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.

'It sort of has the feel of being at home': Mixed-methods evaluation of a pilot community-based palliative end-of-life service in a regional setting.

OBJECTIVE: To evaluate the acceptability and effectiveness of a small community-based hospice on the end-of-life experiences of patients and families. METHODS: Mixed-methods study. DESIGN: Patient admission data were used to assess utilisation of the hospice. Open-ended interviews with hospice patients and their families/carers were used to understand the emotional effects of the service. SETTING: A small palliative end-of-life hospice in a rural town in NSW, Australia, during a 12-month trial period that began in March 2019. Data were collected in October-November 2019. PARTICIPANTS: Patients, families and carers who used the hospice during the trial period, as well as staff working at the hospice. MAIN OUTCOME MEASURE(S): Quantitative measures included the number of patients admitted to the hospice, the average length of stay and the overall occupancy rate of the hospice. Quantitative interviews were used to explore the experiences of patients and families who used the hospice, and whether the hospice met their end-of-life needs. RESULTS: During the trial, 58 patients were admitted to the hospice. The majority of admissions were less than 7 days. Two patients and nine family members were interviewed about their experiences, and six staff completed interviews. Experiences were consistently positive, with the community setting of the hospice contributing to a peaceful and home-like end-of-life experience. Interviewees described meaningful relationships with staff, a pleasant physical environment and the comprehensive care provided were key elements of this experience. CONCLUSION: This model, embedding end-of-life care within a residential aged care facility, facilitated a positive end-of-life experience for residents of this regional community. The development of local models to meet local needs is essential to enabling people nearing the end of life to remain in their location of choice, and ensure that their needs are met at this vulnerable time.

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.

Prognostic Impact of KRAS G12C Mutation in Patients With NSCLC: Results From the European Thoracic Oncology Platform Lungscape Project.

INTRODUCTION: KRAS mutations, the most frequent gain-of-function alterations in NSCLC, are currently emerging as potential predictive therapeutic targets. The role of KRAS-G12C (Kr_G12C) is of special interest after the recent discovery and preclinical analyses of two different Kr_G12C covalent inhibitors (AMG-510, MRTX849). METHODS: KRAS mutations were evaluated in formalin-fixed, paraffin-embedded tissue sections by a microfluidic-based multiplex polymerase chain reaction platform as a component of the previously published European Thoracic Oncology Platform Lungscape 003 Multiplex Mutation study, of clinically annotated, resected, stage I to III NSCLC. In this study, -Kr_G12C mutation prevalence and its association with clinicopathologic characteristics, molecular profiles, and postoperative patient outcome (overall survival, relapse-free survival, time-to-relapse) were explored. RESULTS: KRAS gene was tested in 2055 Lungscape cases (adenocarcinomas: 1014 [49%]) with I or II or III stage respective distribution of 53% or 24% or 22% and median follow-up of 57 months. KRAS mutation prevalence in the adenocarcinoma cohort was 38.0% (95% confidence interval (CI): 35.0% to 41.0%), with Kr_G12C mutation representing 17.0% (95% CI: 14.7% to 19.4%). In the "histologic-subtype" cohort, Kr_G12C prevalence was 10.5% (95% CI: 9.2% to 11.9%). When adjusting for clinicopathologic characteristics, a significant negative prognostic effect of Kr_G12C presence versus other KRAS mutations or nonexistence of KRAS mutation was identified in the adenocarcinoma cohort alone and in the "histologic-subtype" cohort. For overall survival in adenocarcinomas, hazard ratio (HR)G12C versus other KRAS is equal to 1.39 (95% CI: 1.03 to 1.89, p = 0.031) and HRG12C versus no KRAS is equal to 1.32 (95% CI: 1.03 to 1.69, p = 0.028) (both also significant in the "histologic-subtype" cohort). For time-to-relapse, HRG12C versus other KRAS is equal to 1.41 (95% CI: 1.03 to 1.92, p = 0.030). In addition, among all patients, for relapse-free survival, HRG12C versus no KRAS is equal to 1.27 (95% CI: 1.04 to 1.54, p = 0.017). CONCLUSIONS: In this large, clinically annotated stage I to III NSCLC cohort, the specific Kr_G12C mutation is significantly associated with poorer prognosis (adjusting for clinicopathologic characteristics) among adenocarcinomas and in unselected NSCLCs.

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.