Effects of specific disease mutations in non-muscle myosin 2A on its structure and function.

Non-muscle myosin 2A (NM2A), a widely expressed class 2 myosin, is important for organizing actin filaments in cells. It cycles between a compact inactive 10S state in which its regulatory light chain (RLC) is dephosphorylated and a filamentous state in which the myosin heads interact with actin, and the RLC is phosphorylated. Over 170 missense mutations in MYH9, the gene that encodes the NM2A heavy chain, have been described. These cause MYH9 disease, an autosomal-dominant disorder that leads to bleeding disorders, kidney disease, cataracts, and deafness. Approximately two-thirds of these mutations occur in the coiled-coil tail. These mutations could destabilize the 10S state and/or disrupt filament formation or both. To test this, we determined the effects of six specific mutations using multiple approaches, including circular dichroism to detect changes in secondary structure, negative stain electron microscopy to analyze 10S and filament formation in vitro, and imaging of GFP-NM2A in fixed and live cells to determine filament assembly and dynamics. Two mutations in D1424 (D1424G and D1424N) and V1516M strongly decrease 10S stability and have limited effects on filament formation in vitro. In contrast, mutations in D1447 and E1841K, decrease 10S stability less strongly but increase filament lengths in vitro. The dynamic behavior of all mutants was altered in cells. Thus, the positions of mutated residues and their roles in filament formation and 10S stabilization are key to understanding their contributions to NM2A in disease.

The biochemically defined super relaxed state of myosin-A paradox.

The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (Ki, 0.32 and 1.76 µM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.

Pseudorabies virus usurps non-muscle myosin heavy chain IIA to dampen viral DNA recognition by cGAS for antagonism of host antiviral innate immunity.

Alphaherpesvirus pseudorabies virus (PRV) causes severe economic losses to the global pig industry and has garnered increasing attention due to its broad host range including humans. PRV has developed a variety of strategies to antagonize host antiviral innate immunity. However, the underlying mechanisms have not been fully elucidated. In our previous work, we demonstrated that non-muscle myosin heavy chain IIA (NMHC-IIA), a multifunctional cytoskeleton protein, attenuates innate immune responses triggered by RNA viruses. In the current study, we reported a previously unrecognized role of NMHC-IIA in counteracting PRV-induced cyclic GMP-AMP synthase (cGAS)-dependent type I interferon (IFN-I) production. Mechanistically, PRV infection led to an elevation of NMHC-IIA, strengthening the interaction between poly (ADP-ribose) polymerase 1 (PARP1) and cGAS. This interaction impeded cGAS recognition of PRV DNA and hindered downstream signaling activation. Conversely, inhibition of NMHC-IIA by Blebbistatin triggered innate immune responses and enhanced resistance to PRV proliferation both in vitro and in vivo. Taken together, our findings unveil that PRV utilizes NMHC-IIA to antagonize host antiviral immune responses via impairing DNA sensing by cGAS. This in-depth understanding of PRV immunosuppression not only provides insights for potential PRV treatment strategies but also highlights NMHC-IIA as a versatile immunosuppressive regulator usurped by both DNA and RNA viruses. Consequently, NMHC-IIA holds promise as a target for the development of broad-spectrum antiviral drugs.IMPORTANCECyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis plays a vital role in counteracting alphaherpesvirus infections. Alphaherpesviruses exploit various strategies for antagonizing cGAS-STING-mediated antiviral immune responses. However, limited examples of pseudorabies virus (PRV)-caused immunosuppression have been documented. Our findings reveal a novel role of non-muscle myosin heavy chain IIA (NMHC-IIA) in suppressing PRV-triggered innate immune responses to facilitate viral propagation both in vitro and in vivo. In detail, NMHC-IIA recruits poly (ADP-ribose) polymerase 1 (PARP1) to augment its interaction with cGAS, which impairs cGAS recognition of PRV DNA. Building on our previous demonstration of NMHC-IIA's immunosuppressive role during RNA virus infections, these findings indicate that NMHC-IIA acts as a broad-spectrum suppressor of host antiviral innate immunity in response to both DNA and RNA viruses. Therefore, NMHC-IIA will be a promising target for the development of comprehensive antiviral strategies.

A brain specific alternatively spliced isoform of nonmuscle myosin IIA lacks its mechanoenzymatic activities.

Recent genomic studies reported that 90 to 95% of human genes can undergo alternative splicing, by which multiple isoforms of proteins are synthesized. However, the functional consequences of most of the isoforms are largely unknown. Here, we report a novel alternatively spliced isoform of nonmuscle myosin IIA (NM IIA), called NM IIA2, which is generated by the inclusion of 21 amino acids near the actin-binding region (loop 2) of the head domain of heavy chains. Expression of NM IIA2 is found exclusively in the brain tissue, where it reaches a maximum level at 24 h during the circadian rhythm. The actin-dependent Mg2+-ATPase activity and in vitro motility assays reveal that NM IIA2 lacks its motor activities but localizes with actin filaments in cells. Interestingly, NM IIA2 can also make heterofilaments with NM IIA0 (noninserted isoform of NM IIA) and can retard the in vitro motility of NM IIA, when the two are mixed. Altogether, our findings provide the functional importance of a previously unknown alternatively spliced isoform, NM IIA2, and its potential physiological role in regulating NM IIA activity in the brain.

Phosphorylation of myosin A regulates gliding motility and is essential for Plasmodium transmission.

Malaria-causing parasites rely on an actin-myosin-based motor for the invasion of different host cells and tissue traversal in mosquitoes and vertebrates. The unusual myosin A of Plasmodium spp. has a unique N-terminal extension, which is important for red blood cell invasion by P. falciparum merozoites in vitro and harbors a phosphorylation site at serine 19. Here, using the rodent-infecting P. berghei we show that phosphorylation of serine 19 increases ookinete but not sporozoite motility and is essential for efficient transmission of Plasmodium by mosquitoes as S19A mutants show defects in mosquito salivary gland entry. S19A along with E6R mutations slow ookinetes and salivary gland sporozoites in both 2D and 3D environments. In contrast to data from purified proteins, both E6R and S19D mutations lower force generation by sporozoites. Our data show that the phosphorylation cycle of S19 influences parasite migration and force generation and is critical for optimal migration of parasites during transmission from and to the mosquito.

Endothelial cell invasion is controlled by dactylopodia.

Sprouting angiogenesis is fundamental for development and contributes to cancer, diabetic retinopathy, and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on how tip cells invade into tissues. Here, we show that endothelial tip cells use dactylopodia as the main cellular protrusion for invasion into nonvascular extracellular matrix. We show that dactylopodia and filopodia protrusions are balanced by myosin IIA (NMIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of NMIIA promotes excessive dactylopodia formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents dactylopodia development and leads to excessive filopodia formation. We further show that NMIIA inhibits Rac1-dependent activation of Arp2/3 by regulating the maturation state of focal adhesions. Our discoveries establish a comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way toward strategies to block invasive tip cells during sprouting angiogenesis.

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.

Knocking down the expression of the molecular motors, myosin A, C and F genes in Toxoplasma gondii to decrease the parasite virulence.

Toxoplasmosis is a serious parasitic infection and novel therapeutic options are highly demanded to effectively eliminate it. In current study, Toxoplasma gondii myosin A, C and F genes were knocked down using small interference RNA (siRNA) method and the parasite survival and virulence was evaluated in vitro and in vivo. The parasites were transfected with specific siRNA, virtually designed for myosin mRNAs, and co-cultured with human foreskin fibroblasts. The transfection rate and the viability of the transfected parasites were measured using flow cytometry and methyl thiazole tetrazolium (MTT) assays, respectively. Finally, the survival of BALB/c mice infected with siRNAs-transfected T. gondii was assessed. It was demonstrated that a transfection rate of 75.4% existed for siRNAs, resulting in 70% (P = 0.032), 80.6% (P = 0.017) and 85.5% (P = 0.013) gene suppression for myosin A, C and F in affected parasites, respectively, which was subsequently confirmed by Western blot analysis. Moreover, lower parasite viability was observed in those with knocked down myosin C with 80% (P = 0.0001), followed by 86.15% (P = 0.004) for myosin F and 92.3% (P = 0.083) for myosin A. Considerably higher mouse survival (about 40 h) was, also, demonstrated in mice challenged with myosin siRNA-transfected T. gondii, in comparison with control group challenged with wild-type parasites. In conclusion, myosin proteins knock down proposes a promising therapeutic strategy to combat toxoplasmosis.

Microbial tryptophan metabolites regulate gut barrier function via the aryl hydrocarbon receptor.

Inflammatory bowel diseases (IBDs), including Crohn's disease and ulcerative colitis, are associated with dysbiosis of the gut microbiome. Emerging evidence suggests that small-molecule metabolites derived from bacterial breakdown of a variety of dietary nutrients confer a wide array of host benefits, including amelioration of inflammation in IBDs. Yet, in many cases, the molecular pathways targeted by these molecules remain unknown. Here, we describe roles for three metabolites-indole-3-ethanol, indole-3-pyruvate, and indole-3-aldehyde-which are derived from gut bacterial metabolism of the essential amino acid tryptophan, in regulating intestinal barrier function. We determined that these metabolites protect against increased gut permeability associated with a mouse model of colitis by maintaining the integrity of the apical junctional complex and its associated actin regulatory proteins, including myosin IIA and ezrin, and that these effects are dependent on the aryl hydrocarbon receptor. Our studies provide a deeper understanding of how gut microbial metabolites affect host defense mechanisms and identify candidate pathways for prophylactic and therapeutic treatments for IBDs.

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.

Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum.

All symptoms of malaria disease are associated with the asexual blood stages of development, involving cycles of red blood cell (RBC) invasion and egress by the Plasmodium spp. merozoite. Merozoite invasion is rapid and is actively powered by a parasite actomyosin motor. The current accepted model for actomyosin force generation envisages arrays of parasite myosins, pushing against short actin filaments connected to the external milieu that drive the merozoite forwards into the RBC. In Plasmodium falciparum, the most virulent human malaria species, Myosin A (PfMyoA) is critical for parasite replication. However, the precise function of PfMyoA in invasion, its regulation, the role of other myosins and overall energetics of invasion remain unclear. Here, we developed a conditional mutagenesis strategy combined with live video microscopy to probe PfMyoA function and that of the auxiliary motor PfMyoB in invasion. By imaging conditional mutants with increasing defects in force production, based on disruption to a key PfMyoA phospho-regulation site, the absence of the PfMyoA essential light chain, or complete motor absence, we define three distinct stages of incomplete RBC invasion. These three defects reveal three energetic barriers to successful entry: RBC deformation (pre-entry), mid-invasion initiation, and completion of internalisation, each requiring an active parasite motor. In defining distinct energetic barriers to invasion, these data illuminate the mechanical challenges faced in this remarkable process of protozoan parasitism, highlighting distinct myosin functions and identifying potential targets for preventing malaria pathogenesis.

Confinement-optimized three-dimensional T cell amoeboid motility is modulated via myosin IIA-regulated adhesions.

During trafficking through tissues, T cells fine-tune their motility to balance the extent and duration of cell-surface contacts versus the need to traverse an entire organ. Here we show that in vivo, myosin IIA-deficient T cells had a triad of defects, including overadherence to high-endothelial venules, less interstitial migration and inefficient completion of recirculation through lymph nodes. Spatiotemporal analysis of three-dimensional motility in microchannels showed that the degree of confinement and myosin IIA function, rather than integrin adhesion (as proposed by the haptokinetic model), optimized motility rate. This motility occurred via a myosin IIA-dependent rapid 'walking' mode with multiple small and simultaneous adhesions to the substrate, which prevented spurious and prolonged adhesions. Adhesion discrimination provided by myosin IIA is thus necessary for the optimization of motility through complex tissues.

Megakaryocyte migration defects due to nonmuscle myosin IIA mutations underlie thrombocytopenia in MYH9-related disease.

Megakaryocytes (MKs), the precursor cells for platelets, migrate from the endosteal niche of the bone marrow (BM) toward the vasculature, extending proplatelets into sinusoids, where circulating blood progressively fragments them into platelets. Nonmuscle myosin IIA (NMIIA) heavy chain gene (MYH9) mutations cause macrothrombocytopenia characterized by fewer platelets with larger sizes leading to clotting disorders termed myosin-9-related disorders (MYH9-RDs). MYH9-RD patient MKs have proplatelets with thicker and fewer branches that produce fewer and larger proplatelets, which is phenocopied in mouse Myh9-RD models. Defective proplatelet formation is considered to be the principal mechanism underlying the macrothrombocytopenia phenotype. However, MYH9-RD patient MKs may have other defects, as NMII interactions with actin filaments regulate physiological processes such as chemotaxis, cell migration, and adhesion. How MYH9-RD mutations affect MK migration and adhesion in BM or NMIIA activity and assembly prior to proplatelet production remain unanswered. NMIIA is the only NMII isoform expressed in mature MKs, permitting exploration of these questions without complicating effects of other NMII isoforms. Using mouse models of MYH9-RD (NMIIAR702C+/-GFP+/-, NMIIAD1424N+/-, and NMIIAE1841K+/-) and in vitro assays, we investigated MK distribution in BM, chemotaxis toward stromal-derived factor 1, NMIIA activity, and bipolar filament assembly. Results indicate that different MYH9-RD mutations suppressed MK migration in the BM without compromising bipolar filament formation but led to divergent adhesion phenotypes and NMIIA contractile activities depending on the mutation. We conclude that MYH9-RD mutations impair MK chemotaxis by multiple mechanisms to disrupt migration toward the vasculature, impairing proplatelet release and causing macrothrombocytopenia.

HBx-upregulated MAFG-AS1 promotes cell proliferation and migration of hepatoma cells by enhancing MAFG expression and stabilizing nonmuscle myosin IIA.

To identify hepatitis B virus (HBV)-related lncRNA(s), we previously examined the transcription profiles of the HBV-transgenic cell line HepG2-4D14 and parental HepG2 cells by RNA deep sequencing and identified 38 upregulated long noncoding RNAs (lncRNAs). In the present study, the lncRNA MAFG-AS1 is investigated in detail because its gene is located adjacent to the MAFG gene, which is an important transcription factor involved in cell proliferation. The level of MAFG-AS1 is significantly higher in HCC tissue than in nontumor tissues. TCGA data show that the expression level of MAFG-AS1 is negatively correlated with survival of HCC patients. GEO cohort data show that compared with healthy tissues, the expression level of MAFG-AS1 is significantly higher in HBV-infected liver tissues. Real-time PCR and luciferase reporter assay data show that HBx can enhance the transcription of MAFG-AS1. Gain-of-function and loss-of-function experiments indicate that MAFG-AS1 promotes proliferation, migration, and invasion of HCC cells. Tumor formation assay results demonstrate that knockdown of MAFG-AS1 significantly inhibits cell proliferation in nude mice. Furthermore, MAFG-AS1 enhances the transcription of adjacent MAFG via E2F1. Additionally, MAFG-AS1 interacts with three subunits (MYH9, MYL12B, and MYL6) of nonmuscle myosin IIA (NM IIA). Knockdown of MAFG-AS1 inhibits ATPase activity of MYH9, interaction of NM IIA subunits, and cell cycle progression. Thus, the lncRNA MAFG-AS1 is upregulated by HBV and promotes proliferation and migration of HCC cells. Our findings suggest that MAFG-AS1 is a potential oncogene that may contribute to HBV-related HCC development.

A functional role of S100A4/non-muscle myosin IIA axis for pro-tumorigenic vascular functions in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most aggressive form of brain tumor and has vascular-rich features. The S100A4/non-muscle myosin IIA (NMIIA) axis contributes to aggressive phenotypes in a variety of human malignancies, but little is known about its involvement in GBM tumorigenesis. Herein, we examined the role of the S100A4/NMIIA axis during tumor progression and vasculogenesis in GBM. METHODS: We performed immunohistochemistry for S100A4, NMIIA, and two hypoxic markers, hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase 9 (CA9), in samples from 94 GBM cases. The functional impact of S100A4 knockdown and hypoxia were also assessed using a GBM cell line. RESULTS: In clinical GBM samples, overexpression of S100A4 and NMIIA was observed in both non-pseudopalisading (Ps) and Ps (-associated) perinecrotic lesions, consistent with stabilization of HIF-1α and CA9. CD34(+) microvascular densities (MVDs) and the interaction of S100A4 and NMIIA were significantly higher in non-Ps perinecrotic lesions compared to those in Ps perinecrotic areas. In non-Ps perinecrotic lesions, S100A4(+)/HIF-1α(-) GBM cells were recruited to the surface of preexisting host vessels in the vascular-rich areas. Elevated vascular endothelial growth factor A (VEGFA) mRNA expression was found in S100A4(+)/HIF-1α(+) GBM cells adjacent to the vascular-rich areas. In addition, GBM patients with high S100A4 protein expression had significantly worse OS and PFS than did patients with low S100A4 expression. Knockdown of S100A4 in the GBM cell line KS-1 decreased migration capability, concomitant with decreased Slug expression; the opposite effects were elicited by blebbistatin-dependent inhibition of NMIIA. CONCLUSION: S100A4(+)/HIF-1α(-) GBM cells are recruited to (and migrate along) preexisting vessels through inhibition of NMIIA activity. This is likely stimulated by extracellular VEGF that is released by S100A4(+)/HIF-1α(+) tumor cells in non-Ps perinecrotic lesions. In turn, these events engender tumor progression via acceleration of pro-tumorigenic vascular functions. Video abstract.

Distinct roles of two myosins in C. elegans spermatid differentiation.

During spermatogenesis, interconnected haploid spermatids segregate undesired cellular contents into residual bodies (RBs) before detaching from RBs. It is unclear how this differentiation process is controlled to produce individual spermatids or motile spermatozoa. Here, we developed a live imaging system to visualize and investigate this process in C. elegans. We found that non-muscle myosin 2 (NMY-2)/myosin II drives incomplete cytokinesis to generate connected haploid spermatids, which are then polarized to segregate undesired cellular contents into RBs under the control of myosin II and myosin VI. NMY-2/myosin II extends from the pseudo-cleavage furrow formed between two haploid spermatids to the spermatid poles, thus promoting RB expansion. In the meantime, defective spermatogenesis 15 (SPE-15)/myosin VI migrates from spermatids towards the expanding RB to promote spermatid budding. Loss of myosin II or myosin VI causes distinct cytoplasm segregation defects, while loss of both myosins completely blocks RB formation. We found that the final separation of spermatids from RBs is achieved through myosin VI-mediated cytokinesis, while myosin II is dispensable at this step. SPE-15/myosin VI and F-actin form a detergent-resistant actomyosin VI ring that undergoes continuous contraction to promote membrane constriction between spermatid and RB. We further identified that RGS-GAIP-interacting protein C terminus (GIPC)-1 and GIPC-2 cooperate with myosin VI to regulate contractile ring formation and spermatid release. Our study reveals distinct roles of myosin II and myosin VI in spermatid differentiation and uncovers a novel myosin VI-mediated cytokinesis process that controls spermatid release.

Dominantly inherited myosin IIa myopathy caused by aberrant splicing of MYH2.

BACKGROUND: Myosin heavy chain (MyHC) isoforms define the three major muscle fiber types in human extremity muscles. Slow beta/cardiac MyHC (MYH7) is expressed in type 1 muscle fibers. MyHC IIa (MYH2) and MyHC IIx (MYH1) are expressed in type 2A and 2B fibers, respectively. Whereas recessive MyHC IIa myopathy has been described in many cases, myopathy caused by dominant MYH2 variants is rare and has been described with clinical manifestations and muscle pathology in only one family and two sporadic cases. METHODS: We investigated three patients from one family with a dominantly inherited myopathy by clinical investigation, whole-genome sequencing, muscle biopsy, and magnetic resonance imaging (MRI). RESULTS: Three siblings, one woman and two men now 54, 56 and 66 years old, had experienced muscle weakness initially affecting the lower limbs from young adulthood. They have now generalized proximal muscle weakness affecting ambulation, but no ophthalmoplegia. Whole-genome sequencing identified a heterozygous MYH2 variant, segregating with the disease in the three affected individuals: c.5673 + 1G > C. Analysis of cDNA confirmed the predicted splicing defect with skipping of exon 39 and loss of residues 1860-1891 in the distal tail of the MyHC IIa, largely overlapping with the filament assembly region (aa1877-1905). Muscle biopsy in two of the affected individuals showed prominent type 1 muscle fiber predominance with only a few very small, scattered type 2A fibers and no type 2B fibers. The small type 2A fibers were frequently hybrid fibers with either slow MyHC or embryonic MyHC expression. The type 1 fibers showed variation in fiber size, internal nuclei and some structural alterations. There was fatty infiltration, which was also demonstrated by MRI. CONCLUSION: Dominantly inherited MyHC IIa myopathy due to a splice defect causing loss of amino acids 1860-1891 in the distal tail of the MyHC IIa protein including part of the assembly competence domain. The myopathy is manifesting with slowly progressive muscle weakness without overt ophthalmoplegia and markedly reduced number and size of type 2 fibers.

Myosin IIA suppresses glioblastoma development in a mechanically sensitive manner.

The ability of glioblastoma to disperse through the brain contributes to its lethality, and blocking this behavior has been an appealing therapeutic approach. Although a number of proinvasive signaling pathways are active in glioblastoma, many are redundant, so targeting one can be overcome by activating another. However, these pathways converge on nonredundant components of the cytoskeleton, and we have shown that inhibiting one of these-the myosin II family of cytoskeletal motors-blocks glioblastoma invasion even with simultaneous activation of multiple upstream promigratory pathways. Myosin IIA and IIB are the most prevalent isoforms of myosin II in glioblastoma, and we now show that codeleting these myosins markedly impairs tumorigenesis and significantly prolongs survival in a rodent model of this disease. However, while targeting just myosin IIA also impairs tumor invasion, it surprisingly increases tumor proliferation in a manner that depends on environmental mechanics. On soft surfaces myosin IIA deletion enhances ERK1/2 activity, while on stiff surfaces it enhances the activity of NFκB, not only in glioblastoma but in triple-negative breast carcinoma and normal keratinocytes as well. We conclude myosin IIA suppresses tumorigenesis in at least two ways that are modulated by the mechanics of the tumor and its stroma. Our results also suggest that inhibiting tumor invasion can enhance tumor proliferation and that effective therapy requires targeting cellular components that drive both proliferation and invasion simultaneously.

Colocation of Tpm3.1 and myosin IIa heads defines a discrete subdomain in stress fibres.

Co-polymers of tropomyosin and actin make up a major fraction of the actin cytoskeleton. Tropomyosin isoforms determine the function of an actin filament by selectively enhancing or inhibiting the association of other actin binding proteins, altering the stability of an actin filament and regulating myosin activity in an isoform-specific manner. Previous work has implicated specific roles for at least five different tropomyosin isoforms in stress fibres, as depletion of any of these five isoforms results in a loss of stress fibres. Despite this, most models of stress fibres continue to exclude tropomyosins. In this study, we investigate tropomyosin organisation in stress fibres by using super-resolution light microscopy and electron microscopy with genetically tagged, endogenous tropomyosin. We show that tropomyosin isoforms are organised in subdomains within the overall domain of stress fibres. The isoforms Tpm3.1 and 3.2 (hereafter Tpm3.1/3.2, encoded by TPM3) colocalise with non-muscle myosin IIa and IIb heads, and are in register, but do not overlap, with non-muscle myosin IIa and IIb tails. Furthermore, perturbation of Tpm3.1/3.2 results in decreased myosin IIa in stress fibres, which is consistent with a role for Tpm3.1 in maintaining myosin IIa localisation in stress fibres.

Non-cell-autonomous migration of RasV12-transformed cells towards the basal side of surrounding normal cells.

Oncogenic transformation enables cells to behave differently from their neighboring normal cells. Both cancer and normal cells recognize each other, often promoting the extrusion of the former from the epithelial cell layer. Here, we show that RasV12-transformed normal rat kidney 52E (NRK-52E) cells are extruded towards the basal side of the surrounding normal cells, which is concomitant with enhanced motility. The active migration of the basally extruded RasV12 cells is observed when surrounded by normal cells, indicating a non-cell-autonomous mechanism. Furthermore, specific inhibitor treatment and knockdown experiments elucidate the roles of PI3K and myosin IIA in the basal extrusion of Ras cells. Our findings reveal a new aspect of cancer cell invasion mediated by functional interactions with surrounding non-transformed cells.