Publications by authors named "Richard E Cheney"

38 Publications

MYO10 drives genomic instability and inflammation in cancer.

Sci Adv 2021 Sep 15;7(38):eabg6908. Epub 2021 Sep 15.

Department of Pharmacology, Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.

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http://dx.doi.org/10.1126/sciadv.abg6908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8443186PMC
September 2021

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

Elife 2021 09 14;10. Epub 2021 Sep 14.

Department of Pharmacology & Therapeutics, University of Florida College of Medicine, Gainesville, United States.

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 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.
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http://dx.doi.org/10.7554/eLife.72419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8500716PMC
September 2021

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

Elife 2021 02 11;10. Epub 2021 Feb 11.

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, United States.

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 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.
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http://dx.doi.org/10.7554/eLife.61432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968926PMC
February 2021

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

iScience 2020 Dec 13;23(12):101802. Epub 2020 Nov 13.

Department of Cancer Biology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA.

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.
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http://dx.doi.org/10.1016/j.isci.2020.101802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702012PMC
December 2020

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

Genetics 2020 12 16;216(4):905-930. Epub 2020 Oct 16.

Department of Genetics, University of North Carolina, Chapel Hill, North Carolina 27599.

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 O and 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.
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http://dx.doi.org/10.1534/genetics.120.303596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768238PMC
December 2020

A new light chain for myosin-7.

J Biol Chem 2020 07;295(28):9297-9298

Dept. of Cell Biology and Physiology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA

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 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 also report that CALML4 can bind to myosin-7a, this work also has important implications for research on myosin-7a and hereditary deaf-blindness.
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http://dx.doi.org/10.1074/jbc.H120.014595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363122PMC
July 2020

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

Anaerobe 2019 Oct 3;59:205-211. Epub 2019 Aug 3.

Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.anaerobe.2019.102080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785396PMC
October 2019

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

Sci Rep 2017 12 11;7(1):17354. Epub 2017 Dec 11.

Department of Cell Biology and Physiology and Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.

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 ), floxed (Myo10 ), and KO mice (Myo10 ). 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 ) 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.
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http://dx.doi.org/10.1038/s41598-017-17638-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725431PMC
December 2017

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

Infect Immun 2017 09 18;85(9). Epub 2017 Aug 18.

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, USA

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 , 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 630. Our studies reveal that 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 increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to 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.
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http://dx.doi.org/10.1128/IAI.00347-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563577PMC
September 2017

Myosin-X and disease.

Exp Cell Res 2015 May 27;334(1):10-5. Epub 2015 Mar 27.

Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, United States; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.yexcr.2015.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433855PMC
May 2015

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

J Biol Chem 2014 Nov 16;289(48):33513-28. Epub 2014 Oct 16.

From the Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523,

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.
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http://dx.doi.org/10.1074/jbc.M114.578948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246105PMC
November 2014

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

J Cell Sci 2013 Oct 13;126(Pt 20):4756-68. Epub 2013 Aug 13.

Department of Biological Sciences and Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, Tennessee 37235, USA.

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.
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http://dx.doi.org/10.1242/jcs.132969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795341PMC
October 2013

DPP6 regulation of dendritic morphogenesis impacts hippocampal synaptic development.

Nat Commun 2013 ;4:2270

Molecular Neurophysiology and Biophysics Section, Program in Developmental Neuroscience, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

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.
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http://dx.doi.org/10.1038/ncomms3270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3775611PMC
February 2014

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

Cell Microbiol 2013 Mar 13;15(3):353-367. Epub 2012 Nov 13.

Department of Medicine, Division of Infectious Diseases, University of Florida College of Medicine, Gainesville, FL, USA.

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.
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http://dx.doi.org/10.1111/cmi.12051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070382PMC
March 2013

Myosins in cell junctions.

Bioarchitecture 2012 Sep-Oct;2(5):158-70. Epub 2012 Sep 1.

Department of Cell and Molecular Physiology; School of Medicine; University of North Carolina at Chapel Hill; Chapel Hill, NC USA.

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.
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http://dx.doi.org/10.4161/bioa.21791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696060PMC
December 2014

Headless Myo10 is a negative regulator of full-length Myo10 and inhibits axon outgrowth in cortical neurons.

J Biol Chem 2012 Jul 31;287(30):24873-83. Epub 2012 May 31.

Curriculum in Neurobiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

Myo10 is an unconventional myosin that localizes to and induces filopodia, structures that are critical for growing axons. In addition to the ~240-kDa full-length Myo10, brain expresses a ~165 kDa isoform that lacks a functional motor domain and is known as headless Myo10. We and others have hypothesized that headless Myo10 acts as an endogenous dominant negative of full-length Myo10, but this hypothesis has not been tested, and the function of headless Myo10 remains unknown. We find that cortical neurons express both headless and full-length Myo10 and report the first isoform-specific localization of Myo10 in brain, which shows enrichment of headless Myo10 in regions of proliferating and migrating cells, including the embryonic ventricular zone and the postnatal rostral migratory stream. We also find that headless and full-length Myo10 are expressed in embryonic and neuronal stem cells. To directly test the function of headless and full-length Myo10, we used RNAi specific to each isoform in mouse cortical neuron cultures. Knockdown of full-length Myo10 reduces axon outgrowth, whereas knockdown of headless Myo10 increases axon outgrowth. To test whether headless Myo10 antagonizes full-length Myo10, we coexpressed both isoforms in COS-7 cells, which revealed that headless Myo10 suppresses the filopodia-inducing activity of full-length Myo10. Together, these results demonstrate that headless Myo10 can function as a negative regulator of full-length Myo10 and that the two isoforms of Myo10 have opposing roles in axon outgrowth.
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http://dx.doi.org/10.1074/jbc.M112.369173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408153PMC
July 2012

Myosin-X functions in polarized epithelial cells.

Mol Biol Cell 2012 May 14;23(9):1675-87. Epub 2012 Mar 14.

Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein-Myo10 localizes to lateral membrane cell-cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis.
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http://dx.doi.org/10.1091/mbc.E11-04-0358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338435PMC
May 2012

Myosin-X: a MyTH-FERM myosin at the tips of filopodia.

J Cell Sci 2011 Nov;124(Pt 22):3733-41

Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545, USA.

Myosin-X (Myo10) is an unconventional myosin with MyTH4-FERM domains that is best known for its striking localization to the tips of filopodia and its ability to induce filopodia. Although the head domain of Myo10 enables it to function as an actin-based motor, its tail contains binding sites for several molecules with central roles in cell biology, including phosphatidylinositol (3,4,5)-trisphosphate, microtubules and integrins. Myo10 also undergoes fascinating long-range movements within filopodia, which appear to represent a newly recognized system of transport. Myo10 is also unusual in that it is a myosin with important roles in the spindle, a microtubule-based structure. Exciting new studies have begun to reveal the structure and single-molecule properties of this intriguing myosin, as well as its mechanisms of regulation and induction of filopodia. At the cellular and organismal level, growing evidence demonstrates that Myo10 has crucial functions in numerous processes ranging from invadopodia formation to cell migration.
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http://dx.doi.org/10.1242/jcs.023549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225264PMC
November 2011

Myosin X regulates sealing zone patterning in osteoclasts through linkage of podosomes and microtubules.

J Biol Chem 2010 Mar 17;285(13):9506-9515. Epub 2010 Jan 17.

Department of Physiology and Cell Biology, The Ohio State University College of Medicine, Columbus, Ohio 43210. Electronic address:

Osteoclasts use actin-rich attachment structures in place of focal adhesions for adherence to bone and non-bone substrates. On glass, osteoclasts generate podosomes, foot-like processes containing a core of F-actin and regulatory proteins that undergo high turnover. To facilitate bone resorption, osteoclasts generate an actin-rich sealing zone composed of densely packed podosome-like units. Patterning of both podosomes and sealing zones is dependent upon an intact microtubule system. A role for unconventional myosin X (Myo10), which can bind actin, microtubules, and integrins, was examined in osteoclasts. Immunolocalization showed Myo10 to be associated with the outer edges of immature podosome rings and sealing zones, suggesting a possible role in podosome and sealing zone positioning. Further, complexes containing both Myo10 and beta-tubulin were readily precipitated from osteoclasts lysates. RNAi-mediated suppression of Myo10 led to decreased cell and sealing zone perimeter, along with decreased motility and resorptive capacity. Further, siRNA-treated cells could not properly position podosomes following microtubule disruption. Osteoclasts overexpressing dominant negative Myo10 microtubule binding domains (MyTH4) showed a similar phenotype. Conversely, overexpression of full-length Myo10 led to increased formation of podosome belts along with larger sealing zones and enhanced bone resorptive capacity. These studies suggest that Myo10 plays a role in osteoclast attachment and podosome positioning by direct linkage of actin to the microtubule network.
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http://dx.doi.org/10.1074/jbc.M109.017269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2843201PMC
March 2010

Human Myo19 is a novel myosin that associates with mitochondria.

Curr Biol 2009 Dec 26;19(23):2008-13. Epub 2009 Nov 26.

Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Mitochondria are pleomorphic organelles that have central roles in cell physiology. Defects in their localization and dynamics lead to human disease. Myosins are actin-based motors that power processes such as muscle contraction, cytokinesis, and organelle transport. Here we report the initial characterization of myosin-XIX (Myo19), the founding member of a novel class of myosin that associates with mitochondria. The 970 aa heavy chain consists of a motor domain, three IQ motifs, and a short tail. Myo19 mRNA is expressed in multiple tissues, and antibodies to human Myo19 detect an approximately 109 kDa band in multiple cell lines. Both endogenous Myo19 and GFP-Myo19 exhibit striking localization to mitochondria. Deletion analysis reveals that the Myo19 tail is necessary and sufficient for mitochondrial localization. Expressing full-length GFP-Myo19 in A549 cells reveals a remarkable gain of function where the majority of the mitochondria move continuously. Moving mitochondria travel for many micrometers with an obvious leading end and distorted shape. The motility and shape change are sensitive to latrunculin B, indicating that both are actin dependent. Expressing the GFP-Myo19 tail in CAD cells resulted in decreased mitochondrial run lengths in neurites. These results suggest that this novel myosin functions as an actin-based motor for mitochondrial movement in vertebrate cells.
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http://dx.doi.org/10.1016/j.cub.2009.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805763PMC
December 2009

Myosin Vc is a molecular motor that functions in secretory granule trafficking.

Mol Biol Cell 2009 Nov 9;20(21):4471-88. Epub 2009 Sep 9.

Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Class V myosins are actin-based motor proteins that have critical functions in organelle trafficking. Of the three class V myosins expressed in mammals, relatively little is known about Myo5c except that it is abundant in exocrine tissues. Here we use MCF-7 cells to identify the organelles that Myo5c associates with, image the dynamics of Myo5c in living cells, and test the functions of Myo5c. Endogenous Myo5c localizes to two distinct compartments: small puncta and slender tubules. Myo5c often exhibits a highly polarized distribution toward the leading edge in migrating cells and is clearly distinct from the Myo5a or Myo5b compartments. Imaging with GFP-Myo5c reveals that Myo5c puncta move slowly (approximately 30 nm/s) and microtubule independently, whereas tubules move rapidly (approximately 440 nm/s) and microtubule dependently. Myo5c puncta colocalize with secretory granule markers such as chromogranin A and Rab27b, whereas Myo5c tubules are labeled by Rab8a. TIRF imaging indicates that the granules can be triggered to undergo secretion. To test if Myo5c functions in granule trafficking, we used the Myo5c tail as a dominant negative and found that it dramatically perturbs the distribution of granule markers. These results provide the first live-cell imaging of Myo5c and indicate that Myo5c functions in secretory granule trafficking.
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http://dx.doi.org/10.1091/mbc.e08-08-0865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2770936PMC
November 2009

A novel form of motility in filopodia revealed by imaging myosin-X at the single-molecule level.

Curr Biol 2009 Jun 23;19(11):967-73. Epub 2009 Apr 23.

Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Although many proteins, receptors, and viruses are transported rearward along filopodia by retrograde actin flow, it is less clear how molecules move forward in filopodia. Myosin-X (Myo10) is an actin-based motor hypothesized to use its motor activity to move forward along actin filaments to the tips of filopodia. Here we use a sensitive total internal reflection fluorescence (TIRF) microscopy system to directly visualize the movements of GFP-Myo10. This reveals a novel form of motility at or near the single-molecule level in living cells wherein extremely faint particles of Myo10 move in a rapid and directed fashion toward the filopodial tip. These fast forward movements occur at approximately 600 nm/s over distances of up to approximately 10 microm and require Myo10 motor activity and actin filaments. As expected for imaging at the single-molecule level, the faint particles of GFP-Myo10 are diffraction limited, have an intensity range similar to single GFP molecules, and exhibit stepwise bleaching. Faint particles of GFP-Myo5a can also move toward the filopodial tip, but at a slower characteristic velocity of approximately 250 nm/s. Similar movements were not detected with GFP-Myo1a, indicating that not all myosins are capable of intrafilopodial motility. These data indicate the existence of a novel system of long-range transport based on the rapid movement of myosin molecules along filopodial actin filaments.
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http://dx.doi.org/10.1016/j.cub.2009.03.067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817954PMC
June 2009

Transduced viral IL-10 is exocytosed from lacrimal acinar secretory vesicles in a myosin-dependent manner in response to carbachol.

Exp Eye Res 2009 Mar 13;88(3):467-78. Epub 2008 Nov 13.

Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, USA.

The purpose of this study was to determine the intracellular trafficking and release pathways for the therapeutic protein, viral IL-10 (vIL-10), from transduced acinar epithelial cells from rabbit lacrimal gland. Primary cultured rabbit lacrimal gland acinar cells (LGACs) were transduced with adenovirus serotype 5 containing viral interleukin-10 (AdvIL-10). The distribution of vIL-10 was assessed by confocal fluorescence microscopy. Carbachol (CCH)-stimulated release of vIL-10 was quantified by ELISA. vIL-10 localization and exocytosis was probed in response to treatments with agents modulating actin- and myosin-based transport. vIL-10 immunoreactivity was detected in large intracellular vesicles in transduced LGAC. vIL-10 was partially co-localized with biosynthetic but not endosomal compartment markers. vIL-10 release was sensitive to CCH, and the kinetics of release showed an initial burst phase that was similar but not identical to that of the secretory protein, beta-hexosaminidase. Disassembly of actin filaments with latrunculin B significantly increased CCH-stimulated vIL-10 secretion, suggesting that vIL-10 was released from stores sequestered beneath the subapical actin barrier. That release required the activity of actin-dependent myosin motors previously implicated in secretory vesicle exocytosis was confirmed by findings that CCH-stimulated vIL-10 release was reduced by inhibition of non-muscle myosin 2 and myosin 5c function, using ML-7 and overexpression of dominant negative myosin 5c, respectively. These results suggest that the majority of vIL-10 transgene product is packaged into a subpopulation of secretory vesicles that utilize actin-dependent myosin motors for aspects of actin coat assembly, compound fusion and exocytosis at the apical plasma membrane in response to CCH stimulation.
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http://dx.doi.org/10.1016/j.exer.2008.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2656410PMC
March 2009

The class V myosin motor, myosin 5c, localizes to mature secretory vesicles and facilitates exocytosis in lacrimal acini.

Am J Physiol Cell Physiol 2008 Jul 23;295(1):C13-28. Epub 2008 Apr 23.

Department Pharmacology and Pharmaceutical Sciences, USC School of Pharmacy, Los Angeles, CA 90033, USA.

We investigated the role of the actin-based myosin motor, myosin 5c (Myo5c) in vesicle transport in exocrine secretion. Lacrimal gland acinar cells (LGAC) are the major source for the regulated secretion of proteins from the lacrimal gland into the tear film. Confocal fluorescence and immunogold electron microscopy revealed that Myo5c was associated with secretory vesicles in primary rabbit LGAC. Upon stimulation of secretion with the muscarinic agonist, carbachol, Myo5c was also detected in association with actin-coated fusion intermediates. Adenovirus-mediated expression of green fluorescent protein (GFP) fused to the tail domain of Myo5c (Ad-GFP-Myo5c-tail) showed that this protein was localized to secretory vesicles. Furthermore, its expression induced a significant (P < or = 0.05) decrease in carbachol-stimulated release of two secretory vesicle content markers, secretory component and syncollin-GFP. Adenovirus-mediated expression of GFP appended to the full-length Myo5c (Ad-GFP-Myo5c-full) was used in parallel with adenovirus-mediated expression of GFP-Myo5c-tail in LGAC to compare various parameters of secretory vesicles labeled with either GFP-labeled protein in resting and stimulated LGAC. These studies revealed that the carbachol-stimulated increase in secretory vesicle diameter associated with compound fusion of secretory vesicles that was also exhibited by vesicles labeled with GFP-Myo5c-full was impaired in vesicles labeled with GFP-Myo5c-tail. A significant decrease in GFP labeling of actin-coated fusion intermediates was also seen in carbachol-stimulated LGAC transduced with GFP-Myo5c-tail relative to LGAC transduced with GFP-Myo5c-full. These results suggest that Myo5c participates in apical exocytosis of secretory vesicles.
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http://dx.doi.org/10.1152/ajpcell.00330.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2493552PMC
July 2008

Human myosin Vc is a low duty ratio, nonprocessive molecular motor.

J Biol Chem 2008 Mar 16;283(13):8527-37. Epub 2008 Jan 16.

Laboratory of Molecular Physiology, NHLBI, NIH, Bethesda, MD 20892-8015, USA.

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a V(max) of 1.8 +/- 0.3 s(-1) and a K(ATPase) of 43 +/- 11 microm. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (~1.5 s(-1)), rather than the ADP release step (~12.0-16.0 s(-1)). Nevertheless, the ADP affinity of actomyosin Vc (K(d) = 0.25 +/- 0.02 microm) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was approximately 10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of approximately 24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration.
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http://dx.doi.org/10.1074/jbc.M709150200DOI Listing
March 2008

Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors.

J Cell Biol 2007 Dec 24;179(7):1569-82. Epub 2007 Dec 24.

Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, NC 27599, USA.

Endothelial cell migration is an important step during angiogenesis, and its dysregulation contributes to aberrant neovascularization. The bone morphogenetic proteins (BMPs) are potent stimulators of cell migration and angiogenesis. Using microarray analyses, we find that myosin-X (Myo10) is a BMP target gene. In endothelial cells, BMP6-induced Myo10 localizes in filopodia, and BMP-dependent filopodial assembly decreases when Myo10 expression is reduced. Likewise, cellular alignment and directional migration induced by BMP6 are Myo10 dependent. Surprisingly, we find that Myo10 and BMP6 receptor ALK6 colocalize in a BMP6-dependent fashion. ALK6 translocates into filopodia after BMP6 stimulation, and both ALK6 and Myo10 possess intrafilopodial motility. Additionally, Myo10 is required for BMP6-dependent Smad activation, indicating that in addition to its function in filopodial assembly, Myo10 also participates in a requisite amplification loop for BMP signaling. Our data indicate that Myo10 is required to guide endothelial migration toward BMP6 gradients via the regulation of filopodial function and amplification of BMP signals.
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http://dx.doi.org/10.1083/jcb.200704010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373493PMC
December 2007

Myosin Vb is required for trafficking of the cystic fibrosis transmembrane conductance regulator in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.

J Biol Chem 2007 Aug 26;282(32):23725-36. Epub 2007 Apr 26.

Department of Physiology, Dartmouth Medical School, Dartmouth College, Hanover, New Hampshire 03755, USA.

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl(-) channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and DeltaF508-CFTR in the apical membrane and decreased CFTR-mediated Cl(-) secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.
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http://dx.doi.org/10.1074/jbc.M608531200DOI Listing
August 2007

Cdc42 and ARP2/3-independent regulation of filopodia by an integral membrane lipid-phosphatase-related protein.

J Cell Sci 2007 Jan 2;120(Pt 2):340-52. Epub 2007 Jan 2.

Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, USA.

Filopodia are dynamic cell surface protrusions that are required for proper cellular development and function. We report that the integral membrane protein lipid-phosphatase-related protein 1 (LPR1) localizes to and promotes the formation of actin-rich, dynamic filopodia, both along the cell periphery and the dorsal cell surface. Regulation of filopodia by LPR1 was not mediated by cdc42 or Rif, and is independent of the Arp2/3 complex. We found that LPR1 can induce filopodia formation in the absence of the Ena/Vasp family of proteins, suggesting that these molecules are not essential for the development of the protrusions. Mutagenesis experiments identified residues and regions of LPR1 that are important for the induction of filopodia. RNA interference experiments in an ovarian epithelial cancer cell line demonstrated a role for LPR1 in the maintenance of filopodia-like membrane protrusions. These observations, and our finding that LPR1 is a not an active lipid phosphatase, suggest that LPR1 may be a novel integral membrane protein link between the actin core and the surrounding lipid layer of a nascent filopodium.
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http://dx.doi.org/10.1242/jcs.03335DOI Listing
January 2007

Budding of Marburgvirus is associated with filopodia.

Cell Microbiol 2007 Apr 28;9(4):939-51. Epub 2006 Nov 28.

Institute of Virology, Marburg Philipps University, Marburg, Germany.

Viruses exploit the cytoskeleton of host cells to transport their components and spread to neighbouring cells. Here we show that the actin cytoskeleton is involved in the release of Marburgvirus (MARV) particles. We found that peripherally located nucleocapsids and envelope precursors of MARV are located either at the tip or at the side of filopodial actin bundles. Importantly, viral budding was almost exclusively detected at filopodia. Inhibiting actin polymerization in MARV-infected cells significantly diminished the amount of viral particles released into the medium. This suggested that dynamic polymerization of actin in filopodia is essential for efficient release of MARV. The viral matrix protein VP40 plays a key role in the release of MARV particles and we found that the intracellular localization of recombinant VP40 and its release in form of virus-like particles were strongly influenced by overexpression or inhibition of myosin 10 and Cdc42, proteins important in filopodia formation and function. We suggest that VP40, which is capable of interacting with viral nucleocapsids, provides an interface of MARV subviral particles and filopodia. As filopodia are in close contact with neighbouring cells, usurpation of these structures may facilitate spread of MARV to adjacent cells.
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http://dx.doi.org/10.1111/j.1462-5822.2006.00842.xDOI Listing
April 2007

Myosin-X is a molecular motor that functions in filopodia formation.

Proc Natl Acad Sci U S A 2006 Aug 7;103(33):12411-6. Epub 2006 Aug 7.

Department of Cell and Molecular Physiology, Medical Biomolecular Research Building (MBRB), Room 5314, 103 Mason Farm Road, University of North Carolina, Chapel Hill, NC 27599-7545, USA.

Despite recent progress in understanding lamellipodia extension, the molecular mechanisms regulating filopodia formation remain largely unknown. Myo10 is a MyTH4-FERM myosin that localizes to the tips of filopodia and is hypothesized to function in filopodia formation. To determine whether endogenous Myo10 is required for filopodia formation, we have used scanning EM to assay the numerous filopodia normally present on the dorsal surfaces of HeLa cells. We show here that siRNA-mediated knockdown of Myo10 in HeLa cells leads to a dramatic loss of dorsal filopodia. Overexpressing the coiled coil region from Myo10 as a dominant- negative also leads to a loss of dorsal filopodia, thus providing independent evidence that Myo10 functions in filopodia formation. We also show that expressing Myo10 in COS-7 cells, a cell line that normally lacks dorsal filopodia, leads to a massive induction of dorsal filopodia. Because the dorsal filopodia induced by Myo10 are not attached to the substrate, Myo10 can promote filopodia by a mechanism that is independent of substrate attachment. Consistent with this observation, a Myo10 construct that lacks the FERM domain, the region that binds to integrin, retains the ability to induce dorsal filopodia. Deletion of the MyTH4-FERM region, however, completely abolishes Myo10's filopodia-promoting activity, as does deletion of the motor domain. Additional experiments on the mechanism of Myo10 action indicate that it acts downstream of Cdc42 and can promote filopodia in the absence of VASP proteins. Together, these data demonstrate that Myo10 is a molecular motor that functions in filopodia formation.
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http://dx.doi.org/10.1073/pnas.0602443103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1567893PMC
August 2006
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