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.

Mechanotransduction in response to ECM stiffening impairs cGAS immune signaling in tumor cells.

The tumor microenvironment (TME) plays decisive roles in disabling T cell-mediated antitumor immunity, but the immunoregulatory functions of its biophysical properties remain elusive. Extracellular matrix (ECM) stiffening is a hallmark of solid tumors. Here, we report that the stiffened ECM contributes to the immunosuppression in TME via activating the Rho-associated coiled-coil-containing protein kinase (ROCK)-myosin IIA-filamentous actin (F-actin) mechanosignaling pathway in tumor cells to promote the generation of TRIM14-scavenging nonmuscle myosin heavy chain IIA (NMHC-IIA)-F-actin stress fibers, thus accelerating the autophagic degradation of cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) to deprive tumor cyclic GMP-AMP (cGAMP) and further attenuating tumor immunogenicity. Pharmacological inhibition of myosin IIA effector molecules with blebbistatin (BLEB) or the RhoA upstream regulator of this pathway with simvastatin (SIM) restored tumor-intrinsic cGAS-mediated cGAMP production and enhanced antitumor immunity. Our work identifies that ECM stiffness is an important biophysical cue to regulate tumor immunogenicity via the ROCK-myosin IIA-F-actin axis and that inhibiting this mechanosignaling pathway could boost immunotherapeutic efficacy for effective solid tumor treatment.

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.

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.

Nanoparticle Spikes Enhance Cellular Uptake via Regulating Myosin IIA Recruitment.

Spike-like nanostructures are omnipresent in natural and artificial systems. Although biorecognition of nanostructures to cellular receptors has been indicated as the primary factor for virus infection pathways, how the spiky morphology of DNA-modified nanoparticles affects their cellular uptake and intracellular fate remains to be explored. Here, we design dually emissive gold nanoparticles with varied spikiness (from 0 to 2) to probe the interactions of spiky nanoparticles with cells. We discovered that nanospikes at the nanoparticle regulated myosin IIA recruitment at the cell membrane during cellular uptake, thereby enhancing cellular uptake efficiency, as revealed by dual-modality (plasmonic and fluorescence) imaging. Furthermore, the spiky nanoparticles also exhibited facilitated endocytosis dynamics, as revealed by real-time dark-field microscopy (DFM) imaging and colorimetry-based classification algorithms. These findings highlight the crucial role of the spiky morphology in regulating the intracellular fate of nanoparticles, which may shed light on engineering theranostic nanocarriers.

Nonmuscle myosin IIB is a driver of cellular reprogramming.

Nonmuscle myosin IIB (NMIIB) is considered a primary force generator during cell motility. Yet many cell types, including motile cells, do not necessarily express NMIIB. Given the potential of cell engineering for the next wave of technologies, adding back NMIIB could be a strategy for creating supercells with strategically altered cell morphology and motility. However, we wondered what unforeseen consequences could arise from such an approach. Here, we leveraged pancreatic cancer cells, which do not express NMIIB. We generated a series of cells where we added back NMIIB and strategic mutants that increase the ADP-bound time or alter the phosphorylation control of bipolar filament assembly. We characterized the cellular phenotypes and conducted RNA-seq analysis. The addition of NMIIB and the different mutants all have specific consequences for cell morphology, metabolism, cortical tension, mechanoresponsiveness, and gene expression. Major modes of ATP production are shifted, including alterations in spare respiratory capacity and the dependence on glycolysis or oxidative phosphorylation. Several metabolic and growth pathways undergo significant changes in gene expression. This work demonstrates that NMIIB is highly integrated with many cellular systems and simple cell engineering has a profound impact that extends beyond the primary contractile activity presumably being added to the cells.

Nonmuscle myosin IIA promotes the internalization of influenza A virus and regulates viral polymerase activity through interacting with nucleoprotein in human pulmonary cells.

Influenza A virus (IAV), responsible for seasonal epidemics and recurring pandemics, represents a global threat to public health. Given the risk of a potential IAV pandemic, it is increasingly important to better understand virus-host interactions and develop new anti-viral strategies. Here, we reported nonmuscle myosin IIA (MYH9)-mediated regulation of IAV infection. MYH9 depletion caused a profound inhibition of IAV infection by reducing viral attachment and internalization in human lung epithelial cells. Surprisingly, overexpression of MYH9 also led to a significant reduction in viral productive infection. Interestingly, overexpression of MYH9 retained viral attachment, internalization, or uncoating, but suppressed the viral ribonucleoprotein (vRNP) activity in a minigenome system. Further analyses found that excess MYH9 might interrupt the formation of vRNP by interacting with the viral nucleoprotein (NP) and result in the reduction of the completed vRNP in the nucleus, thereby inhibiting subsequent viral RNA transcription and replication. Together, we discovered that MYH9 can interact with IAV NP protein and engage in the regulation of vRNP complexes, thereby involving viral replication. These findings enlighten new mechanistic insights into the complicated interface of host-IAV interactions, ultimately making it an attractive target for the generation of antiviral drugs.

Helicobacter pylori-infected human neutrophils exhibit impaired chemotaxis and a uropod retraction defect.

Helicobacter pylori is a major human pathogen that colonizes the gastric mucosa and plays a causative role in development of peptic ulcers and gastric cancer. Neutrophils are heavily infected with this organism in vivo and play a prominent role in tissue destruction and disease. Recently, we demonstrated that H. pylori exploits neutrophil plasticity as part of its virulence strategy eliciting N1-like subtype differentiation that is notable for profound nuclear hypersegmentation. We undertook this study to test the hypothesis that hypersegmentation may enhance neutrophil migratory capacity. However, EZ-TAXIScan™ video imaging revealed a previously unappreciated and progressive chemotaxis defect that was apparent prior to hypersegmentation onset. Cell speed and directionality were significantly impaired to fMLF as well as C5a and IL-8. Infected cells oriented normally in chemotactic gradients, but speed and direction were impaired because of a uropod retraction defect that led to cell elongation, nuclear lobe trapping in the contracted rear and progressive narrowing of the leading edge. In contrast, chemotactic receptor abundance, adhesion, phagocytosis and other aspects of cell function were unchanged. At the molecular level, H. pylori phenocopied the effects of Blebbistatin as indicated by aberrant accumulation of F-actin and actin spikes at the uropod together with enhanced ROCKII-mediated phosphorylation of myosin IIA regulatory light chains at S19. At the same time, RhoA and ROCKII disappeared from the cell rear and accumulated at the leading edge whereas myosin IIA was enriched at both cell poles. These data suggest that H. pylori inhibits the dynamic changes in myosin IIA contractility and front-to-back polarity that are essential for chemotaxis. Taken together, our data advance understanding of PMN plasticity and H. pylori pathogenesis.

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.

Complementary crosstalk between palmitoylation and phosphorylation events in MTIP regulates its role during Plasmodium falciparum invasion.

Post-translational modifications (PTMs) including phosphorylation and palmitoylation have emerged as crucial biomolecular events that govern many cellular processes including functioning of motility- and invasion-associated proteins during Plasmodium falciparum invasion. However, no study has ever focused on understanding the possibility of a crosstalk between these two molecular events and its direct impact on preinvasion- and invasion-associated protein-protein interaction (PPI) network-based molecular machinery. Here, we used an integrated in silico analysis to enrich two different catalogues of proteins: (i) the first group defines the cumulative pool of phosphorylated and palmitoylated proteins, and (ii) the second group represents a common set of proteins predicted to have both phosphorylation and palmitoylation. Subsequent PPI analysis identified an important protein cluster comprising myosin A tail interacting protein (MTIP) as one of the hub proteins of the glideosome motor complex in P. falciparum, predicted to have dual modification with the possibility of a crosstalk between the same. Our findings suggested that blocking palmitoylation led to reduced phosphorylation and blocking phosphorylation led to abrogated palmitoylation of MTIP. As a result of the crosstalk between these biomolecular events, MTIP's interaction with myosin A was found to be abrogated. Next, the crosstalk between phosphorylation and palmitoylation was confirmed at a global proteome level by click chemistry and the phenotypic effect of this crosstalk was observed via synergistic inhibition in P. falciparum invasion using checkerboard assay and isobologram method. Overall, our findings revealed, for the first time, an interdependence between two PTM types, their possible crosstalk, and its direct impact on MTIP-mediated invasion via glideosome assembly protein myosin A in P. falciparum. These insights can be exploited for futuristic drug discovery platforms targeting parasite molecular machinery for developing novel antimalarial therapeutics.

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.

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.

Nonmuscle myosin IIA dynamically guides regulatory light chain phosphorylation and assembly of nonmuscle myosin IIB.

Nonmuscle myosin II minifilaments have emerged as central elements for force generation and mechanosensing by mammalian cells. Each minifilament can have a different composition and activity due to the existence of the three nonmuscle myosin II paralogs A, B and C and their respective phosphorylation pattern. We have used CRISPR/Cas9-based knockout cells, quantitative image analysis and mathematical modeling to dissect the dynamic processes that control the formation and activity of heterotypic minifilaments and found a strong asymmetry between paralogs A and B. Loss of NM IIA completely abrogates regulatory light chain phosphorylation and reduces the level of assembled NM IIB. Activated NM IIB preferentially co-localizes with pre-formed NM IIA minifilaments and stabilizes the filament in a force-dependent mechanism. NM IIC is only weakly coupled to these processes. We conclude that NM IIA and B play clearly defined complementary roles during assembly of functional minifilaments. NM IIA is responsible for the formation of nascent pioneer minifilaments. NM IIB incorporates into these and acts as a clutch that limits the force output to prevent excessive NM IIA activity. Together these two paralogs form a balanced system for regulated force generation.

MYH9 binds to dNTPs via deoxyribose moiety and plays an important role in DNA synthesis.

The accepted notion of dNTP transport following cytoplasmic biosynthesis is 'facilitated diffusion'; however, whether this alone is sufficient for moving dNTPs for DNA synthesis remains an open question. The data presented here show that the MYH9 gene encoded heavy chain of non-muscle myosin IIA binds dNTPs potentially serving as a 'reservoir'. Pull-down assays showed that MYH9 present in the cytoplasmic, mitochondrial and nuclear compartments bind to DNA and this interaction is inhibited by dNTPs and 2-deoxyribose-5-phosphate (dRP) suggesting that MYH9-DNA binding is mediated via pentose sugar recognition. Direct dNTP-MYH9 binding was demonstrated by ELISA and a novel PCR-based method, which showed that all dNTPs bind to MYH9 with varying efficiencies. Cellular thermal shift assays showed that MYH9 thermal stability is enhanced by dNTPs. MYH9 siRNA transfection or treatment with myosin II selective inhibitors ML7 or blebbistatin decreased cell proliferation compared to controls. EdU labeling and cell cycle analysis by flow cytometry confirmed MYH9 siRNA and myosin II inhibitors decreased progression to S-phase with accumulation of cells in G0/G1 phase. Taken together, our data suggest a novel role for MYH9 in dNTP binding and DNA synthesis.

Competing Roles of Ca2+ and Nonmuscle Myosin IIA on the Dynamics of the Metastasis-Associated Protein S100A4.

The calcium-binding protein S100A4 plays an important role in a wide range of biological processes such as cell motility, invasion, angiogenesis, survival, differentiation, contractility, and tumor metastasis and interacts with a range of partners. To understand the functional roles and interplay of S100A4 binding partners such as Ca2+ and nonmuscle myosin IIA (NMIIA), we used molecular dynamics simulations to investigate apo S100A4 and four holo S100A4 structures: S100A4 bound to Ca2+, S100A4 bound to NMIIA, S100A4 bound to Ca2+ and NMIIA, and a mutated S100A4 bound to Ca2+ and NMIIA. Our results show that two competing factors, namely, Ca2+-induced activation and NMIIA-induced inhibition, modulate the dynamics of S100A4 in a competitive manner. Moreover, Ca2+ binding results in enhanced dynamics, regulating the interactions of S100A4 with NMIIA, while NMIIA induces asymmetric dynamics between the chains of S100A4. The results also show that in the absence of Ca2+ the S100A4-NMIIA interaction is weak compared to that of between S100A4 bound to Ca2+ and NMIIA, which may offer a quick response to dropping calcium levels. In addition, certain mutations are shown to play a marked role on the dynamics of S100A4. The results described here contribute to understanding the interactions of S100A4 with NMIIA and the functional roles of Ca2+, NMIIA, and certain mutations on the dynamics of S100A4. The results of this study could be interesting for the development of inhibitors that exploit the shift of balance between the competing roles of Ca2+ and NMIIA.

Distinct roles of nonmuscle myosin II isoforms for establishing tension and elasticity during cell morphodynamics.

Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogenously coated surfaces, in collagen gels, and on micropatterned substrates. In contrast to homogenously coated surfaces, a structured environment supports a cellular phenotype with invaginated actin arcs even in the absence of NM IIA-induced contractility. A quantitative shape analysis of cells on micropatterns combined with a scale-bridging mathematical model reveals that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. A dynamic cell stretch/release experiment in a three-dimensional scaffold confirms these conclusions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis.

Mechanical and Thermodynamic Properties of Non-Muscle Contractile Tissues: The Myofibroblast and the Molecular Motor Non-Muscle Myosin Type IIA.

Myofibroblasts are contractile cells found in multiple tissues. They are physiological cells as in the human placenta and can be obtained from bone marrow mesenchymal stem cells after differentiation by transforming growth factor-ß (TGF-ß). They are also found in the stroma of cancerous tissues and can be located in non-muscle contractile tissues. When stimulated by an electric current or after exposure to KCl, these tissues contract. They relax either by lowering the intracellular Ca2+ concentration (by means of isosorbide dinitrate or sildenafil) or by inhibiting actin-myosin interactions (by means of 2,3-butanedione monoxime or blebbistatin). Their shortening velocity and their developed tension are dramatically low compared to those of muscles. Like sarcomeric and smooth muscles, they obey Frank-Starling's law and exhibit the Hill hyperbolic tension-velocity relationship. The molecular motor of the myofibroblast is the non-muscle myosin type IIA (NMIIA). Its essential characteristic is the extreme slowness of its molecular kinetics. In contrast, NMIIA develops a unitary force similar to that of muscle myosins. From a thermodynamic point of view, non-muscle contractile tissues containing NMIIA operate extremely close to equilibrium in a linear stationary mode.

Long noncoding RNA HAR1A regulates oral cancer progression through the alpha-kinase 1, bromodomain 7, and myosin IIA axis.

Studies suggested that long noncoding HAR1A RNA may be a tumor suppressor, but its association with oral cancer remains unclear. Here, we show the functional role and mechanisms of HAR1A in oral cancer progression. Microarray analysis was performed to screen the related candidates of long noncoding RNA (lncRNA) in human monocytes. Following lncRNA HAR1A, the regulation of HAR1A, ALPK1, myosin IIA, and BRD7 was tested using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) in oral cancer cells. The inflammatory and epithelial-to-mesenchymal transition marker expressions were analyzed using enzyme-linked immunosorbent assay and western blot. Phenotypic experiments were verified by colony formation assay, transwell migration assay, and Annexin V-apoptotic assay. In the nuclei of cancer cells, HAR1A functions upstream of signaling pathways and knockdown of HAR1A promoted ALPK1 expression and downregulated BRD7 resulting in inflammation and oral cancer progression. In monocytes, the expressions of TNF-α and CCL2 were increased following HAR1A knockdown and reduced following ALPK1 knockdown. HAR1A knockdown upregulated the expression of ALPK1, slug, vimentin, fibronectin, and N-cadherin but reduced the expression of E-cadherin in oral cancer cells. Myosin IIA was primarily located in the cytoplasm and that its decrease in the nuclei of oral cancer cells was likely to demonstrate suppressive ability in late-stage cancer. Our findings suggest that the HAR1A, BRD7, and myosin IIA are tumor suppressors while ALPK1 has oncogene-like property in the nucleus and is involved in inflammation and oral cancer progression. More research for HAR1A activators or ALPK1 inhibitors is required to develop potential therapeutic agents for advanced oral cancer. KEY MESSAGES: lncRNA HAR1A, BRD7, and myosin IIA are tumor suppressors whereas ALPK1 has an oncogenic-like property in the nucleus. lncRNA HAR1A/ALPK1/BRD7/myosin IIA axis plays a critical role in the progression of oral cancer. lncRNA HAR1A localizes upstream of signaling pathways to inhibit ALPK1 expression and then upregulated BRD7. lncRNA HAR1A and ALPK1 are involved in cancer progression via epithelial-to-mesenchymal transition regulations. ALPK1 inhibitors are potential kinase-targeted therapeutic agents for patients with advanced oral cancer.

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.