Styxl2, a poorly characterized pseudophosphatase, was identified as a transcriptional target of the Jak1-Stat1 pathway during myoblast differentiation in culture. Styxl2 is specifically expressed in vertebrate striated muscles. By gene knockdown in zebrafish or genetic knockout in mice, we found that Styxl2 plays an essential role in maintaining sarcomere integrity in developing muscles. To further reveal the functions of Styxl2 in adult muscles, we generated two inducible knockout mouse models: one with Styxl2 being deleted in mature myofibers to assess its role in sarcomere maintenance, and the other in adult muscle satellite cells (MuSCs) to assess its role in de novo sarcomere assembly. We find that Styxl2 is not required for sarcomere maintenance but functions in de novo sarcomere assembly during injury-induced muscle regeneration. Mechanistically, Styxl2 interacts with non-muscle myosin IIs, enhances their ubiquitination, and targets them for autophagy-dependent degradation. Without Styxl2, the degradation of non-muscle myosin IIs is delayed, which leads to defective sarcomere assembly and force generation. Thus, Styxl2 promotes de novo sarcomere assembly by interacting with non-muscle myosin IIs and facilitating their autophagic degradation.

Neuromuscular junctions (NMJs) are evolutionarily ancient, specialized contacts between neurons and muscles. Axons and NMJs must endure mechanical strain through a lifetime of muscle contraction, making them vulnerable to aging and neurodegenerative conditions. However, cellular strategies for mitigating this mechanical stress remain unknown. In this study, we used Drosophila larval NMJs to investigate the role of actin and myosin (actomyosin)-mediated contractility in generating and responding to cellular forces at the neuron-muscle interface. We identified a new long-lived, low-turnover presynaptic actin core traversing the NMJ, which partly co-localizes with non-muscle myosin II (NMII). Neuronal RNAi of NMII induced disorganization of this core, suggesting that this structure might have contractile properties. Interestingly, neuronal RNAi of NMII also decreased NMII levels in the postsynaptic muscle proximal to neurons, suggesting that neuronal actomyosin rearrangements propagate their effects transsynaptically. We also observed reduced Integrin levels upon NMII knockdown, indicating that neuronal actomyosin disruption triggers rearrangements of Integrin-mediated connections between neurons and surrounding muscle tissue. In summary, our study identifies a previously uncharacterized presynaptic actomyosin subpopulation that upholds the neuronal mechanical continuum, transmits signals to adjacent muscle tissue, and collaborates with Integrin receptors to govern the mechanobiology of the neuromuscular junction.

After endocytosis, many plasma membrane components are recycled via membrane tubules that emerge from early endosomes to form recycling endosomes, eventually leading to their return to the plasma membrane. We previously showed that Syndapin/PACSIN-family protein SDPN-1 is required in vivo for basolateral endocytic recycling in the C. elegans intestine. Here, we document an interaction between the SDPN-1 SH3 domain and a target sequence in PXF-1/PDZ-GEF1/RAPGEF2, a known exchange factor for Rap-GTPases. We found that endogenous mutations engineered into the SDPN-1 SH3 domain, or its binding site in the PXF-1 protein, interfere with recycling in vivo, as does the loss of the PXF-1 target RAP-1. In some contexts, Rap-GTPases negatively regulate RhoA activity, suggesting a potential for Syndapin to regulate RhoA. Our results indicate that in the C. elegans intestine, RHO-1/RhoA is enriched on SDPN-1- and RAP-1-positive endosomes, and the loss of SDPN-1 or RAP-1 elevates RHO-1(GTP) levels on intestinal endosomes. Furthermore, we found that depletion of RHO-1 suppressed sdpn-1 mutant recycling defects, indicating that control of RHO-1 activity is a key mechanism by which SDPN-1 acts to promote endocytic recycling. RHO-1/RhoA is well known for controlling actomyosin contraction cycles, although little is known about the effects of non-muscle myosin II on endosomes. Our analysis found that non-muscle myosin II is enriched on SDPN-1-positive endosomes, with two non-muscle myosin II heavy-chain isoforms acting in apparent opposition. Depletion of nmy-2 inhibited recycling like sdpn-1 mutants, whereas depletion of nmy-1 suppressed sdpn-1 mutant recycling defects, indicating that actomyosin contractility controls recycling endosome function.

The morphogenetic process of apical constriction, which relies on non-muscle myosin II (NMII) generated constriction of apical domains of epithelial cells, is key to the development of complex cellular patterns. Apical constriction occurs in almost all multicellular organisms, but one of the most well-characterized systems is the Folded-gastrulation (Fog)-induced apical constriction that occurs in Drosophila. The binding of Fog to its cognizant receptors Mist/Smog results in a signaling cascade that leads to the activation of NMII-generated contractility. Despite our knowledge of key molecular players involved in Fog signaling, we sought to explore whether other proteins have an undiscovered role in its regulation. We developed a computational method to predict unidentified candidate NMII regulators using a network of pairwise protein-protein interactions called an interactome. We first constructed a Drosophila interactome of over 500,000 protein-protein interactions from several databases that curate high-throughput experiments. Next, we implemented several graph-based algorithms that predicted 14 proteins potentially involved in Fog signaling. To test these candidates, we used RNAi depletion in combination with a cellular contractility assay in Drosophila S2R + cells, which respond to Fog by contracting in a stereotypical manner. Of the candidates we screened using this assay, two proteins, the serine/threonine phosphatase Flapwing and the putative guanylate kinase CG11811 were demonstrated to inhibit cellular contractility when depleted, suggestive of their roles as novel regulators of the Fog pathway.

Liver sinusoidal endothelial cells (LSECs) facilitate the efficient transport of macromolecules and solutes between the blood and hepatocytes. The efficiency of this transport is realized via transcellular nanopores, called fenestrations. The mean fenestration size is 140 ± 20 nm, with the range from 50 nm to 350 nm being mostly below the limits of diffraction of visible light. The cellular mechanisms controlling fenestrations are still poorly understood. In this study, we tested a hypothesis that both Rho kinase (ROCK) and myosin light chain (MLC) kinase (MLCK)-dependent phosphorylation of MLC regulates fenestrations. We verified the hypothesis using a combination of several molecular inhibitors and by applying two high-resolution microscopy modalities: structured illumination microscopy (SIM) and scanning electron microscopy (SEM). We demonstrated precise, dose-dependent, and reversible regulation of the mean fenestration diameter within a wide range from 120 nm to 220 nm and the fine-tuning of the porosity in a range from ~0% up to 12% using the ROCK pathway. Moreover, our findings indicate that MLCK is involved in the formation of new fenestrations-after inhibiting MLCK, closed fenestrations cannot be reopened with other agents. We, therefore, conclude that the Rho-ROCK pathway is responsible for the control of the fenestration diameter, while the inhibition of MLCK prevents the formation of new fenestrations.

Tissue fusion frequently requires the removal of an epithelium that intervenes distinct primordia to form one continuous structure. In the mammalian secondary palate, a midline epithelial seam (MES) forms between two palatal shelves and must be removed to allow mesenchymal confluence. Abundant apoptosis and cell extrusion support their importance in MES removal. However, genetically disrupting the intrinsic apoptotic regulators BAX and BAK within the MES results in complete loss of cell death and cell extrusion, but successful removal of the MES. Novel static- and live-imaging approaches reveal that the MES is removed through streaming migration of epithelial trails and islands to reach the oral and nasal epithelial surfaces. Epithelial trail cells that express the basal epithelial marker ΔNp63 begin to express periderm markers, suggesting that migration is concomitant with differentiation. Live imaging reveals anisotropic actomyosin contractility within epithelial trails, and genetic ablation of actomyosin contractility results in dispersion of epithelial collectives and failure of normal MES migration. These findings demonstrate redundancy between cellular mechanisms of morphogenesis, and reveal a crucial and unique form of collective epithelial migration during tissue fusion.

Antigen (Ag)-triggered B-cell receptor (BCR) signaling initiates antibody responses. However, prolonged or uncontrolled BCR signaling is associated with the development of self-reactive B-cells and autoimmune diseases. We previously showed that actin-mediated B-cell contraction on Ag-presenting surfaces negatively regulates BCR signaling. Non-muscle myosin II (NMII), an actin motor, is involved in B-cell development and antibody responses by mediating B-cell migration, cytokinesis, and Ag extraction from Ag-presenting cells. However, whether and how NMII regulates humoral responses through BCR signaling remains elusive. Utilizing a B-cell-specific, partial NMIIA knockout (cIIAKO) mouse model and NMII inhibitors, this study examined the role of NMII in BCR signaling. Upon BCR binding to antibody-coated planar lipid bilayers (PLB), NMIIA was recruited to the B-cell contact membrane and formed a ring-like structure during B-cell contraction. NMII recruitment depended on phosphatidylinositol 5-phosphatase (SHIP1), an inhibitory signaling molecule. NMII inhibition by cIIAKO did not affect B-cell spreading on PLB but delayed B-cell contraction and altered BCR clustering. Surface BCR \"cap\" formation induced by soluble stimulation was enhanced in cIIAKO B-cells. Notably, NMII inhibition by cIIAKO and inhibitors up-regulated BCR signaling in response to both surface-associated and soluble stimulation, increasing phosphorylated tyrosine, CD79a, BLNK, and Erk and decreasing phosphorylated SHIP1. While cIIAKO did not affect B-cell development, the number of germinal center B-cells was significantly increased in unimmunized cIIAKO mice, compared to control mice. While cIIAKO mice mounted similar antibody responses when compared to control mice upon immunization, the percentages of high-affinity antibodies, Ag-specific germinal center B-cells and isotype switched B-cells were significantly lower in cIIAKO mice than in control mice. Furthermore, autoantibody levels were elevated in cIIAKO mice, compared to control mice. Collectively, our results reveal that NMII exerts a B-cell-intrinsic inhibition on BCR signaling by regulating B-cell membrane contraction and surface BCR clustering, which curtails the activation of non-specific and self-reactive B-cells.

Apical constriction, or a reduction in size of the apical domain, underlies many morphogenetic events during development. Actomyosin complexes play an essential role in apical constriction; however, the detailed analysis of molecular mechanisms is still pending. Here, we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells. Lmo7 is primarily localized at apical junctions and promotes the formation of the dense circumferential actomyosin belt. Strikingly, Lmo7 binds non-muscle myosin II (NMII) and recruits it to apical junctions and the apical cortex. This NMII recruitment is essential for Lmo7-mediated apical constriction. Lmo7 knockdown decreases NMIIA localization at apical junctions and delays neural tube closure in Xenopus embryos. Our findings suggest that Lmo7 serves as a scaffold that regulates actomyosin contractility and apical domain size.

Studies on neural development and neuronal regeneration after injury are mainly based on animal models. The establishment of pluripotent stem cell (PSC) technology, however, opened new perspectives for better understanding these processes in human models by providing unlimited cell source for hard-to-obtain human tissues. Here, we aimed at identifying the molecular factors that confine and modulate an early step of neural regeneration, the formation of neurites in human neural progenitor cells (NPCs). Enhanced green fluorescent protein (eGFP) was stably expressed in NPCs differentiated from human embryonic and induced PSC lines, and the neurite outgrowth was investigated under normal and injury-related conditions using a high-content screening system. We found that inhibitors of the non-muscle myosin II (NMII), blebbistatin and its novel, non-toxic derivatives, initiated extensive neurite outgrowth in human NPCs. The extracellular matrix components strongly influenced the rate of neurite formation but NMII inhibitors were able to override the inhibitory effect of a restrictive environment. Non-additive stimulatory effect on neurite generation was also detected by the inhibition of Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), the upstream regulator of NMII. In contrast, inhibition of c-Jun N-terminal kinases (JNKs) had only a negligible effect, suggesting that the ROCK1 signal is dominantly manifested by actomyosin activity. In addition to providing a reliable cell-based in vitro model for identifying intrinsic mechanisms and environmental factors responsible for impeded axonal regeneration in humans, our results demonstrate that NMII and ROCK1 are important pharmacological targets for the augmentation of neural regeneration at the progenitor level. These studies may open novel perspectives for development of more effective pharmacological treatments and cell therapies for various neurodegenerative disorders.

Changes in plasma membrane curvature and intracellular ionic strength are two key features of cell volume perturbations. In this hypothesis we present a model of the responsible molecular apparatus which is assembled of two molecular motors [non-muscle myosin II (NMMII) and protrusive actin polymerization], a spring [a complex between the plasma membrane (PM) and the submembrane actin-based cytoskeleton (smACSK) which behaves like a viscoelastic solid] and the associated signaling proteins. We hypothesize that this apparatus senses changes in both the plasma membrane curvature and the ionic strength and in turn activates signaling pathways responsible for regulatory volume increase (RVI) and regulatory volume decrease (RVD). During cell volume changes hydrostatic pressure (HP) changes drive alterations in the cell membrane curvature. HP difference has opposite directions in swelling versus shrinkage, thus allowing distinction between them. By analogy with actomyosin contractility that appears to sense stiffness of the extracellular matrix we propose that NMMII and actin polymerization can actively probe the transmembrane gradient in HP. Furthermore, NMMII and protein-protein interactions in the actin cortex are sensitive to ionic strength. Emerging data on direct binding to and regulating activities of transmembrane mechanosensors by NMMII and actin cortex provide routes for signal transduction from transmembrane mechanosensors to cell volume regulatory mechanisms.