In vivo Interactions between myosin XI, vesicles, and filamentous actin are fast and transient

Abstract

The actin cytoskeleton and active membrane trafficking machinery are essential for polarized cell growth. To understand the interactions between myosin XI, vesicles, and actin filament in vivo, we performed Fluorescence Recovery After Photobleaching and showed that the dynamics of myosin XIa at the tip of Physcomitrella patens caulonemal cells are actin-dependent and that approximately half of myosin XI is bound to vesicles. To obtain single-particle information, we used Variable Angle Epifluorescence Microscopy in protoplasts to demonstrate that myosin XIa and VAMP72-labeled vesicles localize in time and space for periods lasting only a few seconds. Using tracking data with Hidden Markov Modeling, we showed that myosin XIa and VAMP72-labeled vesicles exhibit short runs of actin-dependent directed transport. We also found that the interaction of myosin XI with vesicles is short-lived. Together, this bound fraction, fast off-rate, and short runlengths are expected to be critical for the dynamic oscillations observed at the tip, and may be vital for the regulation and recycling of the exocytosis machinery; while simultaneously promoting the vesicle focusing and secretion at the tip, necessary for cell wall expansion.