title: Filopodia formation and endosome clustering induced by mutant plus-end-directed myosin VI.
abstract: Myosin VI (MYO6) is the only myosin known to move toward the minus end of actin filaments. It has roles in numerous cellular processes, including maintenance of stereocilia structure, endocytosis, and autophagosome maturation. However, the functional necessity of minus-end-directed movement along actin is unclear as the underlying architecture of the local actin network is often unknown. To address this question, we engineered a mutant of MYO6, MYO6+, which undergoes plus-end-directed movement while retaining physiological cargo interactions in the tail. Expression of this mutant motor in HeLa cells led to a dramatic reorganization of cortical actin filaments and the formation of actin-rich filopodia. MYO6 is present on peripheral adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling endosomes and MYO6+ expression causes a dramatic relocalization and clustering of this endocytic compartment in the cell cortex. MYO6+ and its adaptor GAIP interacting protein, C terminus (GIPC) accumulate at the tips of these filopodia, while APPL1 endosomes accumulate at the base. A combination of MYO6+ mutagenesis and siRNA-mediated depletion of MYO6 binding partners demonstrates that motor activity and binding to endosomal membranes mediated by GIPC and PI(4,5)P2are crucial for filopodia formation. A similar reorganization of actin is induced by a constitutive dimer of MYO6+, indicating that multimerization of MYO6 on endosomes through binding to GIPC is required for this cellular activity and regulation of actin network structure. This unique engineered MYO6+ offers insights into both filopodia formation and MYO6 motor function at endosomes and at the plasma membrane.
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom fb207@cam.ac.uk tam55@cam.ac.uk.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Masters
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom fb207@cam.ac.uk tam55@cam.ac.uk.
authors.forename: Folma
authors.initials: F
authors.lastname: Buss
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D009154:Mutation; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011554:Pseudopodia / Q000235:genetics / Q000378:metabolism*; D059008:Time-Lapse Imaging / Q000379:methods
chemical_list: C464801:APPL1 protein, human; D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C089785:myosin VI; D018995:Myosin Heavy Chains
keywords: actin dynamics; endosomes; filopodia; motor–cargo complexes; unconventional myosins