UBR2 targets myosin heavy chain IIb and IIx for degradation: Molecular mechanism essential for cancer-induced muscle wasting.

Cancer cachexia is a lethal metabolic syndrome featuring muscle wasting with preferential loss of fast-twitching muscle mass through an undefined mechanism. Here, we show that cancer induces muscle wasting by selectively degrading myosin heavy chain (MHC) subtypes IIb and IIx through E3 ligase UBR2-mediated ubiquitylation. Induction of MHC loss and atrophy in C2C12 myotubes and mouse tibialis anterior (TA) by murine cancer cells required UBR2 up-regulation by cancer. Genetic gain or loss of UBR2 function inversely altered MHC level and muscle mass in TA of tumor-free mice. UBR2 selectively interacted with and ubiquitylated MHC-IIb and MHC-IIx through its substrate recognition and catalytic domain, respectively, in C2C12 myotubes. Elevation of UBR2 in muscle of tumor-bearing or free mice caused loss of MHC-IIb and MHC-IIx but not MHC-I and MHC-IIa or other myofibrillar proteins, including α-actin, troponin, tropomyosin, and tropomodulin. Muscle-specific knockout of UBR2 spared KPC tumor-bearing mice from losing MHC-IIb and MHC-IIx, fast-twitching muscle mass, cross-sectional area, and contractile force. The rectus abdominis (RA) muscle of patients with cachexia-prone cancers displayed a selective reduction of MHC-IIx in correlation with higher UBR2 levels. These data suggest that UBR2 is a regulator of MHC-IIb/IIx essential for cancer-induced muscle wasting, and that therapeutic interventions can be designed by blocking UBR2 up-regulation by cancer.

Spindle cell neoplasms with novel LTK fusion - Expanding the spectrum of kinase fusion-positive soft tissue tumors.

AIMS: Kinase fusion-positive soft tissue tumors represent an emerging, molecularly defined group of mesenchymal tumors with a wide morphologic spectrum and diverse activating kinases. Here, we present two cases of soft tissue tumors with novel LTK fusions. METHODS AND RESULTS: Both cases presented as acral skin nodules (big toe and middle finger) in pediatric patients (17-year-old girl and 2-year-old boy). The tumors measured 2 and 3 cm in greatest dimension. Histologically, both cases exhibited bland-looking spindle cells infiltrating adipose tissue and accompanied by collagenous stroma. One case additionally displayed perivascular hyalinization and band-like stromal collagen. Both cases exhibited focal S100 staining, and one case had patchy coexpression of CD34. Targeted RNA-seq revealed the presence of novel in-frame MYH9::LTK and MYH10::LTK fusions, resulting in upregulation of LTK expression. Of interest, DNA methylation-based unsupervised clustering analysis in one case showed that the tumor clustered with dermatofibrosarcoma protuberans (DFSP). One tumor was excised with amputation with no local recurrence or distant metastasis at 18-month follow-up. The other case was initially marginally excised with local recurrence after one year, followed by wide local excision, with no evidence of disease at 10 years of follow-up. CONCLUSIONS: This is the first reported case series of soft tissue tumors harboring LTK fusion, expanding the molecular landscape of soft tissue tumors driven by activating kinase fusions. Furthermore, studies involving a larger number of cases and integrated genomic analyses will be warranted to fully elucidate the pathogenesis and classification of these tumors.

Alternative Splicing Mediated by RNA-Binding Protein RBM24 Facilitates Cardiac Myofibrillogenesis in a Differentiation Stage-Specific Manner.

BACKGROUND: Mutations in genes encoding sarcomeric proteins lead to failures in sarcomere assembly, the building blocks of contracting muscles, resulting in cardiomyopathies that are a leading cause of morbidity and mortality worldwide. Splicing variants of sarcomeric proteins are crucial at different stages of myofibrillogenesis, accounting for sarcomeric structural integrity. RBM24 (RNA-binding motif protein 24) is known as a tissue-specific splicing regulator that plays an essential role in cardiogenesis. However, it had been unclear if the developmental stage-specific alternative splicing facilitated by RBM24 contributes to sarcomere assembly and cardiogenesis. Our aim is to study the molecular mechanism by which RBM24 regulates cardiogenesis and sarcomere assembly in a temporal-dependent manner. METHODS: We ablated RBM24 from human embryonic stem cells (hESCs) using CRISPR/Cas9 techniques. RESULTS: Although RBM24-/- hESCs still differentiated into sarcomere-hosting cardiomyocytes, they exhibited disrupted sarcomeric structures with punctate Z-lines due to impaired myosin replacement during early myofibrillogenesis. Transcriptomics revealed >4000 genes regulated by RBM24. Among them, core myofibrillogenesis proteins (eg, ACTN2 [α-actinin 2], TTN [titin], and MYH10 [non-muscle myosin IIB]) were misspliced. Consequently, MYH6 (muscle myosin II) cannot replace nonmuscle myosin MYH10, leading to myofibrillogenesis arrest at the early premyofibril stage and causing disrupted sarcomeres. Intriguingly, we found that the ABD (actin-binding domain; encoded by exon 6) of the Z-line anchor protein ACTN2 is predominantly excluded from early cardiac differentiation, whereas it is consistently included in human adult heart. CRISPR/Cas9-mediated deletion of exon 6 from ACTN2 in hESCs, as well as forced expression of full-length ACTN2 in RBM24-/- hESCs, further corroborated that inclusion of exon 6 is critical for sarcomere assembly. Overall, we have demonstrated that RBM24-facilitated inclusion of exon 6 in ACTN2 at distinct stages of cardiac differentiation is evolutionarily conserved and crucial to sarcomere assembly and integrity. CONCLUSIONS: RBM24 acts as a master regulator to modulate the temporal dynamics of core myofibrillogenesis genes and thereby orchestrates sarcomere organization.

Analysis of Plasmodium falciparum myosin B ATPase activity and structure in complex with the calmodulin-like domain of its light chain MLC-B.

Myosin B (MyoB) is a class 14 myosin expressed in all invasive stages of the malaria parasite, Plasmodium falciparum. It is not associated with the glideosome complex that drives motility and invasion of host cells. During red blood cell invasion, MyoB remains at the apical tip of the merozoite but is no longer observed once invasion is completed. MyoB is not essential for parasite survival, but when it is knocked out, merozoites are delayed in the initial stages of red blood cell invasion, giving rise to a growth defect that correlates with reduced invasion success. Therefore, further characterization is needed to understand how MyoB contributes to parasite invasion. Here, we have expressed and purified functional MyoB with the help of parasite-specific chaperones Hsp90 and Unc45, characterized its binding to actin and its known light chain MLC-B using biochemical and biophysical methods and determined its low-resolution structure in solution using small angle X-ray scattering. In addition to MLC-B, we found that four other putative regulatory light chains bind to the MyoB IQ2 motif in vitro. The purified recombinant MyoB adopted the overall shape of a myosin, exhibited actin-activated ATPase activity, and moved actin filaments in vitro. Additionally, we determined that the ADP release rate was faster than the ATP turnover number, and thus, does not appear to be rate limiting. This, together with the observed high affinity to actin and the specific localization of MyoB, may point toward a role in tethering and/or force sensing during early stages of invasion.

α-Actinin-4 drives invasiveness by regulating myosin IIB expression and myosin IIA localization.

The mechanisms by which the mechanoresponsive actin crosslinking protein α-actinin-4 (ACTN4) regulates cell motility and invasiveness remain incompletely understood. Here, we show that, in addition to regulating protrusion dynamics and focal adhesion formation, ACTN4 transcriptionally regulates expression of non-muscle myosin IIB (NMM IIB; heavy chain encoded by MYH10), which is essential for mediating nuclear translocation during 3D invasion. We further show that an indirect association between ACTN4 and NMM IIA (heavy chain encoded by MYH9) mediated by a functional F-actin cytoskeleton is essential for retention of NMM IIA at the cell periphery and modulation of focal adhesion dynamics. A protrusion-dependent model of confined migration recapitulating experimental observations predicts a dependence of protrusion forces on the degree of confinement and on the ratio of nucleus to matrix stiffness. Together, our results suggest that ACTN4 is a master regulator of cancer invasion that regulates invasiveness by controlling NMM IIB expression and NMM IIA localization. This article has an associated First Person interview with the first author of the paper.

Mutations in non-muscle myosin 2A disrupt the actomyosin cytoskeleton in Sertoli cells and cause male infertility.

Mutations in non-muscle myosin 2A (NM2A) encompass a wide spectrum of anomalies collectively known as MYH9-Related Disease (MYH9-RD) in humans that can include macrothrombocytopenia, glomerulosclerosis, deafness, and cataracts. We previously created mouse models of the three mutations most frequently found in humans: R702C, D1424N, and E1841K. While homozygous R702C and D1424N mutations are embryonic lethal, we found homozygous mutant E1841K mice to be viable. However the homozygous male, but not female, mice were infertile. Here, we report that these mice have reduced testis size and defects in actin-associated junctions in Sertoli cells, resulting in inability to form the blood-testis barrier and premature germ cell loss. Moreover, compound double heterozygous (R702C/E1841K and D1424/E1841K) males show the same abnormalities in testes as E1841K homozygous males. Conditional ablation of either NM2A or NM2B alone in Sertoli cells has no effect on fertility and testis size, however deletion of both NM2A and NM2B in Sertoli cells results in infertility. Isolation of mutant E1841K Sertoli cells reveals decreased NM2A and F-actin colocalization and thicker NM2A filaments. Furthermore, AE1841K/AE1841K and double knockout Sertoli cells demonstrate microtubule disorganization and increased tubulin acetylation, suggesting defects in the microtubule cytoskeleton. Together, these results demonstrate that NM2A and 2B paralogs play redundant roles in Sertoli cells and are essential for testes development and normal fertility.

Heterozygous variants in MYH10 associated with neurodevelopmental disorders and congenital anomalies with evidence for primary cilia-dependent defects in Hedgehog signaling.

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.

Dysregulated myosin in Hermansky-Pudlak syndrome lung fibroblasts is associated with increased cell motility.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by improper biogenesis of lysosome-related organelles (LROs). Lung fibrosis is the leading cause of death among adults with HPS-1 and HPS-4 genetic types, which are associated with defects in the biogenesis of lysosome-related organelles complex-3 (BLOC-3), a guanine exchange factor (GEF) for a small GTPase, Rab32. LROs are not ubiquitously present in all cell types, and specific cells utilize LROs to accomplish dedicated functions. Fibroblasts are not known to contain LROs, and the function of BLOC-3 in fibroblasts is unclear. Here, we report that lung fibroblasts isolated from patients with HPS-1 have increased migration capacity. Silencing HPS-1 in normal lung fibroblasts similarly leads to increased migration. We also show that the increased migration is driven by elevated levels of Myosin IIB. Silencing HPS1 or RAB32 in normal lung fibroblasts leads to increased MYOSIN IIB levels. MYOSIN IIB is downstream of p38-MAPK, which is a known target of angiotensin receptor signaling. Treatment with losartan, an angiotensin receptor inhibitor, decreases MYOSIN IIB levels and impedes HPS lung fibroblast migration in vitro. Furthermore, pharmacologic inhibition of angiotensin receptor with losartan seemed to decrease migration of HPS lung fibroblasts in vivo in a zebrafish xenotransplantation model. Taken together, we demonstrate that BLOC-3 plays an important role in MYOSIN IIB regulation within lung fibroblasts and contributes to fibroblast migration.

Actin assembly and non-muscle myosin activity drive dendrite retraction in an UNC-6/Netrin dependent self-avoidance response.

Dendrite growth is constrained by a self-avoidance response that induces retraction but the downstream pathways that balance these opposing mechanisms are unknown. We have proposed that the diffusible cue UNC-6(Netrin) is captured by UNC-40(DCC) for a short-range interaction with UNC-5 to trigger self-avoidance in the C. elegans PVD neuron. Here we report that the actin-polymerizing proteins UNC-34(Ena/VASP), WSP-1(WASP), UNC-73(Trio), MIG-10(Lamellipodin) and the Arp2/3 complex effect dendrite retraction in the self-avoidance response mediated by UNC-6(Netrin). The paradoxical idea that actin polymerization results in shorter rather than longer dendrites is explained by our finding that NMY-1 (non-muscle myosin II) is necessary for retraction and could therefore mediate this effect in a contractile mechanism. Our results also show that dendrite length is determined by the antagonistic effects on the actin cytoskeleton of separate sets of effectors for retraction mediated by UNC-6(Netrin) versus outgrowth promoted by the DMA-1 receptor. Thus, our findings suggest that the dendrite length depends on an intrinsic mechanism that balances distinct modes of actin assembly for growth versus retraction.

MYH10 Governs Adipocyte Function and Adipogenesis through Its Interaction with GLUT4.

Adipogenesis is dependent on cytoskeletal remodeling that determines and maintains cellular shape and function. Cytoskeletal proteins contribute to the filament-based network responsible for controlling the shape of adipocytes and promoting the intracellular trafficking of cellular components. Currently, the understanding of these mechanisms and their effect on differentiation and adipocyte function remains incomplete. In this study, we identified the non-muscle myosin 10 (MYH10) as a novel regulator of adipogenesis and adipocyte function through its interaction with the insulin-dependent glucose transporter 4 (GLUT4). MYH10 depletion in preadipocytes resulted in impaired adipogenesis, with knockdown cells exhibiting an absence of morphological alteration and molecular signals. MYH10 was shown in a complex with GLUT4 in adipocytes, an interaction regulated by insulin induction. The missing adipogenic capacity of MYH10 knockdown cells was restored when the cells took up GLUT4 vesicles from neighbor wildtype cells in a co-culture system. This signaling cascade is regulated by the protein kinase C ζ (PKCζ), which interacts with MYH10 to modify the localization and interaction of both GLUT4 and MYH10 in adipocytes. Overall, our study establishes MYH10 as an essential regulator of GLUT4 translocation, affecting both adipogenesis and adipocyte function, highlighting its importance in future cytoskeleton-based studies in adipocytes.

Methamphetamine Learning Induces Persistent and Selective Nonmuscle Myosin II-Dependent Spine Motility in the Basolateral Amygdala.

Nonmuscle myosin II inhibition (NMIIi) in the basolateral amygdala (BLA), but not dorsal hippocampus (CA1), selectively disrupts memories associated with methamphetamine (METH) days after learning, without retrieval. However, the molecular mechanisms underlying this selective vulnerability remain poorly understood. A known function of NMII is to transiently activate synaptic actin dynamics with learning. Therefore, we hypothesized that METH-associated learning perpetuates NMII-driven actin dynamics in synapses, leading to an extended window of vulnerability for memory disruption. We used time-lapse two-photon imaging of dendritic spine motility in acutely prepared brain slices from female and male mice following METH-associated learning as a readout of actin-myosin dynamics. Spine motility was persistently increased in the BLA, but not in CA1. Consistent with the memory disrupting effect of intra-BLA NMII inhibition, METH-induced changes to BLA spine dynamics were reversed by a single systemic injection of an NMII inhibitor. Intra-CA1 NMII inhibition, on the other hand, did not disrupt METH-associated memory. Thus, we report identification of a previously unknown ability for spine actin dynamics to persist days after stimulation and that this is under the control of NMII. Further, these perpetual NMII-driven spine actin dynamics in BLA neurons may contribute to the unique susceptibility of METH-associated memories.SIGNIFICANCE STATEMENT There are no Food and Drug Administration-approved pharmacotherapies to prevent relapse to the use of stimulants, such as methamphetamine (METH). Environmental cues become associated with drug use, such that the memories can elicit strong motivation to seek the drug during abstinence. We previously reported that the storage of METH-associated memories is uniquely vulnerable to immediate, retrieval-independent, and lasting disruption by direct actin depolymerization or by inhibiting the actin driver nonmuscle myosin II (NMII) in the BLA or systemically. Here we report a potential structural mechanism responsible for the unique vulnerability of METH-associated memories and METH-seeking behavior to NMII inhibition within the BLA.

Down-regulation of MYH10 driven by chromosome 17p13.1 deletion promotes hepatocellular carcinoma metastasis through activation of the EGFR pathway.

Somatic copy number alterations (CNAs) are a genomic hallmark of cancers. Among them, the chromosome 17p13.1 deletions are recurrent in hepatocellular carcinoma (HCC). Here, utilizing an integrative omics analysis, we screened out a novel tumour suppressor gene within 17p13.1, myosin heavy chain 10 (MYH10). We observed frequent deletions (~38%) and significant down-regulation of MYH10 in primary HCC tissues. Deletion or decreased expression of MYH10 was a potential indicator of poor outcomes in HCC patients. Knockdown of MYH10 significantly promotes HCC cell migration and invasion in vitro, and overexpression of MYH10 exhibits opposite effects. Further, inhibition of MYH10 markedly potentiates HCC metastasis in vivo. We preliminarily elucidated the mechanism by which loss of MYH10 promotes HCC metastasis by facilitating EGFR pathway activation. In conclusion, our study suggests that MYH10, a candidate target gene for 17p13 deletion, acts as a tumour suppressor and may serve as a potential prognostic indicator for HCC patients.

Filamentous nestin and nonmuscle myosin IIB are associated with a migratory phenotype in neonatal rat cardiomyocytes.

Cardiomyocyte migration represents a requisite event of cardiogenesis and the regenerative response of the injured adult zebrafish and neonatal rodent heart. The present study tested the hypothesis that the appearance of the intermediate filament protein nestin in neonatal rat ventricular cardiomyocytes (NNVMs) was associated in part with the acquisition of a migratory phenotype. The cotreatment of NNVMs with phorbol 12,13-dibutyrate (PDBu) and the p38α/ß mitogen-activated protein kinase inhibitor SB203580 led to the de novo synthesis of nestin. The intermediate filament protein was subsequently reorganized into a filamentous pattern and redistributed to the leading edge of elongated membrane protrusions translating to significant lengthening of NNVMs. PDBu/SB203580 treatment concomitantly promoted the reorganization of nonmuscle myosin IIB (NMIIB) located predominantly at the periphery of the plasma membrane of NNVMs to a filamentous phenotype extending to the leading edge of elongated membrane protrusions. Coimmunoprecipitation assay revealed a physical interaction between NMIIB and nestin after PDBu/SB203580 treatment of NNVMs. In wild-type and heterozygous NMIIB embryonic hearts at E11.5-E14.5 days, nestin immunoreactivity was identified in a subpopulation of cardiomyocytes elongating perpendicular to the compact myocardium, at the leading edge of nascent trabeculae and during thickening of the compact myocardium. In mutant embryonic hearts lacking NMIIB protein expression, trabeculae formation was reduced, the compact myocardium significantly thinner and nestin immunoreactivity undetectable in cardiomyocytes at E14.5 days. These data suggest that NMIIB and nestin may act in a coordinated fashion to facilitate the acquisition of a migratory phenotype in neonatal and embryonic cardiomyocytes.

Comparative proteome analysis of form-deprivation myopia in sclera with iTRAQ-based quantitative proteomics.

Objective: Scleral remodeling plays a key role in axial elongation in myopia. The aim of the present study was to identify the proteomics changes and specific signaling networks to gain insight into the molecular basis of scleral remodeling in myopic eyes. Methods: Guinea pig form-deprivation myopia was induced with a translucent diffuser on a random eye for 4 weeks, while the other eye served as the contralateral control group. The axial length and refraction were measured at the beginning and end of the treatment. The proteins were extracted from the sclerae of each group and prepared for quantitative isobaric tags for relative and absolute quantification (iTRAQ) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The coexpression networks and protein functions were analyzed using Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA). Quantitative real-time PCR (qRT-PCR) and western blotting were performed to confirm the authenticity and accuracy of the iTRAQ results. Results: After 4 weeks, the form-deprivation eyes developed significant degrees of myopia, and the axial length increased statistically significantly (p<0.05). A total of 2,579 unique proteins with <1% false discovery rate (FDR) were identified. Furthermore, 56 proteins were found to be upregulated, and 84 proteins were found to be downregulated, with a threshold of a 1.2-fold change and p<0.05 in the myopia group, when compared to the control group. Further bioinformatics analysis indicated that 44 of 140 differentially expressed proteins were involved in cellular movement and cellular assembly and organization. The qRT-PCR or western blotting results confirmed that myosin IIB, ACTIN3, and cellular cytoskeletons were downregulated, while RhoA and RAP1A were upregulated in the sclera in myopic eyes. These results were consistent with the proteomics results. Conclusions: Proteomics and bioinformatics results can be helpful for identifying proteins and providing new insights for better understanding of the molecular mechanism underlying scleral remodeling. These results revealed that the proteins associated with cellular movement and cellular assembly and organization are altered during the development of myopia. Furthermore, RhoA plays a key role in the pathways involved in cellular movement and cellular assembly and organization.

Changes in the expression and functional activities of Myosin II isoforms in human hyperplastic prostate.

Benign prostatic hyperplasia (BPH) is a common disease among aging males with the etiology remaining unclear. We recently found myosin II was abundantly expressed in rat and cultured human prostate cells with permissive roles in the dynamic and static components. The present study aimed to explore the expression and functional activities of myosin II isoforms including smooth muscle (SM) myosin II (SMM II) and non-muscle myosin II (NMM II) in the hyperplastic prostate. Human prostate cell lines and tissues from normal human and BPH patients were used. Hematoxylin and Eosin (H&E), Masson's trichrome, immunohistochemical staining, in vitro organ bath, RT-polymerase chain reaction (PCR) and Western-blotting were performed. We further created cell models with NMM II isoforms silenced and proliferation, cycle, and apoptosis of prostate cells were determined by cell counting kit-8 (CCK-8) assay and flow cytometry. Hyperplastic prostate SM expressed more SM1 and LC17b isoforms compared with their alternatively spliced counterparts, favoring a slower more tonic-type contraction and greater force generation. For BPH group, blebbistatin (BLEB, a selective myosin II inhibitor), exhibited a stronger effect on relaxing phenylephrine (PE) pre-contracted prostate strips and inhibiting PE-induced contraction. Additionally, NMMHC-A and NMMHC-B were up-regulated in hyperplastic prostate with no change in NMMHC-C. Knockdown of NMMHC-A or NMMHC-B inhibited prostate cell proliferation and induced apoptosis, with no changes in cell cycle. Our novel data demonstrate that expression and functional activities of myosin II isoforms are altered in human hyperplastic prostate, suggesting a new pathological mechanism for BPH. Thus, the myosin II system may provide potential new therapeutic targets for BPH/lower urinary tract symptoms (LUTS).

Network-based analysis reveals novel gene signatures in the peripheral blood of patients with sporadic nonsyndromic thoracic aortic aneurysm.

Thoracic aortic aneurysm (TAA), a serious cardiovascular disease that causes morbidity and mortality worldwide. At present, few biomarkers can accurately diagnose the appearance of TAA before dissection or rupture. Our research has the intention to investigate the developing applicable biomarkers for TAA promising clinically diagnostic biomarkers or probable regulatory targets for TAA. In our research, we built correlation networks utilizing the expression profile of peripheral blood mononuclear cell obtained from a public microarray data set (GSE9106). Furthermore, we chose the turquoise module, which has the strongest significance with TAA and was further analyzed. Fourteen genes that overlapped with differentially expressed proteins in the medial aortic layer were obtained. Subsequently, we verified the results applying quantitative polymerase chain reaction (Q-PCR) to our clinical specimen. In general, the Q-PCR results coincide with the majority of the expression profile. Fascinatingly, a notable change occurred in CLU, DES, MYH10, and FBLN5. In summary, using weighted gene coexpression analysis, our study indicates that CLU, DES, MYH10, and FBLN5 were identified and validated to be related to TAA and might be candidate biomarkers or therapeutic targets for TAA.

Differential contributions of nonmuscle myosin IIA and IIB to cytokinesis in human immortalized fibroblasts.

Nonmuscle myosin II (NMII) plays an important role in cytokinesis by constricting a contractile ring. However, it is poorly understood how NMII isoforms contribute to cytokinesis in mammalian cells. Here, we investigated the roles of the two major NMII isoforms, NMIIA and NMIIB, in cytokinesis using a WI-38 VA13 cell line (human immortalized fibroblast). In this cell line, NMIIB tended to localize to the contractile ring more than NMIIA. The expression level of NMIIA affected the localization of NMIIB. Most NMIIB accumulated at the cleavage furrow in NMIIA-knockout (KO) cells, and most NMIIA was displaced from this location in exogenous NMIIB-expressing cells, indicating that NMIIB preferentially localizes to the contractile ring. Specific KO of each isoform elicited opposite effects. The rate of furrow ingression was decreased and increased in NMIIA-KO and NMIIB-KO cells, respectively. Meanwhile, the length of NMII-filament stacks in the contractile ring was increased and decreased in NMIIA-KO and NMIIB-KO cells, respectively. Moreover, NMIIA helped to maintain cortical stiffness during cytokinesis. These findings suggest that appropriate ratio of NMIIA and NMIIB in the contractile ring is important for proper cytokinesis in specific cell types. In addition, two-photon excitation spinning-disk confocal microscopy enabled us to image constriction of the contractile ring in live cells in a three-dimensional manner.

Non-muscle myosin IIB (Myh10) is required for epicardial function and coronary vessel formation during mammalian development.

The coronary vasculature is an essential vessel network providing the blood supply to the heart. Disruptions in coronary blood flow contribute to cardiac disease, a major cause of premature death worldwide. The generation of treatments for cardiovascular disease will be aided by a deeper understanding of the developmental processes that underpin coronary vessel formation. From an ENU mutagenesis screen, we have isolated a mouse mutant displaying embryonic hydrocephalus and cardiac defects (EHC). Positional cloning and candidate gene analysis revealed that the EHC phenotype results from a point mutation in a splice donor site of the Myh10 gene, which encodes NMHC IIB. Complementation testing confirmed that the Myh10 mutation causes the EHC phenotype. Characterisation of the EHC cardiac defects revealed abnormalities in myocardial development, consistent with observations from previously generated NMHC IIB null mouse lines. Analysis of the EHC mutant hearts also identified defects in the formation of the coronary vasculature. We attribute the coronary vessel abnormalities to defective epicardial cell function, as the EHC epicardium displays an abnormal cell morphology, reduced capacity to undergo epithelial-mesenchymal transition (EMT), and impaired migration of epicardial-derived cells (EPDCs) into the myocardium. Our studies on the EHC mutant demonstrate a requirement for NMHC IIB in epicardial function and coronary vessel formation, highlighting the importance of this protein in cardiac development and ultimately, embryonic survival.

[Overexpression of miR-130a-3p attenuates cardiomyocyte hypertrophy].

This study aimed to explore the role of miR-130a-3p in cardiomyocyte hypertrophy and its underlying mechanisms. Pressure-overload induced myocardial hypertrophy mice model was constructed by thoracic aortic constriction (TAC). In vitro, norepinephrine (NE) was used to stimulate neonatal rat cardiomyocytes (NRCMs) and H9c2 rat cardiomyocytes to induce hypertrophic phenotypes. The expression of miR-130a-3p was detected in mice hypertrophic myocardium, hypertrophic NRCMs and H9c2 cells. The mimics and inhibitors of miR-130a-3p were transfected into H9c2 cells to observe the role of miR-130a-3p on the hypertrophic phenotype change of cardiomyocytes separately. Furthermore, whether miR-130a-3p regulated hypertrophic related signaling pathways was explored. The results showed that the expression of miR-130a-3p was significantly decreased in hypertrophic myocardium, hypertrophic NRCMs and H9c2 cells. After transfection of miR-130a-3p mimics, the expression of hypertrophic marker genes, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and ß-myosin heavy chain (ß-MHC), and the cell surface area were notably down-regulated compared with the control group (mimics N.C. + NE group). But after transfection of miR-130a-3p inhibitor, the expression of ANP, BNP and ß-MHC in H9c2 cells increased significantly, and the cell area increased further. By Western blot, it was found that the protein phosphorylation level of Akt and mTOR were down-regulated after over-expression of miR-130a-3p. These results suggest that miR-130a-3p mimics may alleviate the degree of cardiomyocyte hypertrophy, meanwhile its inhibitor can further aggravate cardiomyocyte hypertrophy. Over-expression of miR-130a-3p may attenuate cardiomyocytes hypertrophy by affecting the Akt pathway.

Multiple S100 protein isoforms and C-terminal phosphorylation contribute to the paralog-selective regulation of nonmuscle myosin 2 filaments.

Nonmuscle myosin 2 (NM2) has three paralogs in mammals, NM2A, NM2B, and NM2C, which have both unique and overlapping functions in cell migration, formation of cell-cell adhesions, and cell polarity. Their assembly into homo- and heterotypic bipolar filaments in living cells is primarily regulated by phosphorylation of the N-terminally bound regulatory light chain. Here, we present evidence that the equilibrium between these filaments and single NM2A and NM2B molecules can be controlled via S100 calcium-binding protein interactions and phosphorylation at the C-terminal end of the heavy chains. Furthermore, we show that in addition to S100A4, other members of the S100 family can also mediate disassembly of homotypic NM2A filaments. Importantly, these proteins can selectively remove NM2A molecules from heterotypic filaments. We also found that tail phosphorylation (at Ser-1956 and Ser-1975) of NM2B by casein kinase 2, as well as phosphomimetic substitutions at sites targeted by protein kinase C (PKC) and transient receptor potential cation channel subfamily M member 7 (TRPM7), down-regulates filament assembly in an additive fashion. Tail phosphorylation of NM2A had a comparatively minor effect on filament stability. S100 binding and tail phosphorylation therefore preferentially disassemble NM2A and NM2B, respectively. These two distinct mechanisms are likely to contribute to the temporal and spatial sorting of the two NM2 paralogs within heterotypic filaments. The existence of multiple NM2A-depolymerizing S100 paralogs offers the potential for diverse regulatory inputs modulating NM2A filament disassembly in cells and provides functional redundancy under both physiological and pathological conditions.