Mechanisms underlying Myosin 10's contribution to the maintenance of mitotic spindle bipolarity.

Myosin 10 (Myo10) couples microtubules and integrin-based adhesions to movement along actin filaments via its microtubule-binding MyTH4 domain and integrin-binding FERM domain, respectively. Here we show that Myo10-depleted HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in Myo10-depleted MEFs and in Myo10-depleted HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates y-tubulin-positive acentriolar foci that serve as extra spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both microtubules and integrins to promote PCM/pole integrity. Conversely, Myo10 only needs interact with integrins to promote supernumerary centrosome clustering. Importantly, images of metaphase Halo-Myo10 knockin cells show that the myosin localizes exclusively to the spindle and the tips of adhesive retraction fibers. We conclude that Myo10 promotes PCM/pole integrity in part by interacting with spindle microtubules, and that it promotes supernumerary centrosome clustering by supporting retraction fiber-based cell adhesion, which likely serves to anchor the microtubule-based forces driving pole focusing.

Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, are able to invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis and here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the ß1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. Spatially, sEV were secreted via filopodia-like cellular protrusions at the leading edge of migrating cells. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI. In vitro assays identified collagen VI as playing the key role in cell adhesion and invasion. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate migration and invasion by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction forces to enable VSMC movement within the complex vascular ECM network.

Myosin 10 uses its MyTH4 and FERM domains differentially to support two aspects of spindle pole biology required for mitotic spindle bipolarity.

Myosin 10 (Myo10) has the ability to link actin filaments to integrin-based adhesions and to microtubules by virtue of its integrin-binding FERM domain and microtubule-binding MyTH4 domain, respectively. Here we used Myo10 knockout cells to define Myo10's contribution to the maintenance of spindle bipolarity, and complementation to quantitate the relative contributions of its MyTH4 and FERM domains. Myo10 knockout HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in knockout MEFs and knockout HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates γ-tubulin-positive acentriolar foci that serve as additional spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both integrins and microtubules to promote PCM/pole integrity. Conversely, Myo10's ability to promote the clustering of supernumerary centrosomes only requires that it interact with integrins. Importantly, images of Halo-Myo10 knock-in cells show that the myosin localizes exclusively within adhesive retraction fibers during mitosis. Based on these and other results, we conclude that Myo10 promotes PCM/pole integrity at a distance, and that it facilitates supernumerary centrosome clustering by promoting retraction fiber-based cell adhesion, which likely provides an anchor for the microtubule-based forces driving pole focusing.

Changes in cell surface excess are coordinated with protrusion dynamics during 3D motility.

To facilitate rapid changes in morphology without endangering cell integrity, each cell possesses a substantial amount of cell surface excess (CSE) that can be promptly deployed to cover cell extensions. CSE can be stored in different types of small surface projections such as filopodia, microvilli, and ridges, with rounded bleb-like projections being the most common and rapidly achieved form of storage. We demonstrate that, similar to rounded cells in 2D culture, rounded cells in 3D collagen contain large amounts of CSE and use it to cover developing protrusions. Upon retraction of a protrusion, the CSE this produces is stored over the cell body similar to the CSE produced by cell rounding. We present high-resolution imaging of F-actin and microtubules (MTs) for different cell lines in a 3D environment and demonstrate the correlated changes between CSE and protrusion dynamics. To coordinate CSE storage and release with protrusion formation and motility, we expect cells to have specific mechanisms for regulating CSE, and we hypothesize that MTs play a substantial role in this mechanism by reducing cell surface dynamics and stabilizing CSE. We also suggest that different effects of MT depolymerization on cell motility, such as inhibiting mesenchymal motility and enhancing amoeboid, can be explained by this role of MTs in CSE regulation.

MYO10 drives genomic instability and inflammation in cancer.

Genomic instability is a hallmark of human cancer; yet the underlying mechanisms remain poorly understood. Here, we report that the cytoplasmic unconventional Myosin X (MYO10) regulates genome stability, through which it mediates inflammation in cancer. MYO10 is an unstable protein that undergoes ubiquitin-conjugating enzyme H7 (UbcH7)/ß-transducin repeat containing protein 1 (ß-TrCP1)­dependent degradation. MYO10 is upregulated in both human and mouse tumors and its expression level predisposes tumor progression and response to immune therapy. Overexpressing MYO10 increased genomic instability, elevated the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING)­dependent inflammatory response, and accelerated tumor growth in mice. Conversely, depletion of MYO10 ameliorated genomic instability and reduced the inflammation signaling. Further, inhibiting inflammation or disrupting Myo10 significantly suppressed the growth of both human and mouse breast tumors in mice. Our data suggest that MYO10 promotes tumor progression through inducing genomic instability, which, in turn, creates an immunogenic environment for immune checkpoint blockades.

Filopodia powered by class x myosin promote fusion of mammalian myoblasts.

Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multinucleated mammalian muscle formation.

Cytoneme delivery of Sonic Hedgehog from ligand-producing cells requires Myosin 10 and a Dispatched-BOC/CDON co-receptor complex.

Morphogens function in concentration-dependent manners to instruct cell fate during tissue patterning. The cytoneme morphogen transport model posits that specialized filopodia extend between morphogen-sending and responding cells to ensure that appropriate signaling thresholds are achieved. How morphogens are transported along and deployed from cytonemes, how quickly a cytoneme-delivered, receptor-dependent signal is initiated, and whether these processes are conserved across phyla are not known. Herein, we reveal that the actin motor Myosin 10 promotes vesicular transport of Sonic Hedgehog (SHH) morphogen in mouse cell cytonemes, and that SHH morphogen gradient organization is altered in neural tubes of Myo10-/- mice. We demonstrate that cytoneme-mediated deposition of SHH onto receiving cells induces a rapid, receptor-dependent signal response that occurs within seconds of ligand delivery. This activity is dependent upon a novel Dispatched (DISP)-BOC/CDON co-receptor complex that functions in ligand-producing cells to promote cytoneme occurrence and facilitate ligand delivery for signal activation.

During development, cells must work together and talk to each other to build the organs and tissues of the growing embryo. To communicate precisely with long-distance targets, cells can project a series of thin finger-like structures known as cytonemes. Cells use these miniature highways to exchange cargo and signals, such as the protein sonic hedgehog (SHH for short). Alterations to the way SHH is exchanged during development predispose to cancer and lead to disorders of the nervous system. Yet, the mechanisms by which cytonemes work in mammals remain to be fully elucidated. In particular, it is still unclear how the structures start to form, and how the proteins are loaded and transported from one end to another. A 'molecular motor' called myosin 10, which can carry cargo along the internal skeleton of cells, may be involved in these processes. To find out, Hall et al. used fluorescent probes to track both myosin 10 and SHH in mouse cells, showing that myosin 10 carries SHH from the core of the signal-producing cell to the tips of cytonemes. There, the protein is passed to the target cell upon contact, triggering a quick response. SHH also appeared to be more than just passive cargo, interacting with another group of proteins in the signal-emitting cell before reaching its target. This mechanism then encourages the signalling cells to produce more cytonemes towards their neighbours. SHH is crucial during development, but also after birth: in fact, changes to SHH transport in adulthood can also disrupt tissue balance and hinder healing. Understanding how healthy tissues send this signal may reveal why and how disease emerges.

Myosin 10 Regulates Invasion, Mitosis, and Metabolic Signaling in Glioblastoma.

Invasion and proliferation are defining phenotypes of cancer, and in glioblastoma blocking one stimulates the other, implying that effective therapy must inhibit both, ideally through a single target that is also dispensable for normal tissue function. The molecular motor myosin 10 meets these criteria. Myosin 10 knockout mice can survive to adulthood, implying that normal cells can compensate for its loss; its deletion impairs invasion, slows proliferation, and prolongs survival in murine models of glioblastoma. Myosin 10 deletion also enhances tumor dependency on the DNA damage and the metabolic stress responses and induces synthetic lethality when combined with inhibitors of these processes. Our results thus demonstrate that targeting myosin 10 is active against glioblastoma by itself, synergizes with other clinically available therapeutics, may have acceptable side effects in normal tissues, and has potential as a heretofore unexplored therapeutic approach for this disease.

Content and Performance of the MiniMUGA Genotyping Array: A New Tool To Improve Rigor and Reproducibility in Mouse Research.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.

A new light chain for myosin-7.

Recent research has revealed that an adhesion complex based on cadherins and the motor protein myosin-7b (MYO7B) links the tips of intestinal microvilli. Choi et al. now report that a largely uncharacterized protein known as calmodulin-like protein 4 (CALML4) is a component of this adhesion complex and functions as a light chain for myosin-7b. Because the intermicrovillar adhesion complex is homologous to the myosin-7a (MYO7A)-based Usher syndrome complex and Choi et al. also report that CALML4 can bind to myosin-7a, this work also has important implications for research on myosin-7a and hereditary deaf-blindness.

Single cell analysis of nutrient regulation of Clostridioides (Clostridium) difficile motility.

Regulation of bacterial motility to maximize nutrient acquisition or minimize exposure to harmful substances plays an important role in microbial proliferation and host colonization. The technical difficulties of performing high-resolution live microscopy on anaerobes have hindered mechanistic studies of motility in Clostridioides (formerly Clostridium) difficile. Here, we present a widely applicable protocol for live cell imaging of anaerobic bacteria that has allowed us to characterize C. difficile swimming at the single-cell level. This accessible method for anaerobic live cell microscopy enables inquiry into previously inaccessible aspects of C. difficile physiology and behavior. We present the first report that vegetative C. difficile are capable of regulated motility in the presence of different nutrients. We demonstrate that the epidemic C. difficile strain R20291 exhibits regulated motility in the presence of multiple nutrient sources by modulating its swimming velocity. This is a powerful illustration of the ability of single-cell studies to explain population-wide phenomena such as dispersal through the environment.

Myosin-X knockout is semi-lethal and demonstrates that myosin-X functions in neural tube closure, pigmentation, hyaloid vasculature regression, and filopodia formation.

Myosin-X (Myo10) is an unconventional myosin best known for its striking localization to the tips of filopodia. Despite the broad expression of Myo10 in vertebrate tissues, its functions at the organismal level remain largely unknown. We report here the generation of KO-first (Myo10 tm1a/tm1a ), floxed (Myo10 tm1c/tm1c ), and KO mice (Myo10 tm1d/tm1d ). Complete knockout of Myo10 is semi-lethal, with over half of homozygous KO embryos exhibiting exencephaly, a severe defect in neural tube closure. All Myo10 KO mice that survive birth exhibit a white belly spot, all have persistent fetal vasculature in the eye, and ~50% have webbed digits. Myo10 KO mice that survive birth can breed and produce litters of KO embryos, demonstrating that Myo10 is not absolutely essential for mitosis, meiosis, adult survival, or fertility. KO-first mice and an independent spontaneous deletion (Myo10 m1J/m1J ) exhibit the same core phenotypes. During retinal angiogenesis, KO mice exhibit a ~50% decrease in endothelial filopodia, demonstrating that Myo10 is required to form normal numbers of filopodia in vivo. The Myo10 mice generated here demonstrate that Myo10 has important functions in mammalian development and provide key tools for defining the functions of Myo10 in vivo.

A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase.

The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile, c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability.

Myosin-X and disease.

Myosin-X (Myo10) is a motor protein best known for its role in filopodia formation. New research implicates Myo10 in a number of disease states including cancer metastasis and pathogen infection. This review focuses on these developments with emphasis on the emerging roles of Myo10 in formation of cancer cell protrusions and metastasis. A number of aggressive cancers show high levels of Myo10 expression and knockdown of Myo10 has been shown to dramatically limit cancer cell motility in 2D and 3D systems. Myo10 knockdown also limits spread of intracellular pathogens marburgvirus and Shigella flexneri. Consideration is given to how these properties might arise and potential paths of future research.

Myosin vc interacts with Rab32 and Rab38 proteins and works in the biogenesis and secretion of melanosomes.

Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion.

DPP6 regulation of dendritic morphogenesis impacts hippocampal synaptic development.

Dipeptidyl-peptidase 6 is an auxiliary subunit of Kv4-mediated A-type K(+) channels that, in addition to enhancing channel surface expression, potently accelerates their kinetics. The dipeptidyl-peptidase 6 gene has been associated with a number of human central nervous system disorders including autism spectrum disorders and schizophrenia. Here we employ knockdown and genetic deletion of dipeptidyl-peptidase 6 to reveal its importance for the formation and stability of dendritic filopodia during early neuronal development. We find that the hippocampal neurons lacking dipeptidyl-peptidase 6 show a sparser dendritic branching pattern along with fewer spines throughout development and into adulthood. In electrophysiological and imaging experiments, we show that these deficits lead to fewer functional synapses and occur independently of the potassium channel subunit Kv4.2. We report that dipeptidyl-peptidase 6 interacts with a filopodia-associated myosin as well as with fibronectin in the extracellular matrix. dipeptidyl-peptidase 6 therefore has an unexpected but important role in cell adhesion and motility, impacting the hippocampal synaptic development and function.

Myosin X and its motorless isoform differentially modulate dendritic spine development by regulating trafficking and retention of vasodilator-stimulated phosphoprotein.

Myosin X (Myo10) is an unconventional myosin with two known isoforms: full-length (FL)-Myo10 that has motor activity, and a recently identified brain-expressed isoform, headless (Hdl)-Myo10, which lacks most of the motor domain. FL-Myo10 is involved in the regulation of filopodia formation in non-neuronal cells; however, the biological function of Hdl-Myo10 remains largely unknown. Here, we show that FL- and Hdl-Myo10 have important, but distinct, roles in the development of dendritic spines and synapses in hippocampal neurons. FL-Myo10 induces formation of dendritic filopodia and modulates filopodia dynamics by trafficking the actin-binding protein vasodilator-stimulated phosphoprotein (VASP) to the tips of filopodia. By contrast, Hdl-Myo10 acts on dendritic spines to enhance spine and synaptic density as well as spine head expansion by increasing the retention of VASP in spines. Thus, this study demonstrates a novel biological function for Hdl-Myo10 and an important new role for both Myo10 isoforms in the development of dendritic spines and synapses.

Myosin-X facilitates Shigella-induced membrane protrusions and cell-to-cell spread.

The intracellular pathogen Shigella flexneri forms membrane protrusions to spread from cell to cell. As protrusions form, myosin-X (Myo10) localizes to Shigella. Electron micrographs of immunogold-labelled Shigella-infected HeLa cells reveal that Myo10 concentrates at the bases and along the sides of bacteria within membrane protrusions. Time-lapse video microscopy shows that a full-length Myo10 GFP-construct cycles along the sides of Shigella within the membrane protrusions as these structures progressively lengthen. RNAi knock-down of Myo10 is associated with shorter protrusions with thicker stalks, and causes a >80% decrease in confluent cell plaque formation. Myo10 also concentrates in membrane protrusions formed by another intracellular bacteria, Listeria, and knock-down of Myo10 also impairs Listeria plaque formation. In Cos7 cells (contain low concentrations of Myo10), the expression of full-length Myo10 nearly doubles Shigella-induced protrusion length, and lengthening requires the head domain, as well as the tail-PH domain, but not the FERM domain. The GFP-Myo10-HMM domain localizes to the sides of Shigella within membrane protrusions and the GFP-Myo10-PH domain localizes to host cell membranes. We conclude thatMyo10 generates the force to enhance bacterial-induced protrusions by binding its head region to actin filaments and its PH tail domain to the peripheral membrane.

Myosins in cell junctions.

The development of cell-cell junctions was a fundamental step in metazoan evolution, and human health depends on the formation and function of cell junctions. Although it has long been known that actin and conventional myosin have important roles in cell junctions, research has begun to reveal the specific functions of the different forms of conventional myosin. Exciting new data also reveals that a growing number of unconventional myosins have important roles in cell junctions. Experiments showing that cell junctions act as mechanosensors have also provided new impetus to understand the functions of myosins and the forces they exert. In this review we will summarize recent developments on the roles of myosins in cell junctions.