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 filaments in vivo, we performed fluorescence recovery after photobleaching and showed that the dynamics of myosin XIa at the tip of the spreading earthmoss Physcomitrella patens caulonemal cells are actin-dependent and that 50% of myosin XI is bound to vesicles. To obtain single-particle information, we used variable-angle epifluorescence microscopy in protoplasts to demonstrate that protein myosin XIa and VAMP72-labeled vesicles localize in time and space over periods lasting only a few seconds. By 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 vesicle-bound fraction, fast off-rate and short average distance traveled seem be crucial for the dynamic oscillations observed at the tip, and might be vital for regulation and recycling of the exocytosis machinery, while simultaneously promoting vesicle focusing and vesicle secretion at the tip, necessary for cell wall expansion.
Highlights
Polarized cell growth, a mechanism by which plasma membrane and cell wall material are deposited to a defined region of the cell, is widespread across the plant kingdom
At early times during fluorescence recovery, we found that myosin XIa could be detected recovering at the cell apex before VAMP72-labeled vesicles (Figure 1B)
We have shown for the first time to the best of our knowledge, that myosin XIa and VAMP72-labeled vesicles co-localize in vivo, and exhibit actin dependent mobility that was observed as short persistent trajectories
Summary
A mechanism by which plasma membrane and cell wall material are deposited to a defined region of the cell, is widespread across the plant kingdom. To better understand how tip growing cells selforganize their cytoplasmic components to achieve and maintain polarized growth, it is critical to determine the function of each component separately as well as their interactions Because of this limited information, quantitative modeling efforts must coarse grain the function of cytoskeleton as directed secretion fluxes (Campàs and Mahadevan, 2009; Dumais et al, 2006; Fayant et al, 2010; Kroeger et al, 2011; Luo et al, 2017; Rojas et al, 2011). Modeling efforts that incorporate the true interactions of the cytoskeleton, its motors, and cargo will help answer questions regarding the establishment and maintenance of polarized secretion
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