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Supported by Deutsche Forschungsgemeinschaft, SFB 863, B6Myosin-XXI is one of only two myosin isoforms found in the genome of the Leishmania parasite. It is involved in the assembly and elongation of the flagellum and in intracellular trafficking. Using mammalian cell transfections, FRAP (fluorescence recovery after photobleaching) measurements and single particle tracking, we elucidated myosin-XXI dynamics in vitro and in vivo. Transfections with myosin-XXI resulted in an accumulation of myosin at the plasma membrane and inside the tips of filopodia. A minimal construct encoding the N-terminal SH3 domain, motor domain, and parts of the tail upto aa 808 was required for this localization pattern. Notably, the myosin-XXI tail contains several lipid binding sites. Live imaging revealed that filopodial myosin accumulations endured length changes of filopodia, pointing toward the possibility of myosin-XXI being stably bound to the plasma membrane. Additionally, we observed an increase in the length and density of filopodia in transfected cells, indicating that the myosin might tether the actin cytoskeleton to the plasma membrane thus stabilizing the architecture of membrane protrusions. To investigate the dynamic behavior of this myosin when bound to membranes, we anchored myosin-XXI to planar supported lipid bilayers via a biotin-streptavidin-biotin link and investigated its diffusion by FRAP and single particle imaging. Furthermore, we studied the myosin's capability of moving filamentous actin when bound to lipids. In filament gliding assays on lipid vesicles, vesicle size appeared to influence filament motility. Accordingly, we tested myosin-XXI for curvature sensitivity employing SLiC (single liposome curvature) assays. |
