title: A Combination of Diffusion and Active Translocation Localizes Myosin 10 to the Filopodial Tip.
abstract: Myosin 10 is an actin-based molecular motor that localizes to the tips of filopodia in mammalian cells. To understand how it is targeted to this distinct region of the cell, we have used total internal reflection fluorescence microscopy to study the movement of individual full-length and truncated GFP-tagged molecules. Truncation mutants lacking the motor region failed to localize to filopodial tips but still bound transiently at the plasma membrane. Deletion of the single α-helical and anti-parallel coiled-coil forming regions, which lie between the motor and pleckstrin homology domains, reduced the instantaneous velocity of intrafilopodial movement but did not affect the number of substrate adherent filopodia. Deletion of the anti-parallel coiled-coil forming region, but not the EKR-rich region of the single α-helical domain, restored intrafilopodial trafficking, suggesting this region is important in determining myosin 10 motility. We propose a model by which myosin 10 rapidly targets to the filopodial tip via a sequential reduction in dimensionality. Molecules first undergo rapid diffusion within the three-dimensional volume of the cell body. They then exhibit periods of slower two-dimensional diffusion in the plane of the plasma membrane. Finally, they move in a unidimensional, highly directed manner along the polarized actin filament bundle within the filopodium becoming confined to a single point at the tip. Here we have observed directly each phase of the trafficking process using single molecule fluorescence imaging of live cells and have quantified our observations using single particle tracking, autocorrelation analysis, and kymographs.
authors: From the Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT and.
authors: Thomas G
authors: TG
authors: Baboolal
authors: The Francis Crick Institute, Mill Hill Laboratory, London NW7 1AA, United Kingdom.
authors: Gregory I
authors: GI
authors: Mashanov
authors: The Francis Crick Institute, Mill Hill Laboratory, London NW7 1AA, United Kingdom.
authors: Tatiana A
authors: TA
authors: Nenasheva
authors: From the Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT and m.peckham@leeds.ac.uk.
authors: Michelle
authors: M
authors: Peckham
authors: The Francis Crick Institute, Mill Hill Laboratory, London NW7 1AA, United Kingdom justin.molloy@crick.ac.uk.
authors: Justin E
authors: JE
authors: Molloy
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D021381:Protein Transport / Q000502:physiology; D011554:Pseudopodia / Q000235:genetics / Q000378:metabolism*
chemical_list: D009218:Myosins
keywords: TIRF; actin; molecular imaging; myosin; single particle analysis; single-molecule biophysics