Nonmuscle Myosin Research Articles

Mitochondrial Micropeptide STMP1 Enhances Mitochondrial Fission to Promote Tumor Metastasis.

This study identifies the mitochondrial micropeptide STMP1 as a regulator of metastasis that promotes mitochondrial fission and tumor cell migration via DRP1 and MYH9.

Orchestrating nonmuscle myosin II filament assembly at the onset of cytokinesis.

Contractile forces in the actomyosin cortex are required for cellular morphogenesis. This includes the invagination of the cell membrane during division, where filaments of nonmuscle myosin II (NMII) are responsible for generating contractile forces in the cortex. However, how NMII heterohexamers form filaments in vivo is not well understood. To quantify NMII filament assembly dynamics, we imaged the cortex of Caenorhabditis elegans embryos at high spatial resolution around the time of the first division. We show that during the assembly of the cytokinetic ring, the number of NMII filaments in the cortex increases and more NMII motors are assembled into each filament. These dynamics are influenced by two proteins in the RhoA GTPase pathway, the RhoA-dependent kinase LET-502 and the myosin phosphatase MEL-11. We find that these two proteins differentially regulate NMII activity at the anterior and at the division site. We show that the coordinated action of these regulators generates a gradient of free NMII in the cytoplasm driving a net diffusive flux of NMII motors toward the cytokinetic ring. Our work highlights how NMII filament assembly and disassembly dynamics are orchestrated over space and time to facilitate the up-regulation of cortical contractility during cytokinesis.

Thick ascending limb specific inactivation of Myh9and Myh10 results in progressive kidney disease with sexually dimorphic sodium transport regulation

Mutations in MYH9 gene that encodes for actin associated molecular motor nonmuscle myosin II (NM2) result in kidney disease in some patients. Based on our previous work that demonstrated a novel role for NM2 motors, we hypothesized that NM2 genes might play a critical role in renal epithelial transport. To test our hypothesis, we generated pan renal tubular conditional knockout (cKO) of Myh9&10 in adult mice that resulted in progressive kidney disease of TAL origin with aberrant transport of TAL cargoes UMOD and NKCC2 (Otterpohl et al; 2020). To confirm the cell‐autonomous role for NM2 proteins in adult TAL epithelium, we generated a TAL‐specific cKO mouse model of Myh9&10using a TAL specific cre transgenic mice, UMOD‐>CreERT2. Cre transgene was induced at 5 weeks of age with tamoxifen injections, age matched control and cKO littermates were part of every cohort. The mice underwent metabolic, histologic, immunohistochemical, and biochemical analysis at varying time points until moribund. The TAL‐specific Myh9&10 cKO mice develop progressive kidney disease indicated by increase in BUN and serum creatinine as well as increasing levels of the tubular injury marker, NGAL. Our results show that Myh9&10 TAL‐ cKO females had significant loss of weight at 12 weeks, the female cKO animals became moribund between 13 – 14 weeks, while the male cohorts survive up to 25 weeks. Histology confirmed the tubulointerstitial disease in Myh9&10 TAL‐cKO with dilated tubules and cellular infiltration. Urine pH declined starting at 9‐weeks of age in females and 12‐week in males. Interestingly, female cKO mice have hypernatremia at 12‐weeks of age compared to male mice which maintain normal sodium levels. Since the pan renal tubular Myh9&10 cKO exhibited loss of NKCC2 leading to salt wasting, this result was unexpected. We analyzed NKCC2 protein levels in kidney lysates, loss of NKCC2 was evident in both female and male Myh9&10 TAL‐cKO. We also observe significant decline in serum potassium levels in 7.5‐week females. We speculated that the hypernatremia in TAL‐Myh9&10 cKO females is due to tubular adaptation in the distal nephron and the collecting duct. Characterization of sodium cotransporters and channels in distal nephron and collecting duct segment revealed an increase in NCC/pNCC levels in males and a decrease in females. Immunostaining experiments showed increase in ENaC gamma expression in the cortical collecting ducts of the females. This could potentially explain the hypernatremia and early morbidity observed in Myh9&10 TAL‐cKO females. We speculate that females upregulate ENaC gamma to compensate for loss of NKCC2, instead males upregulate NCC/pNCC that prevents their early lethality. These experiments confirm the critical role for Myh9&10 proteins in TAL tubule and identify a novel, sex specific tubular adaptation mechanisms. NM2 motor regulating the unique, TAL‐specific sodium cotransporter is intriguing and warrants further analysis. Our model also provides a good resource for understanding the sex‐specific differences of sodium handling and potentially clarify mechanisms underlying Type I Bartter Syndrome.

Changes in Cardiac and Vascular Contractile Protein in a Rat Model of Heart Failure with Preserved Ejection Fraction

Heart failure is classified as heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF). Although several therapies have been demonstrated to improve outcomes in patients with HFrEF, none benefit patients with HFpEF. The molecular mechanism that results in HFpEF is unknown and has been impacted by the lack of animal models, and thus, we tested the hypothesis that metabolic and hypertensive stress would result in a rat model of HFpEF. Fischer 344 rats (6mo old) were fed a high fat diet and L‐NAME in the drinking water for 4‐weeks. Invasive and noninvasive hemodynamics demonstrate that the treated animals develop HFpEF; treated animals (n=7) vs controls (n=6) have normal systolic function with LV hypertrophy (20.7±0.4mm v 27.6±2.1mm, p<0.05) and diastolic abnormalities including a decrease in both E (79.4±3.6cm/s v 64.2±3.4cm/s, p=0.003) and dP/dtmin (‐7600±1100mmHg/s v ‐5600±1400mmHg/s, p=0.015), a steeper EDPVR (0.1±1.1mmHg/μl v 3.6±1.7mmHg/μl, p=0.042) and a slower time constant of relaxation (τ; 11±1ms v 16±2ms, p=0.029), as well as edema of the liver and lungs. Compared to controls, in cardiac muscle of HFpEF rats, there is no difference (p>0.05) in the expression and/or phosphorylation of TnI or MyBPC. However, the expression of SERCA2 (p=0.021) and phosphorylation of phospholamban (p=0.046) are reduced, which would be expected to decrease the rate of SR Ca2+ reuptake and slow relaxation. In both the aorta and tertiary mesenteric vessels of HFpEF rats, the expression of nonmuscle myosin was significantly higher and the expression of the leucine zipper positive isoform of myosin light chain phosphatase was reduced, which should increase vascular tone and decrease the sensitivity of the vasculature to NO mediated vasodilatation. This study represents the first rat model of HFpEF and our results define a molecular mechanism that would contribute to both diastolic dysfunction and vascular abnormalities that characterize this disease. This rat HFpEF model could be used in future studies designed to delineate the mechanism(s) that produce the cardiac and vascular abnormalities that produce HFpEF and identify novel targets for rational drug design.

Characterization and application of a novel immortalized murine thick ascending limb (imTAL) cell line to investigate the role of myosin II motor proteins Myh9 and Myh10 in TAL cargo transport

Conditional genetic inactivation of Myh9and Myh10, the genes which encode the heavy chains of nonmuscle myosin IIA and IIB, respectively, in adult murine renal epithelium leads to aberrant trafficking of the thick ascending limb‐specific GPI‐anchored glycoprotein uromodulin (UMOD) and its subcellular accumulation in cells of the thick ascending limb of Henle’s loop (Otterpohl et al. 2020). A novel immortalized murine thick ascending limb (imTAL) cell line harboring doxycycline inducible Myh9and Myh10conditional knockout (cKO) capability was established in our lab to facilitate long term in vitrostudies. We utilized biotinylated anti‐uromodulin antibody to isolate cells from collagenase digested Myh9&10cKO whole mouse kidneys. Characterization of these cells has revealed endogenous expression of TAL‐specific protein UMOD and NKCC2. RT‐qPCR detected mRNA expression of TAL specific proteins. We generated imTAL cells stably expressing GFP‐tagged UMOD using retroviral transduction. GFP‐UMOD expressing cells underwent 72‐hour doxycycline treatment and, along with untreated control cells, were fixed and analyzed via confocal microscope. While GFP‐UMOD is enriched along the plasma membrane in control cells, we observe accumulation of GFP‐UMOD within vesicular structures upon doxycycline‐induced loss of Myh9 and Myh10 proteins. We also utilized imTAL cells stably expressing an GFP‐tagged thermosensitive variant of vesicular stomatitis virus glycoprotein (VSV‐GtsO45‐GFP), which has been widely employed in studying the secretory pathway. The temperature sensitive nature of VSV‐GtsO45 enabled us to perform pulse‐chase type experiments, following its anterograde transport through the ER, Golgi, and ultimately to the plasma membrane. Following incubation at the restrictive temperature (40°C) overnight, cells were moved to permissive temperature (32°C) and followed until 45 minutes. VSV‐GtsO45‐GFP in control cells undergoes ER export immediately (5 minutes) after incubation at the permissive temperature and is transported along the secretory pathway, arriving at the plasma membrane 30‐40 minutes later. Inactivation of Myh9and Myh10 results in sequestration of VSV‐GtsO45‐GFP in perinuclear vesicular and tubular structures. Immunolocalization studies are being performed to identify the vesicular compartment(s) in which VSV‐GtsO45‐GFP is sequestered in the absence of Myh9 and Myh10 proteins. imTAL cells stably expressing NKCC2‐GFP are also being analyzed to assess the anterograde transport of NKCC2 in doxycycline‐treated cells. Taken together, we have established a novel, immortalized, inducible, Myh9&10 cKO murine TAL cell line to enable long term in vitro studies and have expanded the molecular toolbox to elucidate the mechanisms underlying NM2 motor mediated TAL cargo transport.

Microridge-like structures anchor motile cilia

Several tissues contain cells with multiple motile cilia that generate a fluid or particle flow to support development and organ functions; defective motility causes human disease. Developmental cues orient motile cilia, but how cilia are locked into their final position to maintain a directional flow is not understood. Here we find that the actin cytoskeleton is highly dynamic during early development of multiciliated cells (MCCs). While apical actin bundles become increasingly more static, subapical actin filaments are nucleated from the distal tip of ciliary rootlets. Anchorage of these subapical actin filaments requires the presence of microridge-like structures formed during MCC development, and the activity of Nonmuscle Myosin II. Optogenetic manipulation of Ezrin, a core component of the microridge actin-anchoring complex, or inhibition of Myosin Light Chain Kinase interfere with rootlet anchorage and orientation. These observations identify microridge-like structures as an essential component of basal body rootlet anchoring in MCCs.

Myosin II proteins are required for organization of calcium-induced actin networks upstream of mitochondrial division.

The formin INF2 polymerizes a calcium-activated cytoplasmic network of actin filaments, which we refer to as calcium-induced actin polymerization (CIA). CIA plays important roles in multiple cellular processes, including mitochondrial dynamics and vesicle transport. Here, we show that nonmuscle myosin II (NMII) is activated within 60 s of calcium stimulation and rapidly recruited to the CIA network. Knockout of any individual NMII in U2OS cells affects the organization of the CIA network, as well as three downstream effects: endoplasmic-reticulum-to-mitochondrial calcium transfer, mitochondrial Drp1 recruitment, and mitochondrial division. Interestingly, while NMIIC is the least abundant NMII in U2OS cells (>200-fold less than NMIIA and >10-fold less than NMIIB), its knockout is equally deleterious to CIA. On the basis of these results, we propose that myosin II filaments containing all three NMII heavy chains exert organizational and contractile roles in the CIA network. In addition, NMIIA knockout causes a significant decrease in myosin regulatory light chain levels, which might have additional effects.

Non-Muscle Myosin II Is Essential for the Negative Regulation of B-Cell Receptor Signaling and B-Cell Activation.

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

The ascending aorta of male hypertensive bicuspid aortic valve patients preferentially associated with a cellular aneurysmal phenotype

Male sex and hypertension represent risk factors in the progression of an aortic aneurysm. The present study examined the morphological/cellular phenotype of the ascending aorta (AA) of male and female patients diagnosed with a bicuspid aortic valve (BAV) to test the hypothesis that hypertension‐induced remodeling of male BAV patients preferentially recapitulated the expression of a panel of proteins favoring aneurysm formation. The diameter of the AA of hypertensive male (35 ± 6 mm) and female (39 ± 5 mm) BAV patients was comparable to normotensive patients reflecting an early phase of vessel expansion. Morphological/structural remodeling of the medial region of the AA of male normotensive and hypertensive BAV patients were comparable. Protein levels of non‐muscle myosin IIB, the cell cycle inhibitor p27kip1, tumor suppressor p53 and matrix metalloproteinase‐2 and −9 were significantly upregulated in the AA of male hypertensive BAV patients. In female hypertensive BAV patients, collagen content was significantly increased whereas elastin content and medial width of the AA were similar to normotensive BAV patients. In the AA of female hypertensive BAV patients, matrix metalloproteinase‐9 and p27kip1 protein levels were unchanged whereas p53 and matrix metalloproteinase‐2 protein expression was significantly reduced. Nestin protein levels were diminished in the AA of male and female hypertensive BAV patients. Thus, sexual dimorphic remodeling of the AA was prevalent in hypertensive BAV patients. Moreover, during the early phase of vessel expansion, the AA of male hypertensive BAV patients was preferentially associated with the upregulation of a panel of proteins linked to progressive dilatation and potential aneurysm formation.

Critical role for the lung endothelial nonmuscle myosin light-chain kinase isoform in the severity of inflammatory murine lung injury.

Global knockout of the nonmuscle isoform of myosin light‐chain kinase (nmMLCK), a primary cellular regulator of cytoskeletal machinery, is strongly protective in preclinical murine models of inflammatory lung injury. The current study was designed to assess the specific contribution of endothelial cell (EC) nmMLCK to the severity of murine inflammatory lung injury produced by lipopolysaccharide (LPS) and mechanical ventilation ventilator‐induced lung injury or ventilation (VILI). Responses to combined LPS/VILI exposure were assessed in: (i) wild‐type (WT) C57BL/6J mice; (ii) transgenic mice with global deletion of nmMLCK (nmMylk −/−); (iii) transgenic nmMylk −/− mice with overexpression of nmMLCK restricted to the endothelium (nmMylk −/−/ec‐tg+). Lung inflammation indices included lung histology, bronchoalveolar lavage (BAL) polymorphonuclear leukocytes (PMNs), lung protein biochemistry, tissue albumin levels, Evans blue dye (EBD) lung extravasation, and plasma cytokines (interleukin‐6 [IL‐6], keratinocyte chemoattractant [KC]/IL‐8, IL‐1bβ, extracellular nicotinamide phosphoribosyltransferase, tumor necrosis factor‐α). Compared to WT C57BL/6J mice, the severity of LPS/VILI‐induced lung injury was markedly reduced in mice with global nmMLCK deletion reflected by reductions in histologic inflammatory lung injury, BAL PMN counts, mitogen‐activated protein kinase, and NF‐kB pathway activation in lung homogenates, plasma cytokine levels, and parameters of lung permeability (increased BAL protein, tissue albumin levels, EBD lung extravasation). In contrast, mice with restricted overexpression of nmMLCK in EC (nmMylk −/−/ec‐tg+) showed significant persistence of LPS/VILI‐induced lung injury severity compared to WT mice. In conclusion, these studies strongly endorse the role of EC nmMLCK in driving the severity of preclinical inflammatory lung injury. Precise targeting of EC nmMLCK may represent an attractive therapeutic strategy to reduce lung inflammation and both lung and systemic vascular permeability.

A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair.

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell‐matrix adhesions. Although biochemical mechanisms that regulate the activity of non‐muscle myosin II (NM‐II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC‐45A is a cytoskeletal chaperone that is essential for proper folding of NM‐II heavy chains and myofilament assembly. We found abundant expression of UNC‐45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein level of UNC‐45A was decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9‐mediated knock‐out of UNC‐45A in HT‐29cf8 and SK‐CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC‐45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti‐migratory effects of UNC‐45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC‐45A also decreased contractile forces at apical junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC‐45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

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.

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.

Nectin-1 and Non-muscle Myosin Heavy Chain-IIB: Major Mediators of Herpes Simplex Virus-1 Entry Into Corneal Nerves.

Herpes Simplex Virus 1 (HSV-1) invades corneal nerves upon its infection of the cornea and then establishes latency in the trigeminal ganglion (TG). The latent virus in TG is often reactivated and travels back to the cornea, causing recurrent herpes simplex keratitis (HSK). The entry of HSV-1 into the corneal nerve is considered the initial step of infection resulting in HSV-1 latency and HSK recurrence. Several gD and gB receptors have been identified, including nectin-1, herpes virus entry medium (HVEM) and 3-O-sulfated heparan sulfate (3-OS-HS) as gD receptors, and non-muscle myosin heavy chain IIA (NMHC-IIA), NMHC-IIB and myelin-associated glycoprotein (MAG) as gB receptors. However, which receptors contribute to the entry of HSV-1 into corneal nerves are yet to be determined. This study observed that receptors nectin-1, HVEM, 3-OS-HS, NMHC-IIA, and NMHC-IIB, not MAG, were expressed in healthy corneal nerves. Further, we cultured TG neurons extracted from mice in vitro to screen for functional gD/gB receptors. Both in vitro siRNA knockdown and in vivo antibody blocking of either nectin-1 or NMHC-IIB reduced the entry and the replication of HSV-1 as shown by qPCR analysis and immunofluorescence measure, respectively. Also, we observed that the re-localization and the upregulation expression of NMHC-IIB after HSV-1 exposure were inhibited when gD receptor nectin-1 was knocked down. These data suggest that nectin-1 was the main gD receptor and NMHC-IIB was the main gB receptor in mediating HSV-1 entry and hold promise as therapeutic targets for resolving HSV-1 latency and HSK recurrence.

Optogenetic inhibition of actomyosin reveals mechanical bistability of the mesoderm epithelium during Drosophila mesoderm invagination.

Apical constriction driven by actin and non-muscle myosin II (actomyosin) provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of actomyosin, we find that during Drosophila mesoderm invagination, actomyosin contractility is critical to prevent tissue relaxation during the early, 'priming' stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. This binary response suggests that Drosophila mesoderm is mechanically bistable during gastrulation. Computer modeling analysis demonstrates that the binary tissue response to actomyosin inhibition can be recapitulated in the simulated epithelium that undergoes buckling-like deformation jointly mediated by apical constriction in the mesoderm and in-plane compression generated by apicobasal shrinkage of the surrounding ectoderm. Interestingly, comparison between wild-type and snail mutants that fail to specify the mesoderm demonstrates that the lateral ectoderm undergoes apicobasal shrinkage during gastrulation independently of mesoderm invagination. We propose that Drosophila mesoderm invagination is achieved through an interplay between local apical constriction and mechanical bistability of the epithelium that facilitates epithelial buckling.

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.

Abstract P5-10-05: HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers

Abstract HER2 is amplified in about 20% of breast cancers. HER3 is as essential as HER2 for maintaining cell viability in HER2+ breast cancer cells. Inhibition of HER2 tyrosine kinase activity results in upregulation of HER3 transcription and phosphorylation. We sought to identify HER3 binding partners upon pharmacological inhibition of HER2 using the irreversible pan HER inhibitor neratinib. We immunoaffinity-purified HER3 from HER2+ BT474 cells treated ± neratinib. Following immunoprecipitation using a HER3 antibody, binding partners were released under reducing conditions. Mass spectrometry experiments identified non-muscle myosin IIA (NMIIA) increased upon inhibition of HER2 with neratinib and decreased under DMSO control treatment from HER3 immunoprecipitates. To validate the presence of NMIIA, we performed immunoprecipitation experiments in HER2+ BT474 and MDA-MB-453 cells using a HER3 antibody. Immunoblots showed increased NMIIA levels upon treatment with 200nM neratinib for 24 hours in both cell lines. Myosin heavy chain 9 (MYH9) gene encodes the protein NMIIA. NMIIA localizes to actin stress fibers and has been implicated in regulation of cell contractility and stress fiber organization.To test if there is an interaction between HER3 and NMIIA, we immunoprecipitated NMIIA from BT474 and MDA-MB-453 cells using an NMIIA antibody. The products were analyzed using immunoblots. The results indicated that HER3 levels were increased upon inhibition of HER2 with 200 nM neratinib for 24 hours.We next examined long term overall survival of primary breast cancer patients who have high and low levels of gene expression for MYH9 from the METABRIC cohort. We observed that patients with high levels of MYH9 have a statistically significant worse overall survival versus patients who express low levels of MYH9.We next sought to examine overall levels of HER3 and MYH9 mRNA and protein upon treatment with neratinib in HER2+ breast cancer whole cell lysates. We observed that mRNA and protein levels of both HER3 and MYH9 increased upon HER2 inhibition with 24 hours of neratinib treatment. To further investigate the role of NMIIA, we knocked out NMIIA in BT474 and MDA-MB-453 using inducible lentiviral shRNA targeting MYH9. This system utilizes the Tet-On 3G induction system and is a highly controlled system that consists of an inducible RNA polymerase II promoter. In the presence of doxycycline, the TRE3G promoter is bound and activated by the constitutively expressed Tet-On 3G trans-activator protein. Non-targeting shRNA was used as control and cells were selected in 1-2 ug/ml puromycin. Western blot analysis demonstrated that cells infected with lentiviral shRNA targeting MYH9 treated with doxycycline had reduced protein expression of NMIIA. BT474 and MDA-MB-453 transduced with shMYH9 were used to perform growth, migration, and invasion assays. The results indicated that MYH9 knockdown + neratinib treatment suppresses BT474 and MDA-MB-453 growth, migration, and invasion. Currently, in vivo xenograft experiments examining BT474 and MDA-MB-453 tumor growth ±NMIIA shRNA in the presence or absence of neratinib treatment are underway. In conclusion, we have identified that NMIIA mRNA and protein levels are increased in the presence of neratinib treatment. Our data reveals that HER2 inhibition increases NMIIA to promote HER2+ breast cancer growth. Citation Format: Samar M Alanazi, Rosalin Mishra, Hima Patel, Long Yuan, Mary Kate Kilroy, Joan T Garrett. HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-05.

DDR1 associates with TRPV4 in cell-matrix adhesions to enable calcium-regulated myosin activity and collagen compaction.

Tissue fibrosis manifests as excessive deposition of compacted, highly aligned collagen fibrils, which interfere with organ structure and function. Cells in collagen-rich lesions often exhibit marked overexpression of discoidin domain receptor 1 (DDR1), which is linked to increased collagen compaction through the association of DDR1 with the Ca2+ -dependent nonmuscle myosin IIA (NMIIA). We examined the functional relationship between DDR1 and the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca2+ -permeable ion channel that is implicated in collagen compaction. Fibroblasts expressing high levels of DDR1 were used to model cells in lesions with collagen compaction. In these cells, the expression of the β1 integrin was deleted to simplify studies of DDR1 function. Compared with DDR1 wild-type cells, high DDR1 expression was associated with increased Ca2+ influx through TRPV4, enrichment of TRPV4 in collagen adhesions, and enhanced contractile activity mediated by NMIIA. At cell adhesion sites to collagen, DDR1 associated with TRPV4, which enhanced DDR1-mediated collagen alignment and compaction. We conclude that DDR1 regulates Ca2+ influx through the TRPV4 channel to promote critical, DDR1-mediated processes that are important in lesions with collagen compaction and alignment.

Nonmuscle Myosin II is Required for Larval Shell Formation in a Patellogastropod.

The molecular mechanisms underlying larval shell development in mollusks remain largely elusive. We previously found evident filamentous actin (F-actin) aggregations in the developing shell field of the patellogastropod Lottia goshimai, indicating roles of actomyosin networks in the process. In the present study, we functionally characterized nonmuscle myosin II (NM II), the key molecule in actomyosin networks, in the larval shell development of L. goshimai. Immunostaining revealed general colocalization of phosphorylated NM II and F-actin in the shell field. When inhibiting the phosphorylation of NM II using the specific inhibitor blebbistatin in one- or 2-h periods during shell field morphogenesis (6–8 h post-fertilization, hpf), the larval shell plate was completely lost in the veliger larva (24 hpf). Scanning electron microscopy revealed that the nascent larval shell plate could not be developed in the manipulated larvae (10 hpf). Further investigations revealed that key events in shell field morphogenesis were inhibited by blebbistatin pulses, including invagination of the shell field and cell shape changes and cell rearrangements during shell field morphogenesis. These factors caused the changed morphology of the shell field, despite the roughly retained “rosette” organization. To explore whether the specification of related cells was affected by blebbistatin treatments, we investigated the expression of four potential shell formation genes (bmp2/4, gata2/3, hox1 and engrailed). The four genes did not show evident changes in expression level, indicating unaffected cell specification in the shell field, while the gene expression patterns showed variations according to the altered morphology of the shell field. Together, our results reveal that NM II contributes to the morphogenesis of the shell field and is crucial for the formation of the larval shell plate in L. goshimai. These results add to the knowledge of the mechanisms of molluskan shell development.

The role of NM2 rearrangement during cytokinesis

The cellular distribution of non-muscle myosin 2 (NM2) is essential for plethora of cellular movements, such as axonal growth and cytokinesis. However, it is poorly understood how the role of NM2 in the dynamics of cytokinesis is related to its intracellular distribution and the ATPase inhibition. Like in stress fibers, NM2 has a crucial role in governing the normal progression of cytokinesis via cross-linking actin filaments in the cytokinetic ring. Recently, we showed that partial inhibition of the ATPase activity of NM2 results in increased diffusion rates of NM2 in load-bearing stress fibers while complete inhibition blocks their intracellular diffusion.