Abstract
Myosin-X, (Myo 10), is an unconventional myosin that transports the specific cargos to filopodial tips, and is associated with the mechanism underlying filopodia formation and extension. To clarify the innate motor characteristic, we studied the single molecule movement of a full-length myosin-X construct with leucine zipper at the C-terminal end of the tail (M10FullLZ) and the tail-truncated myosin-X without artificial dimerization motif (BAP-M101–979HMM). M10FullLZ localizes at the tip of filopodia like myosin-X full-length (M10Full). M10FullLZ moves on actin filaments in the presence of PI(3,4,5)P3, an activator of myosin-X. Single molecule motility analysis revealed that the step sizes of both M10FullLZ and BAP-M101–979HMM are widely distributed on single actin filaments that is consistent with electron microscopy observation. M10FullLZ moves on filopodial actin bundles of cells with a mean step size (~36 nm), similar to the step size on single actin filaments (~38 nm). Cartesian plot analysis revealed that M10FullLZ meandered on filopodial actin bundles to both x- and y- directions. These results suggest that the lever-arm of full-length myosin-X is flexible enough to processively steps on different actin filaments within the actin bundles of filopodia. This characteristic of myosin-X may facilitate actin filament convergence for filopodia production.
Highlights
Myosin-X is a member of the myosin superfamily and found in vertebrates and in filasterea[1,2]
It was found that phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) binding at the PH domain of the tail activates the motor activity and induces dimer formation[19]. These results suggest that full-length myosin-X becomes active upon binding PI(3,4,5)P3 and that the endogenous coiled-coil domain can form dimer the stability of the dimer is weak
To check the functional authenticity, we examined whether this myosin-X construct facilitates filopodia formation in mammalian cells (Supplementary Fig. 2)
Summary
Myosin-X is a member of the myosin superfamily and found in vertebrates and in filasterea[1,2]. Sun et al.[15] showed that the tail-truncated forced dimer of myosin-X moves on single actin filaments as well as actin bundles without notable difference in processivity. Bao et al revealed that the tail-truncated forced dimer of myosin-X (1–940 amino acids) takes ~31 nm steps in both single and actin bundles[14]. Takagi et al.[18] recently reported that tail-truncated forced dimer of myosin-X (1–936 amino acids) takes 35 nm forward steps on single actin filaments in single molecule optical tweezers experiments. A question is how myosin-X can be a cargo transporting motor if it is a monomeric myosin Answering this question, it was found that phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) binding at the PH domain of the tail activates the motor activity and induces dimer formation[19]. These results suggest that full-length myosin-X becomes active upon binding PI(3,4,5)P3 and that the endogenous coiled-coil domain can form dimer the stability of the dimer is weak
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