F-Actin Mediated Focusing of Vesicles at the Cell Tip Is Essential for Polarized Growth

Abstract

(L.V.).F-actin has been shown to be essential for tip growth in an array of plant models, including Physcomitrella patens. One hypothesis is that diffusion can transport secretory vesicles, while actin plays a regulatory role during secretion. Alternatively, it is possible that actin-based transport is necessary to overcome vesicle transport limitations to sustain secretion. Therefore, a quantitative analysis of diffusion, secretion kinetics, and cell geometry is necessary to clarify the role of actin in polarized growth. Using fluorescence recovery after photobleaching analysis, we first show that secretory vesicles move toward and accumulate at the tip in an actin-dependent manner. We then depolymerized F-actin to decouple vesicle diffusion from actin-mediated transport and measured the diffusion coefficient and concentration of vesicles. Using these values, we constructed a theoretical diffusion-based model for growth, demonstrating that with fast-enough vesicle fusion kinetics, diffusion could support normal cell growth rates. We further refined our model to explore how experimentally extrapolated vesicle fusion kinetics and the size of the secretion zone limit diffusion-based growth. This model predicts that diffusion-mediated growth is dependent on the size of the region of exocytosis at the tip and that diffusion-based growth would be significantly slower than normal cell growth. To further explore the size of the secretion zone, we used a cell wall degradation enzyme cocktail and determined that the secretion zone is smaller than 6 mm in diameter at the tip. Taken together, our results highlight the requirement for active transport in polarized growth and provide important insight into vesicle secretion during tip growth.

Myosin XI Interacting with a RabE GTPase Is Required for Polarized Growth

“…The observed apical cluster of RabE is similar to the previously observed dynamic compartment of myosin XI, F-actin, and secretory vesicles (Vidali et al, 2009;Vidali et al, 2010;Furt et al, 2013). Furthermore, myosin XI and secretory vesicles are spatiotemporally coincident, with bulk vesicle flow predominantly mediated by F-actin (Bibeau et al, 2018), presumably through myosin XI activity. This led us to hypothesize that RabE functions as a secretory vesicle receptor for myosin XI, in a manner analogous to yeast Sec4 associating with Myo2 for polarized transport (Jin et al, 2011).…”

“…However, MyoB does not colocalize with secretory vesicles or any other known markers (Peremyslov et al, 2015;Kurth et al, 2017), and is absent at the cortical division site where myosin XI (Abu-Abied et al, 2018;Sun et al, 2018), Rab GTPses (Chow et al, 2008;Ahn et al, 2013), and multisubunit tethering complexes (Rybak et al, 2014) are present. Observations of robust correlations between myosin XI and vesicles in cells lacking cytoplasmic streaming (Furt et al, 2013;Bibeau et al, 2018) and well-characterized homologous myosin V-Rab interactions from yeast to human (Hammer and Sellers, 2012), strongly suggest the presence of a homologous myosin-Rab interaction in plants to mediate vesicle association and transport.…”

“…Polarized exocytosis requires precisely coordinated vesicle trafficking to support the essential function of polarized growth across eukaryotes, in addition to more plantspecific functions such as pathogen defense and cell wall biogenesis. (Robatzek, 2007;Bove et al, 2008;Ebine and Ueda, 2015;Bibeau et al, 2018). Plant polarized growth, or tip growth, exploits turgor pressure and active transport of secretory vesicles to drive rapid anisotropic expansion (Hepler et al, 2001;McKenna et al, 2009).…”

“…Polarized delivery of secretory vesicles is the rate limiting step for growth in plants, and is dependent upon the molecular motor myosin XI (Vidali et al, 2010;Madison and Nebenfuhr, 2013;Rounds and Bezanilla, 2013). Our traditional understanding of active transport frames myosin XI as providing the mechanical force to translocate cargo on Factin cables, since diffusion alone is insufficient to support polarized growth (Campas and Mahadevan, 2009;Moscatelli et al, 2012;Bibeau et al, 2018). Recent work demonstrated myosin's vesicular cargo as a site of actin assembly, suggesting myosin and vesicles function as a fluid organizing center of actin nucleation and cargo transport (Schuh, 2011;Furt et al, 2013;Pylypenko et al, 2016).…”

Cellular Dynamics: Cellular Systems in the Time Domain

“…The Update by Szymanski and Staiger (2018) focuses on recent discoveries that advance our understanding of how particular actin filament arrays influence growth and organelle clustering in polarized cell types. In this issue, quantitative live cell imaging and biophysical modeling of organelle diffusion was used to demonstrate an important role for actin filaments in locally increasing vesicle concentration at the apex of tip-growing moss protonema (Bibeau et al, 2018). A major research question in the transport and morphogenesis field is centered on understanding how myosin motors are dynamically coupled to actin filament roadways and cargo.…”