Myosin X is recruited to nascent focal adhesions at the leading edge and induces multi-cycle filopodial elongation

Kangmin He,Yoshikazu Tsukasaki,Mitsuo Ikebe,Tsuyoshi Sakai,Tomonobu M Watanabe

doi:10.1038/s41598-017-06147-6

Abstract

Filopodia protrude from the leading edge of cells and play important roles in cell motility. Here we report the mechanism of myosin X (encoded by Myo10)-induced multi-cycle filopodia extension. We found that actin, Arp2/3, vinculin and integrin-β first accumulated at the cell’s leading edge. Myosin X was then gathered at these sites, gradually clustered by lateral movement, and subsequently initiated filopodia formation. During filopodia extension, we found the translocation of Arp2/3 and integrin-β along filopodia. Arp2/3 and integrin-β then became localized at the tip of filopodia, from where myosin X initiated the second extension of filopodia with a change in extension direction, thus producing long filopodia. Elimination of integrin-β, Arp2/3 and vinculin by siRNA significantly attenuated the myosin-X-induced long filopodia formation. We propose the following mechanism. Myosin X accumulates at nascent focal adhesions at the cell’s leading edge, where myosin X promotes actin convergence to create the base of filopodia. Then myosin X moves to the filopodia tip and attracts integrin-β and Arp2/3 for further actin nucleation. The tip-located myosin X then initiates the second cycle of filopodia elongation to produce the long filopodia.

Highlights

Filopodia are finger-like cell surface projections composed of parallel bundles of actin filaments and are found in a variety of cell types and play sensory or exploratory functions in cell migration, adhesion to the extracellular matrix, guidance towards chemoattractants, neuronal growth-cone path finding and embryonic development[1,2,3]
EGFP-myosin X were transiently co-expressed in COS7 cells together with mCherry-Arp2/3, vinculin-tomato, or mCherry-VASP
With triple-color total internal reflection fluorescence microscopy (TIRFM) imaging, we found that mCherry-Arp2/3 colocalized with EGFP-myosin X at the tip as well as shaft of filopodia (Fig. 1A and B)

Summary

Introduction

Filopodia are finger-like cell surface projections composed of parallel bundles of actin filaments and are found in a variety of cell types and play sensory or exploratory functions in cell migration, adhesion to the extracellular matrix, guidance towards chemoattractants, neuronal growth-cone path finding and embryonic development[1,2,3]. It was postulated that myosin X binds to PtdIns(3,4,5)P3 at the cell’s leading edge to form a dimer, and utilizes its motor function to induce actin filament convergence to initiate filopodia formation[7]. Since the filopodia induced by the tailless myosin X dimer was short and unstable[5], it was thought that transportation of the tail-binding cargo molecules played a role in producing the stable and long filopodia. Supporting this view, actin-regulating proteins (e.g. VASP and integrin-β) have been found to be involved in regulating myosin X-induced filopodia formation[4,21]. The filopodia protrusion is performed by machinery composed of the interactions among actin-associated proteins mediated by myosin X

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