Actin-Based Vesicle Focusing Sustains Tip Growth 2 3

34 35 Filamentous actin has been shown to be essential for tip growth in an array of 36 plant models, including Physcomitrella patens. One hypothesis is that diffusion 37 can transport secretory vesicles, while actin plays a regulatory role during 38 secretion. Alternatively, it is possible that actin-based transport is necessary to 39 overcome vesicle transport limitations to sustain secretion. Therefore, a 40 quantitative analysis of diffusion, secretion kinetics and geometry is necessary to 41 clarify the role of actin in polarized growth. Using FRAP analysis, we first show 42 that secretory vesicles move toward and accumulate at the tip in an actin43 dependent manner. We then depolymerized F-actin to decouple vesicle diffusion 44 from actin-mediated transport, and measured the diffusion coefficient and 45 concentration of vesicles. Using these values, we constructed a theoretical 46 diffusion-based model for growth, demonstrating that with fast-enough vesicle 47 fusion kinetics, diffusion could support normal cell growth rates. We further 48 refined our model to explore how experimentally-extrapolated vesicle fusion 49 kinetics and the size of the secretion zone limit diffusion-based growth. This 50 model predicts that diffusion-mediated growth is dependent on the size of the 51 region of exocytosis at the tip, and that diffusion-based growth would be 52 significantly slower than normal cell growth. To further explore the size of the 53 secretion zone, we used a cell wall-degradation enzyme cocktail, and determined 54 that the secretion zone is smaller than 6 μm in diameter at the tip. Taken together 55 our results highlight the requirement for active transport in polarized growth and 56 provide important insight into vesicle secretion during tip growth. 57 58 www.plantphysiol. org on November 20, 2017 Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved. 3 Introduction 59 60 Precise regulation of exocytosis is essential to maintain polarized cell growth in a 61 variety of plant systems. Cell polarized growth, or tip growth, is a process by 62 which a cell grows in a unidirectional manner and is found ubiquitously 63 throughout the plant kingdom (Hepler et al., 2001). Pollen tubes, root hairs, and 64 moss protonemal cells have all emerged as models for this process (Hepler et 65 al., 2001; Menand et al., 2007). To achieve polar expansion in the presence of 66 uniform turgor pressure, these cells must spatially regulate the extensibility of the 67 cell wall (Winship et al., 2010; Hepler et al., 2013). This is achieved through the 68 polarized exocytosis of various cell wall materials and loosening enzymes (Rojas 69 et al., 2011). 70 How plant cells establish spatially directed exocytosis during polarized 71 growth has been the focus of a number of studies in the past decade (Cardenas 72 et al., 2008; McKenna et al., 2009; Moscatelli et al., 2012). Many of these efforts 73 have heavily implicated the cytoskeleton as a key player in exocytosis. Evidence 74 suggests that myosin XI transports vesicle containing cell wall materials via 75 filamentous actin (F-actin) to the growing tip of the cell (Vidali et al., 2010; 76 Madison and Nebenfuhr, 2013; Madison et al., 2015). In pollen tubes, a cortical 77 actin fringe several microns behind the cell tip has been shown to be essential in 78 polarized growth (Vidali et al., 2001; Lovy-Wheeler et al., 2005; Vidali et al., 79 2009). Rounds et al. have shown that the presence of this fringe is necessary for 80 the focusing of apical pectin deposition (Rounds et al., 2014). However, work 81 with FM dyes supports the hypothesis that exocytosis happens along an annulus 82 behind the cell tip (Bove et al., 2008; Zonia and Munnik, 2008). In moss 83 protonemal cells, the actin cytoskeleton concentrates at the extreme cell apex 84 (Vidali et al., 2009). At this extreme apex, vesicle fluctuations have been shown 85 to predict F-actin fluctuations (Furt et al., 2013). Furthermore Myosin XI, which is 86 essential for tip growth (Vidali et al., 2010), can also anticipate actin fluctuations 87 (Furt et al., 2013). ROP GTPases, which have been thought to initiate tip growth 88 (Lee and Yang, 2008), have been shown to influence apical filamentous actin 89 dynamics and concentrations (Burkart et al., 2015). 90 www.plantphysiol. org on November 20, 2017 Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved. 4 Significant work has been done to probe the molecular players involved in 91 cytoskeletal mediated exocytosis, however, there is a growing need to 92 demonstrate a mechanistic link between F-actin and polarized growth (Rounds 93 and Bezanilla, 2013). Although the actin fringe has been modeled in pollen tubes 94 (Sanati Nezhad et al., 2014), to the best of our knowledge, the role of apical F95 actin in other, slower polar growth systems, is yet to be examined. What are the 96 physical limitations an active transport system like the actin cytoskeleton must 97 overcome to facilitate vesicle exocytosis and sustain polarized growth? Vesicle 98 concentrations and diffusion coefficients, exocytic reaction kinetics, cell growth 99 rates, and the size of the active region of exocytosis, all place specific limitations 100 on the actin-based transport system. Quantifying these fundamental 101 requirements will provide key insight into understanding the requirement for 102 transport in this system. 103 Without a quantitative assessment of the potential physical limitations 104 outlined above, we can hypothesize several functions for the actin cytoskeleton. 105 For example, it could serve as a means to overcome slow vesicle diffusion 106 limitations to drive exocytosis. The actin cytoskeleton could also function to 107 surpass slow reaction kinetics associated with vesicle fusion events on the 108 plasma membrane. It is also possible that exocytosis is confined to a relatively 109 small area on the plasma membrane; the actin cytoskeleton could then function 110 as a means to focus vesicles to this small exotic zone. Finally, it also remains a 111 possibility that F-actin active transport is not required to sustain polarized growth. 112 To better understand how F-actin influences vesicle transport, we 113 fluorescently labeled the v-SNARE, VAMP72 (Sanderfoot, 2007) in the moss 114 Physcomitrella patens (Vidali and Bezanilla, 2012). P. patens was chosen here 115 because it does not exhibit large organelle cytoplasmic streaming (Shimmen, 116 2007) which can complicate the analysis of vesicle transport (Furt et al., 2012). 117 Instead, P. patens only exhibits two modes of vesicle transport, namely diffusion 118 and active transport along the cytoskeleton. We visualized these modes of 119 transport, and performed Fluorescence Recovery After Photobleaching (McNally, 12