable formation. [ABSTRACT FROM AUTHOR]

Currently, the major measures of preventing and controlling microbial infection are vaccinations and drugs. However, the appearance of drug resistance microbial mounts is main obstacle in current anti-microbial therapy. One of the most ubiquitous actin-binding proteins, non-muscle myosin II (NM II) plays a crucial role in a wide range of cellular physiological activities in mammals, including cell adhesion, migration, and division. Nowadays, growing evidence indicates that aberrant expression or activity of NM II can be detected in many diseases caused by microbes, including viruses and bacteria. Furthermore, an important role for NM II in the infection of some microbes is verified. Importantly, modulating the expression of NM II with small hairpin RNA (shRNA) or the activity of it by inhibitors can affect microbial-triggered phenotypes. Therefore, NM II holds the promise to be a potential target for inhibiting the infection of microbes and even treating microbial-triggered discords. In spite of these, a comprehensive view on the functions of NM II in microbial infection and the regulators which have an impact on the roles of NM II in this context, is still lacking. In this review, we summarize our current knowledge on the roles of NM II in microbial-triggered discords and provide broad insights into its regulators. In addition, the existing challenge of investigating the multiple roles of NM II in microbial infection and developing NM II inhibitors for treating these microbial-triggered discords, are also discussed. [ABSTRACT FROM AUTHOR]

Various forms of cell motility critically depend on pushing, pulling, and resistance forces generated by the actin cytoskeleton. Whereas pushing forces largely depend on actin polymerization, pulling forces responsible for cell contractility and resistance forces maintaining the cell shape require interaction of actin filaments with the multivalent molecular motor myosin II. In contrast to muscle-specific myosin II paralogs, nonmuscle myosin II (NMII) functions in virtually all mammalian cells, where it executes numerous mechanical tasks. NMII is expressed in mammalian cells as a tissue-specific combination of three paralogs, NMIIA, NMIIB, and NMIIC. Despite overall similarity, these paralogs differ in their molecular properties, which allow them to play both unique and common roles. Importantly, the three paralogs can also cooperate with each other by mixing and matching their unique capabilities. Through specialization and cooperation, NMII paralogs together execute a great variety of tasks in many different cell types. Here, we focus on mammalian NMII paralogs and review novel aspects of their kinetics, regulation, and functions in cells from the perspective of how distinct features of the three myosin II paralogs adapt them to perform specialized and joint tasks in the cells. [ABSTRACT FROM AUTHOR]

Abstract Nonmuscle myosin II is an actin-based motor that executes numerous mechanical tasks in cells including spatiotemporal organization of the actin cytoskeleton, adhesion, migration, cytokinesis, tissue remodeling, and membrane trafficking. Nonmuscle myosin II is ubiquitously expressed in mammalian cells as a tissue-specific combination of three paralogs. Recent studies reveal novel specific aspects of their kinetics, intracellular regulation and functions. On the other hand, the three paralogs also can copolymerize and cooperate in cells. Here we review the recent advances from the prospective of how distinct features of the three myosin II paralogs adapt them to perform specialized and joint tasks in the cell. Highlights • Nonmuscle myosin II executes numerous mechanical tasks in cells including cytoskeleton organization and cell migration. • Three mammalian nonmuscle myosin II paralogs have distinct kinetic and dynamic properties in vitro and in vivo. • Mammalian nonmuscle myosin II paralogs mix and match their abilities to perform both individual and joint tasks in the cell. [ABSTRACT FROM AUTHOR]

Nonmuscle myosin-IIA (NMHC-IIA) heavy chain phosphorylation has gained recognition as an important feature of myosin-II regulation. In previous work, we showed that phosphorylation on S1943 promotes myosin-IIA filament disassembly in vitro and enhances EGF-stimulated lamellipod extension of breast tumor cells. However, the contribution of NMHC-IIA S1943 phosphorylation to the modulation of invasive cellular behavior and metastasis has not been examined. Stable expression of phosphomimetic (S1943E) or non-phosphorylatable (S1943A) NMHC-IIA in breast cancer cells revealed that S1943 phosphorylation enhances invadopodia function, and is critical for matrix degradation in vitro and experimental metastasis in vivo . These studies demonstrate a novel link between NMHC-IIA S1943 phosphorylation, the regulation of extracellular matrix degradation and tumor cell invasion and metastasis. [ABSTRACT FROM AUTHOR]

Sodium channels play pivotal roles in health and diseases due to their ability to control cellular excitability. The pore-forming α-subunits (sodium channel alpha subunits) of the voltage-sensitive channels (i.e., Nav1.1-1.9) and the nonvoltage-dependent channel (i.e., Nax) share a common structural motif and selectivity for sodium ions. We hypothesized that the actin-based nonmuscle myosin II motor proteins, nonmuscle myosin heavy chain-IIA/myh9, and nonmuscle myosin heavy chain-IIB/myh10 might interact with sodium channel alpha subunits to play an important role in their transport, trafficking, and/or function. Immunochemical and electrophysiological assays were conducted using rodent nervous (brain and dorsal root ganglia) tissues and ND7/23 cells coexpressing Nav subunits and recombinant myosins. Immunoprecipitation of myh9 and myh10 from rodent brain tissues led to the coimmunoprecipitation of Nax, Nav 1.2, and Nav1.3 subunits, but not Nav1.1 and Nav1.6 subunits, expressed there. Similarly, immunoprecipitation of myh9 and myh10 from rodent dorsal root ganglia tissues led to the coimmunoprecipitation of Nav1.7 and Nav1.8 subunits, but not Nav1.9 subunits, expressed there. The functional implication of one of these interactions was assessed by coexpressing myh10 along with Nav1.8 subunits in ND7/23 cells. Myh10 overexpression led to three-fold increase (P<0.01) in the current density of Nav1.8 channels expressed in ND7/23 cells. Myh10 coexpression also hyperpolarized voltage-dependent activation and steady-state fast inactivation of Nav1.8 channels. In addition, coexpression of myh10 reduced (P<0.01) the offset of fast inactivation and the amplitude of the ramp currents of Nav1.8 channels. These results indicate that nonmuscle myosin heavy chain-IIs interact with sodium channel alpha subunits subunits in an isoform-dependent manner and influence their functional properties. [ABSTRACT FROM AUTHOR]

Nonmuscle myosin II (NMII) plays an essential role in directional cell migration. In this study, we investigated the roles of NMII isoforms (NMIIA and NMIIB) in the migration of human embryonic lung fibroblasts, which exhibit directionally persistent migration in an intrinsic manner. NMIIA-knockdown (KD) cells migrated unsteadily, but their direction of migration was approximately maintained. By contrast, NMIIB-KD cells occasionally reversed their direction of migration. Lamellipodium-like protrusions formed in the posterior region of NMIIB-KD cells prior to reversal of the migration direction. Moreover, NMIIB KD led to elongation of the posterior region in migrating cells, probably due to the lack of load-bearing stress fibers in this area. These results suggest that NMIIA plays a role in steering migration by maintaining stable protrusions in the anterior region, whereas NMIIB plays a role in maintenance of front-rear polarity by preventing aberrant protrusion formation in the posterior region. These distinct functions of NMIIA and NMIIB might promote intrinsic and directed migration of normal human fibroblasts. [ABSTRACT FROM AUTHOR]

Nonmusclemyosin 2 (NM-2) powers cell motility and tissue morphogenesis by assembling into bipolar filaments that interact with actin. Although the enzymatic properties of purified NM-2 motor fragments have been determined, the emergent properties of filament ensembles are unknown. Using single myosin filament in vitro motility assays, we report fundamental differences in filaments formed of different NM-2 motors. Filaments consisting of NM2-B moved processively along actin, while under identical conditions, NM2-A filaments did not. By more closely mimicking the physiological milieu, either by increasing solution viscosity or by copolymerization with NM2-B, NM2-A containing filaments moved processively. Our data demonstrate that both the kinetic and mechanical properties of these two myosins, in addition to the stochiometry of NM-2 subunits, can tune filament mechanical output. We propose altering NM- 2 filament composition is a general cellular strategy for tailoring force production of filaments to specific functions, such as maintaining tension or remodeling actin. [ABSTRACT FROM AUTHOR]

Despite a generic, highly conserved motor domain, ATP turnover kinetics and their activation by F-actin vary greatly between myosin-2 isoforms. Here, we present a 2.25 Å pre- powerstroke state (ADP-VO4) crystal structure of the human nonmuscle myosin-2C motor domain, one of the slowest myosins characterized. In combination with integrated mutagenesis, ensemble- solution kinetics, and molecular dynamics simulation approaches, the structure reveals an allosteric communication pathway that connects the distal end of the motor domain with the active site. Disruption of this pathway by mutation of hub residue R788, which forms the center of a cluster of interactions connecting the converter, the SH1-SH2 helix, the relay helix, and the lever, abolishes nonmuscle myosin-2 specific kinetic signatures. Our results provide insights into structural changes in the myosin motor domain that are triggered upon F-actin binding and contribute critically to the mechanochemical behavior of stress fibers, actin arcs, and cortical actin-based structures. [ABSTRACT FROM AUTHOR]

Recent progress in understanding the essential roles of mechanical forces in regulating various cellular processes expands the field of biology to one where interdisciplinary approaches with engineering techniques become indispensable. Contractile forces or contractility-inherently present in proliferative cells due to the activity of ubiquitous nonmuscle myosin II ( NMII)-are one of such mechano-regulators, but because NMII works downstream of diverse signaling pathways, it is often difficult to predict how the inherent cellular forces change upon perturbations to particular molecules. Here, we determine whether the contractility of individual cells is upregulated or downregulated based on an assay analyzing specific deformations of silicone gel substrates. We focus on the effect of mutations in the human MYH9 gene that encodes NMIIA, which have been implicated in the pathogenesis of various diseases including nephritis. Our assay equipped with a high-throughput data analysis capability reveals that a point mutation of E1841K but not I1816V significantly reduces the magnitude of the endogenous forces of human embryonic kidney ( HEK293) cells. Given the increasingly recognized roles of the endogenous forces as a critical mechano-regulator as well as that no apparent morphological changes were induced to cells even by introducing the mutations, our findings suggest a possibility that the detected reduction in the force magnitude at the individual cellular level may underlie the pathogenesis of the kidney disease. [ABSTRACT FROM AUTHOR]

Memories associated with drug use can trigger strong motivation for the drug, which increases relapse vulnerability in substance use disorder (SUD). Currently there are no treatments for relapse to abuse of psychostimulants, such as methamphetamine (METH). We previously reported that storage of memories associated with METH, but not those for fear or food reward, and the concomitant spine density increase are disrupted in a retrieval-independent manner by depolymerizing actin in the basolateral amygdala complex (BLC) of adult male rats and mice. Similar results are achieved in males through intra-BLC or systemic inhibition of nonmuscle myosin II (NMII), a molecular motor that directly drives actin polymerization. Given the substantial differences in physiology between genders, we sought to determine if this immediate and selective disruption of METH-associated memory extends to adult females. A single intra-BLC infusion of the NMII inhibitor Blebbistatin (Blebb) produced a long-lasting disruption of context-induced drug seeking for at least 30 days in female rats that mirrored our prior results in males. Furthermore, a single systemic injection of Blebb prior to testing disrupted METH-associated memory and the concomitant increase in BLC spine density in females. Importantly, as in males, the same manipulation had no effect on an auditory fear memory or associated BLC spine density. In addition, we established that the NMII-based disruption of METH-associated memory extends to both male and female adolescents. These findings provide further support that small molecular inhibitors of NMII have strong therapeutic potential for the prevention of relapse to METH abuse triggered by associative memories. [ABSTRACT FROM AUTHOR]

The neuronal spine is a small, actin-rich dendritic or somatic protrusion that serves as the postsynaptic compartment of the excitatory synapse. The morphology of the spine reflects the activity of the synapse and is regulated by the dynamics of the actin cytoskeleton inside, which is controlled by actin binding proteins such as non-muscle myosin. Previously, we demonstrated that the subcellular localization and function of myosin IIb are regulated by its binding partner, filamin-A interacting protein (FILIP). However, how the subcellular distribution of myosin IIb is controlled by FILIP is not yet known. The objective of this study was to identify potential binding partners of FILIP that contribute to its regulation of non-muscle myosin IIb. Pull-down assays detected a 70-kDa protein that was identified by mass spectrometry to be the chaperone protein Hsc70. The binding of Hsc70 to FILIP was controlled by the adenosine triphosphatase (ATPase) activity of Hsc70. Further, FILIP bound to Hsc70 via a domain that was not required for binding non-muscle myosin IIb. Inhibition of ATPase activity of Hsc70 impaired the effect of FILIP on the subcellular distribution of non-muscle myosin IIb. Further, in primary cultured neurons, an inhibitor of Hsc70 impeded the morphological change in spines induced by FILIP. Collectively, these results demonstrate that Hsc70 interacts with FILIP to mediate its effects on non-muscle myosin IIb and to regulate spine morphology. [ABSTRACT FROM AUTHOR]

Glomerular epithelial cells, podocytes, are insulin responsive and can develop insulin resistance. Here, we demonstrate that the small GTPase septin 7 forms a complex with nonmuscle myosin heavy chain IIA (NMHC-IIA; encoded by MYH9 ), a component of the nonmuscle myosin IIA (NM-IIA) hexameric complex. We observed that knockdown of NMHC-IIA decreases insulin-stimulated glucose uptake into podocytes. Both septin 7 and NM-IIA associate with SNAP23, a SNARE protein involved in GLUT4 storage vesicle (GSV) docking and fusion with the plasma membrane. We observed that insulin decreases the level of septin 7 and increases the activity of NM-IIA in the SNAP23 complex, as visualized by increased phosphorylation of myosin regulatory light chain. Also knockdown of septin 7 increases the activity of NM-IIA in the complex. The activity of NM-IIA is increased in diabetic rat glomeruli and cultured human podocytes exposed to macroalbuminuric sera from patients with type 1 diabetes. Collectively, the data suggest that the activity of NM-IIA in the SNAP23 complex plays a key role in insulin-stimulated glucose uptake into podocytes. Furthermore, we observed that septin 7 reduces the activity of NM-IIA in the SNAP23 complex and thereby hinders GSV docking and fusion with the plasma membrane. [ABSTRACT FROM AUTHOR]

Mammalian cells express three Class II nonmuscle myosins (NM): NM2A, NM2B and NM2C. The three NM2s have well-established essential roles in cell motility, adhesion and cytokinesis, and less well defined roles in vesicle transport and other processes that would require association of NM2s with cell membranes. Previous evidence for the mechanism of NM2-membrane association includes direct interaction of NM2s with membrane lipids and indirect interaction by association of NM2s with membrane-bound F-actin or peripheral membrane proteins. Direct binding of NM2s to phosphatidylserine liposomes, but not to phosphatidylcholine liposomes, has been reported but the molecular basis of the interaction between NM2s and acidic phospholipids has not been previously investigated. We now show that filamentous, full-length NM2A, NM2B and NM2C, and monomeric, non- filamentous heavy meromyosin (HMM) bind to liposomes containing one or more acidic phospholipids (phosphatidylserine, phosphatidylinositol-4,5-diphosphate and phosphatidylinositol-3,4,5-triphosphate) but do not bind to 100% phosphatidylcholine-liposomes. Binding of NM2s to acidic liposomes occurs predominantly through interaction of the liposomes with the regulatory light chain (RLC) binding site in the myosin heavy chain with concomitant dissociation of the RLC. Phosphorylation of myosin-bound RLC by myosin light chain kinase substantially inhibits binding to liposomes of both filamentous NM2 and non-filamentous HMM; addition of excess unbound RLC, but not excess unbound essential light chain, competes with liposome binding. Consistent with the in vitro data, we show that endogenous and expressed NM2A associates with the plasma membrane of HeLa cells and fibrosarcoma cells independently of F-actin. [ABSTRACT FROM AUTHOR]

Expression of adaptor protein, phosphotyrosine interaction, pleckstrin homology domain, and leucine zipper containing 1 (APPL1) promoted glucose transporter 4 (GLUT4) translocation and glucose uptake in adipose and muscle tissues in response to stimulation with insulin, adiponectin, or exercise. In response to mechanical stretch, knockdown of APPL1 in C2C12 myotubes suppressed glucose uptake. APPL1-induced increased glucose uptake was mediated by protein kinase C (PKC) ζ but not AKT, AMPK, or calmodulin-dependent protein kinase. In myotubes overexpressing APPL1, PKCζ was phosphorylated and translocated to the plasma membrane (PM) in response to mechanical stretch. Phosphorylated PKCζ co-immunoprecipitated with protein phosphatase 2A (PP2A) under basal conditions, but dissociated upon myotube stretching. Moreover, stretch-induced phosphorylated PKCζ co-immunoprecipitated with non-muscle myosin IIa. Blebbistatin, an inhibitor of myosin II ATPase activity, suppressed APPL1-mediated stretch-induced glucose uptake and PKCζ translocation. Taken together these data demonstrate that in response to mechanical stretch, APPL1 enhances glucose uptake by modulating the activation and localization of PKCζ, as well as its functional interaction with both PP2A and myosin IIa. These findings support a new function for non-muscle myosin IIa in differentiated myotubes. [ABSTRACT FROM AUTHOR]

MYH9 disorder is a rare autosomal-dominant disorder. We previously reported that it is caused by mutations in the gene for nonmuscle myosin heavy chain IIA (NMMHC-IIA). MYH9 disorder causes congenital macrothrombocytopenia accompanied by progressive sensorineural hearing loss, nephropathy, and cataract. However, there are few reports that describe the audiological features of MYH9 disorder. The objective of this study was to characterize auditory and other phenotypes of patients with MYH9 disorder. We examined nine subjects from one Japanese family. Audiological, ophthalmological, hematological, and imaging examinations were used to assess clinical features. We carried out genetic analysis of the causative gene, MYH9. Five subjects exhibited macrothrombocytopenia and neutrophil cytoplasmic inclusion bodies. Immunofluorescence analysis of neutrophil NMMHC-IIA revealed abnormal type II localization. Two subjects had high-frequency dominant hearing loss, which was adult onset and progressive. Only one subject had cataract. MYH9 sequencing analysis of all thrombocytopenic subjects revealed a heterozygous c.4270G>A mutation in exon 30 (p.D1424N). We identified five patients with MYH9 disorder from the family. The hearing impairment associated with MYH9 disorder in this family was characterized as adult onset, progressive, and high-frequency dominant. Hematological manifestations of MYH9 disorder show complete penetrance, whereas extra-hematological manifestations show incomplete penetrance and variable expressivity in this family. [ABSTRACT FROM AUTHOR]

Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis. [ABSTRACT FROM AUTHOR]

Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher k cat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas K m values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity ( k cat /K m) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. Significance of the study Phosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle. [ABSTRACT FROM AUTHOR]

The Ca2+-binding protein S100A4 interacts with the C terminus of nonmuscle myosin IIA ( NMIIA) causing filament disassembly, which is correlated with an increased metastatic potential of tumor cells. Despite high sequence similarity of the three NMII isoforms, S100A4 discriminates against binding to NMIIB. We searched for structural determinants of this selectivity. Based on paralog scanning using phage display, we identified a single position as major determinant of isoform selectivity. Reciprocal single amino acid replacements showed that at position 1907 ( NMIIA numbering), the NMIIA/ NMIIC-specific alanine provides about 60-fold higher affinity than the NMIIB-specific asparagine. The structural background of this can be explained in part by a communication between the two consecutive α-helical binding segments. This communication is completely abolished by the Ala-to-Asn substitution. Mutual swapping of the disordered tailpieces only slightly affects the affinity of the NMII chimeras. Interestingly, we found that the tailpiece and position 1907 act in a nonadditive fashion. Finally, we also found that the higher stability of the C-terminal coiled-coil region of NMIIB also discriminates against interaction with S100A4. Our results clearly show that the isoform-selective binding of S100A4 is determined at multiple levels in the structure of the three NMII isoforms and the corresponding functional elements of NMII act synergistically with one another resulting in a complex interaction network. The experimental and in silico results suggest two divergent evolutionary pathways: NMIIA and NMIIB evolved to possess S100A4-dependent and -independent regulations, respectively. [ABSTRACT FROM AUTHOR]

ASAP1 regulates F-actin-based structures and functions, including focal adhesions (FAs) and circular dorsal ruffles (CDRs), cell spreading and migration. ASAP1 function requires its N-terminal BAR domain. We discovered that nonmuscle myosin 2A (NM2A) directly bound the BAR-PH tandem of ASAP1 in vitro. ASAP1 and NM2A co-immunoprecipitated and colocalized in cells. Knockdown of ASAP1 reduced colocalization of NM2A and F-actin in cells. Knockdown of ASAP1 or NM2A recapitulated each other's effects on FAs, cell migration, cell spreading and CDRs. The NM2A-interacting BAR domain contributed to ASAP1 control of cell spreading and CDRs. Exogenous expression of NM2A rescued the effect of ASAP1 knockdown on CDRs but ASAP1 did not rescue NM2A knockdown defect in CDRs. Our results support the hypothesis that ASAP1 is a positive regulator of NM2A. Given other binding partners of ASAP1, ASAP1 may directly link signaling and the mechanical machinery of cell migration. [ABSTRACT FROM AUTHOR]

Non-muscle myosin regulatory light chain (nmMLC 20 ) is reported to exert transcriptional function in regulation of gene expression, and NADPH oxidase (NOX)-derived reactive oxygen species contribute to vascular remodeling of pulmonary artery hypertension (PAH). This study aims to determine if nmMLC 20 can promote endothelial progenitor cells (EPCs) senescence and dysfunction through up-regulation of NOX in PAH rats. The rats were exposed to10% hypoxia for 3 weeks to establish a PAH model, which showed an increase in right ventricle systolic pressure, right ventricular and pulmonary vascular remodeling, and the accelerated senescence and impaired functions in EPCs, accompanied by an increase in Rho-kinase (ROCK) and NOX activities, p-nmMLC 20 level, NOX expression and H 2 O 2 content; these phenomena were reversed by fasudil, a selective inhibitor of ROCK. Next, normal EPCs were cultured under hypoxia to induce senescence in vitro. Consistent with the in vivo findings, hypoxia increased the senescence and dysfunction of EPCs concomitant with an increase in ROCK and NOX activities, p-nmMLC 20 level, NOX expression and H 2 O 2 content; these phenomena were reversed by fasudil. Knockdown of nmMLC 20 showed similar results to that of fasudil except no effect on ROCK activity. Based on these observations, we conclude that nmMLC 20 could promote the senescence and dysfunctions of EPCs in PAH through up-regulation of NOX in a phosphorylation-dependent manner. [ABSTRACT FROM AUTHOR]

Non-muscle myosin IIA (NMIIA) has been found to be overexpressed in gastric cancer tissues. However, the roles of NMIIA in the migration and invasion of gastric cancer cells have not yet been elucidated. The aim of the present study was to assess the effects of NMIIA knockdown on the migratory and invasive capacities of gastric cancer cells and to investigate the potential underlying mechanisms in vitro. First, the expression of NMIIA was assessed in gastric cancer tissues and non-cancerous tissues using western blot analysis. The expression levels of NMIIA protein in 51 out of 63 gastric cancer tissue specimens were higher compared to those in their matched non-tumoric gastric tissue specimens, and differences between the two groups were statistically significant (P<0.001). After downregulation of NMIIA using RNA interference, the migratory and invasive abilities of the SGC-7901 and MGC-803 gastric cancer cell lines were observed using a wound-healing assay and a Transwell assay, respectively. Knockdown of NMIIA significantly decreased the amount of wound closure as well as the number of cells which transgressed through the Matrigel (P<0.01). Finally, the levels of c-Jun N-terminal kinase (JNK), phosphorylated (p)-JNK, c-Jun and p-c-Jun were detected using western blot analysis in order to explore the association between NMIIA and the JNK signaling pathway. Knockdown of NMIIA decreased the levels of p-JNK and p-c-Jun in the two gastric cancer cell lines (P<0.05). In conclusion, the present study indicated that knockdown of NMIIA inhibited the migration and invasion of gastric cancer cells, which may be, at least in part, mediated via the JNK signaling pathway. [ABSTRACT FROM AUTHOR]

Platelet-driven blood clot contraction (retraction) is thought to promote wound closure and secure hemostasis while preventing vascular occlusion. Notwithstanding its importance, clot contraction remains a poorly understood process, partially because of the lack ofmethodology to quantify its dynamics and requirements. We used a novel automated optical analyzer to continuously track in vitro changes in the size of contracting clots in whole blood and in variously reconstituted samples. Kinetics of contraction was complemented with dynamic rheometry to characterize the viscoelasticity of contracting clots. This combined approach enabled investigation of the coordinated mechanistic impact of platelets, including nonmusclemyosin II, redbloodcells (RBCs), fibrin(ogen), factorXIIIa (FXIIIa), and thrombin on the kinetics and mechanics of the contraction process. Clot contraction is composed of 3 sequential phases, each characterized by a distinct rate constant. Thrombin, Ca2+, the integrin αIIbβ3, myosin IIa, FXIIIa cross-linking, and platelet count all promote 1 or more phases of the clot contraction process. In contrast, RBCs impair contraction and reduce elasticity, while increasing the overall contractile stress generated by the plateletfibrin meshwork. A better understanding of the mechanisms by which blood cells, fibrin(ogen), and platelet-fibrin interactions modulate clot contractionmay generate novel approaches to reveal and to manage thrombosis and hemostatic disorders. [ABSTRACT FROM AUTHOR]

Vascular interventions are associated with high failure rates from restenosis secondary to negative remodeling and neointimal hyperplasia. Periadventitial delivery of nitric oxide ( NO) inhibits neointimal hyperplasia, preserving lumen patency. With the development of new localized delivery vehicles, NO-based therapies remain a promising therapeutic avenue for the prevention of restenosis. While the time course of events during neointimal development has been well established, a full characterization of the impact of NO donors on the cells that comprise the arterial wall has not been performed. Thus, the aim of our study was to perform a detailed assessment of proliferation, cellularity, inflammation, and phenotypic cellular modulation in injured arteries treated with the short-lived NO donor, PROLI/NO. PROLI/NO provided durable inhibition of neointimal hyperplasia for 6 months after arterial injury. PROLI/NO inhibited proliferation and cellularity in the media and intima at all of the time points studied. However, PROLI/NO caused an increase in adventitial proliferation at 2 weeks, resulting in increased cellularity at 2 and 8 weeks compared to injury alone. PROLI/NO promoted local protein S-nitrosation and increased local tyrosine nitration, without measurable systemic effects. PROLI/NO predominantly inhibited contractile smooth muscle cells in the intima and media, and had little to no effect on vascular smooth muscle cells or myofibroblasts in the adventitia. Finally, PROLI/NO caused a delayed and decreased leukocyte infiltration response after injury. Our results show that a short-lived NO donor exerts durable effects on proliferation, phenotype modulation, and inflammation that result in long-term inhibition of neointimal hyperplasia. [ABSTRACT FROM AUTHOR]

Introduction: Hyperthermic mitomycin (HM) is a novel treatment modality for selected patients with high-risk non-muscle invasive bladder cancer (NMIBC). We sought to determine predictors of response to this therapy. Patients and Methods: A longitudinal, cohort study of 97 patients with high-risk NMIBC treated with ≥4 HM instillations on a prophylactic schedule was conducted. The primary outcome was time-to-progression survival; secondary outcomes were overall survival, cancer-specific survival, and adverse events. Descriptive statistics, Kaplan-Meier survival analyses, Cox proportional hazards modelling, and univariate and multivariable regression were performed. Results: The presence of initial complete response (CR; no evidence of disease at first check video-cystoscopy and urine cytology) post-HM treatment was an independent predictor of good response to HM. Female patients and those without carcinoma in situ (CIS) also appeared to respond better to the intervention. The overall bladder preservation rate at a median of 27 months was 81.4%; 17/97 (17.5%) patients died during the course of the study. Conclusions: High-risk NMIBC patients can be safely treated with HM and have good oncological outcome. However, those without an initial CR have a poor prognosis and should be counselled towards adopting other treatment methodologies such as cystectomy. Female gender and lack of CIS may be good prognostic indicators for response to HM. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The article discusses a study by A. S. Smith and colleagues on nonmuscle myosin (NM2) function in red blood cells (RBCs). Topics include function of NM2 in most cell types, evidence sought by the researchers that NM2A is active in RBCs, and NM2A's physiological significance in RBCs. The magnitude of NM2A's role in the RBC membrane cytoskeleton and its role in most other cellular contexts are also examined.