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In this study, we investigated the influence of galangin on vascular contractibility and to determine the mechanism underlying the relaxation. Isometric contractions of denuded aortic muscles were recorded and combined with western blot analysis which was performed to measure the phosphorylation of phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) and to evaluate the effect of galangin on the RhoA/ROCK/CPI-17 pathway. Galangin significantly inhibited phorbol ester-, fluoride- and thromboxane mimetic-induced vasoconstrictions regardless of endothelial nitric oxide synthesis, suggesting its direct effect on vascular smooth muscle. Galangin significantly inhibited the fluoride-dependent increase in pMYPT1 and pCPI-17 levels and phorbol 12,13-dibutyrate-dependent increase in pERK1/2 level, suggesting repression of ROCK and MEK activity and subsequent phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that galangin-induced relaxation involves myosin phosphatase reactivation and calcium desensitization, which appears to be mediated by CPI-17 dephosphorylation via not PKC but ROCK inactivation.

Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of the Drosophila pupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, we investigate how pattern refinement is regulated. We found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate competence after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. We propose that mechano-chemical feedback leads to competition for the developmental signal which plays a critical role in pattern refinement.

Myosin VI（MYO6） is an unconventional myosin that is vital for auditory and vestibular function. Pathogenic variants in the human MYO6 gene cause autosomal-dominant or -recessive forms of hearing loss. Effective treatments for Myo6 mutation causing hearing loss are limited. We studied whether adeno-associated virus (AAV)-PHP.eB vector-mediated in vivo delivery of Staphylococcus aureus Cas9 (SaCas9-KKH)-single-guide RNA (sgRNA) complexes could ameliorate hearing loss in a Myo6(WT/C442Y) mouse model that recapitulated the phenotypes of human patients. The in vivo editing efficiency of the AAV-SaCas9-KKH-Myo6-g2 system on Myo6(C442Y) is 4.05% on average in Myo6(WT/C442Y) mice, which was ∼17-fold greater than editing efficiency of Myo6(WT) alleles. Rescue of auditory function was observed up to 5 months post AAV-SaCas9-KKH-Myo6-g2 injection in Myo6(WT/C442Y) mice. Meanwhile, shorter latencies of auditory brainstem response (ABR) wave I, lower distortion product otoacoustic emission (DPOAE) thresholds, increased cell survival rates, more regular hair bundle morphology, and recovery of inward calcium levels were also observed in the AAV-SaCas9-KKH-Myo6-g2-treated ears compared to untreated ears. These findings provide further reference for in vivo genome editing as a therapeutic treatment for various semi-dominant forms of hearing loss and other semi-dominant diseases.

In plants, secretion of cell wall components and membrane proteins plays a fundamental role in growth and development as well as survival in diverse environments. Exocytosis, as the last step of the secretory trafficking pathway, is a highly ordered and precisely controlled process involving tethering, docking, and fusion of vesicles at the plasma membrane (PM) for cargo delivery. Although the exocytic process and machinery are well characterized in yeast and animal models, the molecular players and specific molecular events that underpin late stages of exocytosis in plant cells remain largely unknown. Here, by using the delivery of functional, fluorescent-tagged cellulose synthase (CESA) complexes (CSCs) to the PM as a model system for secretion, as well as single-particle tracking in living cells, we describe a quantitative approach for measuring the frequency of vesicle tethering events. Genetic and pharmacological inhibition of cytoskeletal function, reveal that the initial vesicle tethering step of exocytosis is dependent on actin and myosin XI. In contrast, treatments with the microtubule inhibitor, oryzalin, did not significantly affect vesicle tethering or fusion during CSC exocytosis but caused a minor increase of transient or aborted tethering events. With data from this new quantitative approach and improved spatiotemporal resolution of single particle events during secretion, we generate a revised model for the role of the cortical cytoskeleton in CSC trafficking.

The movement of organelles and vesicles in pollen tubes depends on F-actin. However, the molecular mechanism through which plant myosin XI drives the movement of organelles is still controversial, and the relationship between myosin XI and vesicle movement in pollen tubes is also unclear. In this study, we found that the siliques of the myosin xi-b/e mutant were obviously shorter than those of the wild-type (WT) and that the seed set of the mutant was severely deficient. The pollen tube growth of myosin xi-b/e was significantly inhibited both in vitro and in vivo. Fluorescence recovery after photobleaching (FRAP) showed that the velocity of vesicle movement in the pollen tube tip of the myosin xi-b/e mutant was lower than that of the WT. It was also found that peroxisome movement was significantly inhibited in the pollen tubes of the myosin xi-b/e mutant, while the velocities of the Golgi stack and mitochondrial movement decreased relatively less in the pollen tubes of the mutant. The endoplasmic reticulum streaming in the pollen tube shanks was not significantly different between the WT and the myosin xi-b/e mutant. In addition, we found that myosin XI-B-GFP colocalized obviously with vesicles and peroxisomes in the pollen tubes of Arabidopsis. Taken together, these results indicate that myosin XI-B may bind mainly to vesicles and peroxisomes and drive their movement in pollen tubes. These results also suggest that the mechanism by which myosin XI drives organelle movement in plant cells may be evolutionarily conserved compared with other eukaryotic cells.

Epithelial cells use the process of extrusion to promote cell death while preserving a tight barrier. To extrude, a cell and its neighbors contract actin and myosin circumferentially and basolaterally to seamlessly squeeze it out of the epithelium. Recent research highlights how early apical pulsatile contractions within the extruding cell might orchestrate contraction in three dimensions so that a cell extrudes out apically. Along with apical constrictions, studies of ion channels and mathematical modeling reveal how differential contraction between cells helps select specific cells to extrude. In addition, several studies have offered new insights into pathways that use extrusion to eliminate transformed cells or cause an aberrant form of extrusion that promotes cell invasion.

Astrocytes, which can be obtained from neural stem cells (NSCs) by adding serum and/or recombinant proteins in culture media or by passaging NSCs repeatedly, are expected to be applicable in regenerative medicine for the treatment of neurodegenerative diseases. However, astrocytes obtained using existing methods are costly and have poor quality. The stiffness of culture surfaces has been reported to affect astrocytic differentiation of adult NSCs. However, the influence of surface stiffness on astrocytic differentiation of embryonic NSCs has not yet been reported. In this study, we showed that astrocytic differentiation of embryonic NSCs was increased on soft surfaces (1 kPa and 12 kPa) compared with the NSCs on stiff surfaces (2.8 GPa) in serum-free condition. Furthermore, di-phosphorylated myosin regulatory light chain (PP-MRLC) was decreased in embryonic NSCs cultured on the soft surfaces than the cells on the stiff surfaces. Additionally, astrocytic differentiation of embryonic NSCs was induced by a Ras homolog associated kinase (ROCK) inhibitor, which decreased PP-MRLC in NSCs. These results suggest that decreasing the PP-MRLC of embryonic NSCs on soft surfaces or treating NSCs with a ROCK inhibitor is a good method to prepare astrocytes for application in regenerative medicine.

Background: Photoreceptor death and neurodegeneration is the leading cause of irreversible vision loss. The inflammatory response of microglia plays an important role in the process of neurodegeneration. In this study, we examined the involvement of myosin 1f as a key regulator of immune cell activation via the AKT and MAPK pathways in microglia. Methods: We chose retinal detachment as the model of photoreceptor degeneration. Immunofluorescence and Western Blot was performed to confirm the expression and location of myosin 1f in detached retina. The RD mouse model was induced in WT and myosin 1f-/- mice and confirmed by HE and TUNEL staining. The expression of inflammatory cytokine and downstream pathways was assessed via qPCR and WB.Results: Myosin 1f was upregulated after retinal detachment, and it was specifically expressed in microglia. Deficiency of myosin 1f protected against cell death by inhibiting microglia activation. The elimination of microglia can abolish the protective effect of myosin 1f deficiency. After stimulation by LPS, microglia with myosin 1f deficiency showed downregulation of the MAPK and AKT pathways.Conclusions: Myosin 1f plays a crucial role in microglia-induced neuro-inflammation after retinal injury and photoreceptor degeneration by regulating 2 classic pathways, MAPK and AKT, and thereby decreasing the expression of inflammatory cytokines. Myosin 1f can be inhibited to prevent a decline in visual acuity after photoreceptor degeneration.

Proper muscle development and function depend on myosin being properly folded and integrated into the thick filament structure. For this to occur the myosin chaperone UNC-45, or UNC-45B, must be present and able to chaperone myosin. Here we use a combination of in vivo C. elegans experiments and in vitro biophysical experiments to analyze the effects of six missense mutations in conserved regions of UNC-45/UNC-45B. We found that the phenotype of paralysis and disorganized thick filaments in 5/6 of the mutant nematode strains can likely be attributed to both reduced steady state UNC-45 protein levels and reduced chaperone activity. Interestingly, the biophysical assays performed on purified proteins show that all of the mutations result in reduced myosin chaperone activity but not overall protein stability. This suggests that these mutations only cause protein instability in the in vivo setting and that these conserved regions may be involved in UNC-45 protein stability/regulation via posttranslational modifications, protein-protein interactions, or some other unknown mechanism.

In eukaryotes, myosin provides the necessary impetus for a series of physiological processes, including organelle movement, cytoplasmic flow, cell division, and mitosis. Previously, three members of myosin were identified in Magnaporthe oryzae, with class II and class V myosins playing important roles in intracellular transport, fungal growth, and pathogenicity. However, limited is known about the biological function of the class I myosin protein in the rice blast fungus. Here, we found that Momyo1 is highly expressed during conidiation and infection. Functional characterization of this gene via RNA interference (RNAi) revealed that Momyo1 is required for vegetative growth, conidiation, melanin pigmentation, and pathogenicity of M. oryzae. The Momyo1 knockdown mutant is defective in formation of appressorium-like structures (ALS) at the hyphal tips. In addition, Momyo1 also displays defects on cell wall integrity, hyphal hydrophobicity, extracellular enzyme activities, endocytosis, and formation of the Spitzenkorper. Furthermore, Momyo1 was identified to physically interact with the MoShe4, a She4p/Dim1p orthologue potentially involved in endocytosis, polarization of the actin cytoskeleton. Overall, our findings provide a novel insight into the regulatory mechanism of Momyo1 that is involved in fungal growth, cell wall integrity, endocytosis, and virulence of M. oryzae. • Momyo1 is required for vegetative growth and pigmentation of M. oryzae. • Momyo1 is essential for cell wall integrity and endocytosis of M. oryzae. • Momyo1 is involved in hyphal surface hydrophobicity of M. oryzae.

Cell signaling pathways regulating myosin regulatory light chain (LC20) phosphorylation contribute to determining contractile responses in smooth muscles. Following excitation and contraction, phasic smooth muscles, such as the digestive tract and urinary bladder, undergo relaxation due to a decline of cellular Ca2+ concentration and decreased Ca2+ sensitivity of LC20 phosphorylation, named Ca2+ desensitization. Here, we determined the mechanisms underlying the temporal Ca2+ desensitization of LC20 phosphorylation in phasic smooth muscles using permeabilized strips of the mouse ileum and urinary bladder. Upon stimulation with pCa6.0 at 20°C, contraction and LC20 phosphorylation peaked within 30 s and then declined to about 50% of the peak force at 2 min after stimulation. During the relaxation phase after the contraction, LC20 kinase [myosin light chain kinase (MLCK)] was inactivated, but no fluctuation in LC20 phosphatase activity occurred, suggesting that MLCK inactivation is a cause of the Ca2+-induced Ca2+ desensitization of LC20 phosphorylation. MLCK inactivation was associated with phosphorylation at the calmodulin-binding domain of the kinase. Treatment with STO-609 and TIM-063 antagonists for Ca2+/calmodulin (CaM)-dependent protein kinase kinase-β (CaMKKβ) attenuated both the phasic response of the contraction and MLCK phosphorylation, whereas neither CaM kinase II, AMP-activated protein kinase, nor p21-activated kinase induced MLCK inactivation in phasic smooth muscles. Conversely, protein phosphatase 2A inhibition amplified the phasic response. Signaling pathways through CaMKKβ and protein phosphatase 2A may contribute to regulating the phasic response of smooth muscle contraction.

We have created a novel in-vitro platform to study reverse remodeling of engineered heart tissue (EHT) after mechanical unloading. EHTs were created by seeding decellularized porcine myocardial sections with a mixture of primary neonatal rat ventricular myocytes and cardiac fibroblasts. Each end of the ribbon-like constructs was fixed to a plastic clip, allowing the tissues to be statically stretched or slackened. Inelastic deformation was introduced by stretching tissues by 20% of their original length. EHTs were subsequently unloaded by returning tissues to their original, shorter length. Mechanical characterization of EHTs immediately after unloading and at subsequent time points confirmed the presence of a reverse-remodeling process, through which stress-free tissue length was increased after chronic stretch but gradually decreased back to its original value within nine days. When a cardiac myosin inhibitor was applied to tissues after unloading, EHTs failed to completely recover their passive and active mechanical properties, suggesting a role for actomyosin contraction in reverse remodeling. Selectively inhibiting cardiomyocyte contraction or fibroblast activity after mechanical unloading showed that contractile activity of both cell types was required to achieve full remodeling. Similar tests with EHTs formed from human induced pluripotent stem cell-derived cardiomyocytes also showed reverse remodeling that was enhanced when treated with omecamtiv mecarbil, a myosin activator. These experiments suggest essential roles for active sarcomeric contraction and fibroblast activity in reverse remodeling of myocardium after mechanical unloading. Our findings provide a mechanistic rationale for designing potential therapies to encourage reverse remodeling in patient hearts.

B7-H3 (also known as CD276) is associated with aggressive characteristics in various cancers. Meanwhile, in alveolar rhabdomyosarcoma (ARMS), PAX3-FOXO1 fusion protein is associated with increased aggressiveness and poor prognosis. In the present study, we explored the relationship between PAX3-FOXO1 and B7-H3 and the biological roles of B7-H3 in ARMS. Quantitative real time PCR and flow cytometry revealed that PAX3-FOXO1 knockdown downregulated B7-H3 expression in all the selected cell lines (Rh-30, Rh-41, and Rh-28), suggesting that PAX3-FOXO1 positively regulates B7-H3 expression. Gene expression analysis revealed that various genes and pathways involved in chemotaxis, INF-γ production, and myogenic differentiation were commonly affected by the knockdown of PAX3-FOXO1 and B7-H3. Wound healing and transwell migration assays revealed that both PAX3-FOXO1 and B7-H3 were associated with cell migration. Furthermore, knockdown of PAX3-FOXO1 or B7-H3 induced myogenin expression in all cell lines, although myosin heavy chain induction varied depending on the cellular context. Our results indicate that PAX3-FOXO1 regulates B7-H3 expression and that PAX3-FOXO1 and B7-H3 are commonly associated with multiple pathways related to an aggressive phenotype in ARMS, such as cell migration and myogenic differentiation block.

In the three-step myofibrillogenesis model, mature myofibrils are formed through two intermediate structures: premyofibrils and nascent myofibrils. We have recently reported that several inhibitors of the Ubiquitin Proteosome System, for example, MG-132, and DBeQ, reversibly block progression of nascent myofibrils to mature myofibrils. In this investigation, we studied the effects of MG132 and DBeQ on the expression of various myofibrillar proteins including actin, myosin light and heavy chains, tropomyosin, myomesin, and myosin binding protein-C in cultured embryonic quail myotubes by western blotting using two loading controls-α-tubulin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Surprisingly, we found that MG-132 affected the level of expression of GAPDH but DBeQ did not. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription-PCR (qRT-PCR) showed no significant effect of MG-132 on GAPDH transcription. Two-dimensional (2D) western blot analyses with extracts of control and MG-132-treated cells using anti-ubiquitin antibody indicated that MG132-treated myotubes show a stronger emitter-coupled logic signal. However, Spot% and Spot volume calculations for all spots from both western blot film signals and matched Coomassie-stained 2D polyacrylamide gel electrophoresis showed that the intensity of staining in a spot of ~39 kDa protein is 3.5-fold lower in the gel of MG-132-treated extracts. Mass spectrometry analyses identified the ~39 kDa protein as quail GAPDH. Immunohistochemical analysis of fixed MG-132-treated myotubes with anti-GAPDH antibody showed extensive clump formation, which may be analogous to granule formation by stress response factors in MG132-treated cells. This is the first report on in vivo ubiquitination of GAPDH. This may be essential for the moonlighting (Jeffery, 1999) activity of GAPDH for tailoring stress in myotubes.

A large number of studies have shown that polyphenols can regulate skeletal muscle fiber type transformation through AMPK signal. However, the effects and mechanism of naringin (a natural polyphenol) on muscle fiber type transformation still remains unclear. Thus, we hypothesized that naringin would induce the transformation of skeletal muscle fibers from type II to type I by AMPK signaling. C2C12 myotubes and BALB/c mice models were used to test this hypothesis. We found that naringin significantly increased the protein expression of slow myosin heavy chain (MyHC), myoglobin and troponin I type I slow skeletal (Troponin I-SS) and the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH), and significantly decreased fast MyHC protein expression and lactate dehydrogenase (LDH) activity, accompanied by the activation of AMPK and the activity of peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) in mice and C2C12 myotubes. Further inhibition of AMPK activity by compound C showed that the above effects were significantly inhibited in C2C12 myotubes. In conclusion, naringin promotes the transformation of skeletal muscle fibers from type II to type I through AMPK/PGC-1α signaling pathway, which not only enriches the nutritional and physiological functions of naringin, but also provides a theoretical basis for the regulation of muscle fiber type transformation by nutritional approaches.

In the present study, we investigated the possible involvement of the TGF-β/Smad signaling pathway in the osteopontin-derived SVVYGLR (SV) peptide-mediated migratory activities of myogenic cells and evaluated the facilitative effects of the SV peptide on the differentiation of myogenic cells in vitro. The SV peptide-induced migration in both human-derived satellite cells and myoblasts was substantially suppressed by the TGF-β1 receptor inhibitor SB431542 or SB505124. Besides, the expression level of the Smad3 phosphorylation was further enhanced by the addition of the SV peptide in comparison with control groups. Furthermore, an increase in the expression of myogenin-positive nuclei and a higher number of nascent myotubes with myosin heavy chain expression was confirmed in cultured myoblasts supplemented with the SV peptide. These results suggest that the involvement of the TGF-β/Smad signaling pathway in the SV peptide-mediated migration and the facilitative effect of the SV peptide on the differentiation of myogenic cells into myotubes.

Patients with autosomal dominant SPECC1L variants show syndromic malformations, including hypertelorism, cleft palate and omphalocele. These SPECC1L variants largely cluster in the second coiled-coil domain (CCD2), which facilitates association with microtubules. To study SPECC1L function in mice, we first generated a null allele (Specc1lΔEx4) lacking the entire SPECC1L protein. Homozygous mutants for these truncations died perinatally without cleft palate or omphalocele. Given the clustering of human variants in CCD2, we hypothesized that targeted perturbation of CCD2 may be required. Indeed, homozygotes for in-frame deletions involving CCD2 (Specc1lΔCCD2) resulted in exencephaly, cleft palate and ventral body wall closure defects (omphalocele). Interestingly, exencephaly and cleft palate were never observed in the same embryo. Further examination revealed a narrower oral cavity in exencephalic embryos, which allowed palatal shelves to elevate and fuse despite their defect. In the cell, wild-type SPECC1L was evenly distributed throughout the cytoplasm and colocalized with both microtubules and filamentous actin. In contrast, mutant SPECC1L-ΔCCD2 protein showed abnormal perinuclear accumulation with diminished overlap with microtubules, indicating that SPECC1L used microtubule association for trafficking in the cell. The perinuclear accumulation in the mutant also resulted in abnormally increased actin and non-muscle myosin II bundles dislocated to the cell periphery. Disrupted actomyosin cytoskeletal organization in SPECC1L CCD2 mutants would affect cell alignment and coordinated movement during neural tube, palate and ventral body wall closure. Thus, we show that perturbation of CCD2 in the context of full SPECC1L protein affects tissue fusion dynamics, indicating that human SPECC1L CCD2 variants are gain-of-function.

Kinesin-like calmodulin-binding protein (KCBP) is a unique kinesin with half kinesin and half myosin, with kinesin motor domain at C-terminus and myosin tail homology region 4 (MyTH4) and band 4.1, ezrin, radixin, moesin (FERM) domains at N-terminus. The special structure endows KCBP multi-intracellular functions, including cell division, trichome morphogenesis in plants, and flagellar function in algae. However, little is known about the molecular mechanism underlying these functions. Here, we identified a molecular chaperone Hsp90 as a novel binding partner with KCBP in Dunaliella salina using a yeast two-hybrid screen. Further analysis showed that Hsp90 interacted with both the N-terminal and C-terminal of DsKCBP. Since Hsp90 was involved in the stability and proteolytic turnover of numerous proteins, whether Hsp90 regulated the degradation of DsKCBP was investigated. Our results showed that both Hsp90 and DsKCBP presented in the purified proteasome, and the interaction of DsKCBP-Hsp90 was inhibited upon Hsp90 inhibitor geldanamycin treatment. The level of DsKCBP proteins was diminished remarkably indicating that the disassociation of DsKCBP from Hsp90 accelerated the degradation of the former. Furthermore, immunofluorescence results showed that the localization of DsKCBP at basal body and flagella was disappeared by Hsp90 inhibition. The increased mRNA level of DsKCBP during flagellar assembly was not obvious by geldanamycin treatment. These data provided evidence that Hsp90 protected DsKCBP from degradation by proteasome and was involved in the role of DsKCBP in flagellar assembly.

The plasma-membrane marker FM1-43 was employed to reveal the relative significance of different types of endocytic and transcytic mechanisms in outer hair cells (OHCs) of the guinea-pig cochlea. A double-barrel local perfusion system was used to label independently the apical or synaptic pole of the isolated OHC, to study mechanisms of vesicle uptake at the poles and of vesicle trafficking along and across the cell. Treatment with an inhibitor of macropino- and phagocytosis, phenylarsine oxide, or of clathrin-mediated endocytic activity, concanavalin A, significantly reduced the dye uptake at both the apical and the synaptic poles, indicating the presence of both clathrin-independent and clathrin-mediated processes at both poles. However, measurement of uptake speed in the presence of the inhibitors suggested that clathrin-independent processes contribute more extensively to endocytosis at the basal pole than the apical pole. Treatment with an inhibitor of myosin VI, 2,4,6-triiodophenol, significantly delayed both the apicobasal and the basoapical fluorescence signals. However, treatment with an inhibitor of kinesin, monastrol, or of dynein, ciliobrevin D, significantly delayed the signals only in the basoapical direction. The myosin-VI inhibitor, but neither the kinesin nor dynein inhibitors, significantly delayed the signals to the subsurface cisternae. That is, myosin VI carries vesicles in both longitudinal directions as well as radially to the sub-surface cisternae, whereas kinesin and dynein participate primarily in basoapical trafficking. This fundamental information is essential for elucidating recycling mechanisms of specific proteins involved in establishing, controlling, and maintaining the electromechanical action of OHCs and, therefore, vital for understanding auditory perception.

Background Proper muscle function is heavily dependent on highly ordered protein complexes. UNC45 is a USC (named since this region is shared by three proteins UNC45/CRO1/She4P) chaperone that is necessary for myosin incorporation into the thick filaments. UNC45 is expressed throughout the entire Drosophila life cycle and it has been shown to be important during late embryogenesis when initial muscle development occurs. However, the effects of UNC45 manipulation at later developmental times, after muscle development, have not yet been studied. Main results UNC45 was knocked down with RNAi in a manner that permitted survival to the pupal stage, allowing for characterization of sarcomere organization in the well-studied third instar larvae. Second harmonic generation (SHG) microscopy revealed changes in the striated pattern of body wall muscles as well as a reduction of signal intensity. This observation was confirmed with immunofluorescence and electron microscopy imaging, showing diminished UNC45 signal and disorganization of myosin and z-disk proteins. Concomitant alterations in both synaptic physiology and locomotor function were also found. Both nerve-stimulated response and spontaneous vesicle release were negatively affected, while larval movement was impaired. Conclusions This study highlights the dependency of normal sarcomere structure on UNC45 expression. We confirm the known role of UNC45 for myosin localization and further show the I-Z-I complex is also disrupted. This suggests a broad need for UNC45 to maintain sarcomere integrity. Newly discovered changes in synaptic physiology reveal the likely presence of a homeostatic response to partially maintain synaptic strength and muscle function.

International audience; Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N′)-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N′-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N′-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Sphingolipids function as both structural components of cells and intracellular messengers. The role of sphingolipids, including ceramide, in the regulation of muscle plasticity has not been studied. The present work aimed to study the effect of an inhibitor of the catalytic pathway of ceramide formation on the expression of myosin heavy chains (MyHC), atrophy, p-mTOR (Ser2484) and p-p70S6 kinase (Thr389) protein levels in the postural muscle (m. soleus) under conditions of functional unloading. Experiments were carried out on male Wistar rats (180−230 g). To reproduce muscle unloading, the hindlimb suspension (HS) model was used. We determined the signs of muscle atrophy (changes in muscle mass and the Feret’s diameter of muscle fibers), levels of ceramide (thin-layer chromatography) and mTOR signaling system proteins (Western blotting), as well as the expression of fast and slow MyHC isoforms (qPCR, immunofluorescence study). In suspended m. soleus, we found a decrease in p-mTOR (Ser2484) and p-p70S6k (Thr389) levels, which was prevented by the administration of an acid sphingomyelinase (aSMase) inhibitor clomipramine. Clomipramine was also found to abolish a muscle phenotype shift toward the expression of fast MyHC isoforms (MyHC IIB and MyHC IId/x) and to reduce the degree of muscle atrophy. These results indicate the role of the sphingolipid mechanisms in the disruption of mTOR signaling, dysregulation of the expression of fast MyHC isoforms, and development of muscle atrophy caused by functional unloading.

When B cells are exposed to antigens, they use their B cell receptors (BCRs) to transduce this external signal into internal signaling cascades and uptake antigen, which activate transcriptional programs. Signaling activation requires complex cytoskeletal remodeling initiated by BCR signaling. The actin cytoskeletal remodeling drives B cell morphological changes, such as spreading, protrusion, contraction, and endocytosis of antigen by mechanical forces, which in turn affect BCR signaling. Therefore, the relationship between the actin cytoskeleton and BCR signaling is a two-way feedback loop. These morphological changes represent the indirect ways by which the actin cytoskeleton regulates BCR signaling. Recent studies using high spatiotemporal resolution microscopy techniques have revealed that actin also can directly influence BCR signaling. Cortical actin networks directly affect BCR mobility, not only during the resting stage by serving as diffusion barriers, but also at its activation stage by altering BCR diffusivity through enhanced actin flow velocities. Furthermore, the actin cytoskeleton along with myosin enable B cells to sense the physical properties of its environment and generate and transmit forces through the BCR. Consequently, the actin cytoskeleton modulates the signaling threshold of BCR to antigenic stimulation. This review discusses the latest research on the relationship between BCR signaling and actin remodeling, and the research techniques. Exploration of the role of actin in BCR signaling will expand the fundamental understanding of the relationship between cell signaling and the cytoskeleton and the mechanisms underlying cytoskeleton related immune disorders and cancer.

Objective: Skeletal muscle satellite cells (SMSCs) are significant for the growth, regeneration, and maintenance of skeletal muscle after birth. However, currently, few studies have been performed on the isolation, culture and inducing differentiation of goose muscle satellite cells. Previous studies have shown that C1q and tumor necrosis factor-related protein 3 (CTRP3) participated in the process of muscle growth and development, but its role in the goose skeletal muscle development is not yet clear. This study aimed to isolate, culture, and identify the goose SMSCs in vitro. Additionally, to explore the function of CTRP3 in goose SMSCs.Methods: Goose SMSCs were isolated using 0.25% trypsin from leg muscle (LM) of 15 to 20 day fertilized goose eggs. Cell differentiation was induced by transferring the cells to differentiation medium with 2% horse serum and 1% penicillin streptomycin. Immunofluorescence staining of Desmin and Pax7 was used to identify goose SMSCs. Quantitative realtime polymerase chain reaction and western blot were applied to explore developmental expression profile of CTRP3 in LM and the regulation of CTRP3 on myosin heavy chains (MyHC), myogenin (MyoG) expression and Notch signaling pathway related genes expression.Results: The goose SMSCs were successfully isolated and cultured. The expression of Pax7 and Desmin were observed in the isolated cells. The expression of CTRP3 decreased significantly during leg muscle development. Overexpression of CTRP3 could enhance the expression of two myogenic differentiation marker genes, MyHC and MyoG. But knockdown of CTRP3 suppressed their expression. Furthermore, CTRP3 could repress the mRNA level of Notch signaling pathway-related genes, notch receptor 1, notch receptor 2 and hairy/enhancer-of-split related with YRPW motif 1, which previously showed a negative regulation in myoblast differentiation.Conclusion: These findings provide a useful cell model for the future research on goose muscle development and suggest that CTRP3 may play an essential role in skeletal muscle growth of goose.

Lysosomes are involved in nutrient sensing via the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 is tethered to lysosomes by the Ragulator complex, a heteropentamer in which Lamtor1 wraps around Lamtor2–5. Although the Ragulator complex is required for cell migration, the mechanisms by which it participates in cell motility remain unknown. Here, we show that lysosomes move to the uropod in motile cells, providing the platform where Lamtor1 interacts with the myosin phosphatase Rho-interacting protein (MPRIP) independently of mTORC1 and interferes with the interaction between MPRIP and MYPT1, a subunit of myosin light chain phosphatase (MLCP), thereby increasing myosin II–mediated actomyosin contraction. Additionally, formation of the complete Ragulator complex is required for leukocyte migration and pathophysiological immune responses. Together, our findings demonstrate that the lysosomal Ragulator complex plays an essential role in leukocyte migration by activating myosin II through interacting with MPRIP., Myosin II–mediated contractility is required for leukocyte migration. Here, authors show that lysosomes are involved in leukocyte migration by providing the platform where Ragulator complex interacts with the myosin phosphatase Rho-interacting protein (MPRIP) independently of mTORC1 and interferes with the interaction between MPRIP and a subunit of myosin light chain phosphatase (MLCP).