Biology subjects

Cheney, R. E.

Publications and source records attributed to Cheney, R. E..

7 recordsLinked to original sources

bioRxiv2023

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, can 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. Here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the {beta}1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. 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 preventing cellular spreading and adhesion. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI on the sEV surface. In vitro assays identified collagen VI as playing a key role in cell adhesion and invasion directionality. 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 directional invasion, most likely by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction force to enable VSMC movement within the complex vascular ECM network. Figure AbstractVascular smooth muscle cells sense fibronectin via {beta}1 integrin and secrete small extracellular vesicles loaded with collagen VI. These extracellular vesicles are entrapped in the extracellular matrix and induce formation of peripheral focal adhesions presenting adhesion complex ECM proteins including collagen VI, LGALS3BP, EDIL3 and TGFBI. Focal adhesions anchor the extracellular matrix to actin fibrils in the cell. Contraction of the actin fibrils generates the mechanical force for directional cell invasion through the matrix. This figure was created with BioRender (https://biorender.com/). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/551257v3_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1355004org.highwire.dtl.DTLVardef@1186e9forg.highwire.dtl.DTLVardef@106ad30org.highwire.dtl.DTLVardef@1543518_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

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 Myo10s 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 y-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, Myo10s 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.

cell biology↗

bioRxiv2022

Cell surface excess is essential for protrusions and motility in 3D matrix

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 in this paper that cells migrating in 3D collagen use CSE to cover the developing protrusions. After retraction of a protrusion, the CSE this produces is stored over the cell body similar to the CSE produced by cell rounding. For the coordinated process of CSE storage and release, all cells should have specific mechanisms of regulation, and we hypothesize that microtubules (MT) play an important role in this mechanism. We show here that different effects of MT depolymerization on cell motility such as inhibiting mesenchymal motility and enhancing amoeboid, can be explained by the essential role of MT in CSE regulation and dynamics.

cell biology↗

bioRxiv2022

Myosin 10 supports mitotic spindle bipolarity by promoting PCM integrity and supernumerary centrosome clustering

Myosin 10 (Myo10) is a member of the MyTH4/FERM domain family of unconventional, actin-based motor proteins. Studies have implicated Myo10 in supporting cell adhesion via its integrin-binding FERM domain, and spindle positioning and spindle pole integrity via its microtubule-binding MyTH4 domain. Here we characterized Myo10s contribution to mitosis using Myo10 knockout HeLa cells and MEFs isolated from a Myo10 knockout mouse. Most notably, both of these knockout cells 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 PCM fragmentation, which creates y-tubulin-positive, centriole-negative microtubule asters that serve as additional spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing centrosome clustering. These results argue, therefore, that Myo10 supports spindle bipolarity by maintaining PCM integrity in both normal and cancer cells, and by promoting supernumerary centrosome clustering in cancer cells. Finally, we present evidence that the defect in spindle pole integrity in Myo10 knockout cells is likely due to a defect in pole stability rather than pole maturation, and that Myo10 promotes supernumerary centrosome clustering at least in part by promoting cell adhesion during mitosis.

cell biology↗

bioRxiv2021

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. 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 multi-nucleated mammalian muscle formation.

cell biology↗

bioRxiv2020

Myosin 10 and a Cytoneme-Localized Ligand Complex Promote Morphogen Transport

Morphogens function in concentration-dependent manners to instruct cell fate during tissue patterning. Molecular mechanisms by which these signaling gradients are established and reinforced remain enigmatic. The cytoneme transport model posits that specialized filopodia extend between morphogen-sending and responding cells to ensure that appropriate signal activation thresholds are achieved across developing tissues. How morphogens are transported along and deployed from cytonemes is not known. Herein we show that the actin motor Myosin 10 promotes cytoneme-based transport of Sonic Hedgehog (SHH) morphogen to filopodial tips, and that SHH movement within cytonemes occurs by vesicular transport. We demonstrate that cytoneme-mediated deposition of SHH onto receiving cells induces a rapid signal response, and that SHH cytonemes are promoted by a complex containing a ligand-specific deployment protein and associated co-receptor. One-Sentence summaryCytoneme-based delivery of the Sonic Hedgehog activation signal is promoted by Myosin 10 and BOC/CDON co-receptor function.

cell biology↗

bioRxiv2020

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 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 6,899 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, the extension of the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. There is preliminary but striking 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 main stock for a significant action of the genotyped inbred samples. We conclude that MiniMUGA is a valuable platform for genetic QC and important new tool to the increase rigor and reproducibility of mouse research.

genetics↗