F-Actin Meditated Focusing of Vesicles at the Cell …...74 have heavily implicated the cytoskeleton as a key player in exocytosis. Evidence 75 suggests that myosin XI transports vesicle

1

Short title: 1

Actin-Based Vesicle Focusing Sustains Tip Growth 2

3

Jeffrey P. Bibeau1† , James L. Kingsley2†, Fabienne Furt1, Erkan Tüzel2*, Luis 4

Vidali1* 5

† Co-first authors 6

* Co-corresponding authors 7

8

Long title: 9

F-Actin Meditated Focusing of Vesicles at the Cell Tip Is Essential for Polarized 10 Growth 11 12

1 Worcester Polytechnic Institute, Department of Biology & Biotechnology 13

2 Worcester Polytechnic Institute, Department of Physics 14

15

Summary sentence: 16

Quantitative analysis and modeling of vesicle diffusion shows that polarized cell 17 growth rates are sustained by actin-based vesicle clustering at the tip. 18 19

20 Author Contributions: 21 22 J.P.B. performed experiments and differential equation modeling of cell growth 23

and wrote the article with contribution from all authors. J.L.K. created the FRAP 24

model and provided assistance on differential equation modeling of cell growth. 25

F.F. generated cell lines. E.T. supervised modeling and complemented writing. 26

L.V. designed and supervised experiments, and complemented writing. 27

28

Financial Support: 29

This work was supported by National Science Foundation CBET 1309933 to ET 30

and NSF-MCB 1253444 to LV. 31

32

Plant Physiology Preview. Published on October 2, 2017, as DOI:10.1104/pp.17.00753

Copyright 2017 by the American Society of Plant Biologists

www.plantphysiol.orgon March 13, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.











http://www.plantphysiol.org











2

33

Abstract 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 actin-43

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



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



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 F-95

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, 120

2008; Loren et al., 2015), FRAP, during polarized growth. To probe the utility of 121



5

actin-mediated active transport we used the small molecule inhibitor latrunculin B 122

to depolymerize actin and uncouple vesicle diffusion from active transport. With 123

FRAP, and Number and Brightness analysis (Digman et al., 2008; Loren et al., 124

2015)(Kingsley, Bibeau et al., see attached supporting manuscript), we 125

measured vesicle diffusion rates and concentrations, respectively. We then 126

developed a diffusion-limited analytical model, and numerically solved it using 127

these parameters, as well as previously measured cellular growth rates (Furt et 128

al., 2013). Using this model, we quantitatively explored the limiting factors 129

associated with cell growth, and used enzymatic digestions of the cell wall to 130

further constrain the model. 131

132

Results 133

Depolymerization of Actin Stops Cell Growth in Minutes 134 135 Previous evidence in plants suggests that actin filaments are essential for 136

polarized growth (Vidali et al., 2001). It remains unclear, however, if removal of 137

the actin cytoskeleton immediately abolishes growth, or if some growth, 138

supported by diffusion, continues to happen once actin is removed. To address 139

this question, we treated growing protonemal cells with the actin monomer 140

sequestering agent latrunculin B to depolymerize the actin filaments (Vidali et al., 141

2001; Vidali et al., 2009). To visualize vesicle accumulation, we used a cell line 142

expressing 3mEGFP-VAMP72 (Furt et al., 2013). Following latrunculin B 143

treatment, we could not detect any tip growth (Fig. 1A), which is consistent with 144

previous work (Vidali et al., 2001; Vidali et al., 2009). Treatment also abolished 145

tip localization of VAMP72-vesicles (Fig. 1B) and produced an intensity gradient 146

that decreased toward the cell tip (Fig. 1C). Vehicle-treated controls grew at 147

5.5 ± 0.4 𝑛𝑚/𝑠, which is within the range of previously measured growth rates 148

(Furt et al., 2013). Since latrunculin B arrests growth, we concluded that cell 149

growth could not continue without filamentous actin. 150

Vesicles Exhibit Active Transport at the Cell Tip 151

Since the depolymerization of filamentous actin immediately blocks growth, we 152



6

sought to determine if actin plays a role in vesicle transport at the cell tip. To this 153

aim, we performed fluorescence recovery after photobleaching experiments on a 154

cell line expressing the vesicle marker 3mCherry-VAMP72 (Furt et al., 2013) at 155

the cell tip (Fig. 2A), and a region distal to the tip, subsequently referred to as the 156

shank. We chose this cell line because we found that 3mCherry is more sensitive 157

to photobleaching than 3mEGFP. To better understand the differences in 158

molecular flux at the tip and the shank, fluorescence recoveries were corrected 159

for acquisition photobleaching (Supplementary Data, Fig. S1) and normalized to 160

the mean prebleach shank intensity. Since VAMP72-vesicles localize to the cell 161

tip (Fig. 2B, and Supplementary Data Fig. S2) --likely through myosin binding to 162

actin, we expected a reduction in the recovery after photobleaching of vesicles at 163

the cell tip. Instead VAMP72-vesicles exhibited a faster exchange of particles at 164

the tip than the shank (Fig. 2A), demonstrating that the accumulation of vesicles 165

at the cell tip is not simply due to static capture. This indicates that there is a 166

stream of active transport at the cell tip that is faster than the recovery at the 167

shank. 168

To better understand the directionality of this fast active transport, we 169

recorded the movement of VAMP72-vesicles after photobleaching. After 170

averaging several fluorescent recoveries we found that these vesicles moved 171

along the cell cortex toward the tip of the cell (Fig. 2B and Supplementary Data 172

Movie 1). We further quantified this directional recovery by measuring the 173

fluorescence intensity along the perimeter of the photobleaching region of 174

interest (Fig. 2B). At early times (∆𝑡1 = 0.7 − 1.6 𝑠) after photobleaching we 175

found that the maximum fluorescence intensity was cortical but moving toward 176

the cell tip (Fig. 2B and C and Supplementary Data Movie 1). Finally, at longer 177

time intervals (∆𝑡2 = 20 – 40 𝑠) VAMP72-vesicles became localized to the cell 178

tip (Fig. 2B and 2D, and Supplementary Data Movie 1). Since these vesicles 179

exhibited an increased flux at the cell tip when compared to the shank, and 180

because the vesicles recovered along the cell cortex toward the tip, we conclude 181

that active transport drives a significant portion of the movement of VAMP72 182

labeled vesicles. 183



7

Vesicles Undergo Diffusion when Uncoupled from Actin 184

To determine if active vesicle transport is actin dependent, and to quantify vesicle 185

diffusion, we uncoupled vesicle diffusion from actin-mediated transport with the 186

actin depolymerizing agent latrunculin. This treatment abolishes the tip 187

localization of VAMP72-vesicles (Fig. 1 and Supplementary Data Fig. S2), 188

suggesting that untreated VAMP72 labeled vesicles exhibit active transport in an 189

actin dependent manner. To accurately quantify vesicle diffusion, we performed 190

photobleaching at the cell tip and shank, and, as done in the previous section, 191

performed corrections on the recovery curves to remove the effects of acquisition 192

photobleaching and reversible photoswitching (Supplementary Data Fig. S1). 193

Fluorescence recovery curves for the two specific locations were normalized to 194

their respective prebleach intensities. Following photobleaching, the cell tip 195

exhibited a reduced fluorescence recovery rate when compared to the cell shank 196

(Fig. 3A). Because photobleaching at the cell tip was close to the cell membrane, 197

we expected the diffusion based recovery at the tip to be limited by this 198

boundary, and as a consequence slower. This is not the case at the cell shank 199

where fluorescence recovery can happen from all directions. To accurately 200

determine the diffusion coefficient, we used a particle-based computational 201

model of FRAP that incorporates the properties of our confocal imaging system, 202

the three-dimensional moss cell shape, and the thermal fluctuations of the 203

fluorophore (Kingsley, Bibeau et al., see attached supporting manuscript). 204

To reduce the number of free parameters in this FRAP model, we 205

experimentally measured the point spread function of our confocal system, and 206

the bleaching width of the confocal laser (Supplementary Data Fig. S3). The 207

FRAP model recovery curves were also corrected for reversible photobleaching 208

to better match experimental results (see Materials and Methods). The two 209

remaining open parameters in our model were the diffusion coefficient and the 210

photobleaching proportionality constant that relates laser intensity to the number 211

of fluorophores bleached. After generating simulations for an array of these two 212

parameters, we employed a fitting routine to determine the best-fit parameters 213

(see Materials and Methods). This analysis showed that a single diffusion 214



8

coefficient ( 𝐷 = 0.29 ± 0.14 𝜇𝑚2𝑠−1 ) was sufficient to account for the 215

fluorescence recovery seen both at the tip and the shank. 216

To further demonstrate that our best-fit particle-based simulation results 217

accurately represents the motion of VAPM72-vesicles in vivo, we compared the 218

directional fluorescence recovery of the best-fit particle-based simulation (without 219

the reversibly photobleached fraction) to the experimental fluorescence recovery. 220

At early times of fluorescence recovery, an intensity gradient was present within 221

the photobleached regions for both the experiments and simulations (Fig. 3B, 222

and Supplementary Data Movie 2) following limited volume corrections (Fig. S4). 223

The gradient decayed toward the cell tip (Fig. 3C) and flattened at long times 224

(Fig. 3D), which suggests that the cell boundary restricts the spatial recovery of 225

VAMP72-vesicles. This gradient was also observed in our simulations of FRAP at 226

the cell tip (Fig. 3E and 3F), but not at the cell shank (Supplementary Data Fig. 227

S5). This indicates that vesicle recovery at the cell tip is influenced by its position 228

next to the apical plasma membrane, such that material cannot flow in or 229

exchange in all directions. Importantly, this recovery closely matches a diffusion 230

model (Fig. 3E and 3F), further supporting our claim that VAMP72-vesicles 231

exhibit diffusion. 232

Analytical Modeling Demonstrates Cell Growth Is not Entirely Diffusion 233 Limited 234

To determine if diffusion alone can supply enough vesicles to the growing cell 235

edge to sustain polarized growth in P. patens, we initially created a simple 236

analytical model of diffusion-based cell growth in the absence of the actin 237

cytoskeleton. To build this model, we first determined the number of vesicle 238

fusion events per second, 𝜙, needed to support normal cell growth (Bove et al., 239

2008). We assumed that P. patens is cylindrical in shape at the shank with an 240

outer cell radius of 𝑅𝑜 ~ 6 𝜇𝑚, and a wall thickness 𝑑 ~ 250 𝑛𝑚 (Martin et al., 241

2009). As the cell grows, the wall maintains its outer radius Ro and thickness d, 242

and elongates at a rate of �̇�𝑔 = 5.5 ± 0.4 𝑛𝑚/𝑠. The wall volume per second, 𝑉�̇�, 243

needed to sustain this growth can be written as, 244



9

�̇�𝑤 ≡ 𝑑𝑉𝑤/𝑑𝑡 = �̇�𝑔𝜋[𝑅02 − (𝑅0 − 𝑑)2] . (1) 245

Assuming that the cell wall materials within a vesicle do not change in volume 246

once they are incorporated into the wall, the number of vesicle fusion events 247

needed to support growth can be written as, 248

𝜙 =�̇�𝑤

𝑉v . (2) 249

Here 𝜙 is the number of vesicle fusion events as introduced above, and 𝑉v is the 250

volume of a vesicle. This assumption is supported by electron micrographs in 251

pollen tubes that show that the electron densities of vesicles and the cell wall are 252

similar (Lancelle and Hepler, 1992; Derksen et al., 1995). Assuming that 253

secretory vesicles are spherical in shape with a radius of a 40 nm (Lancelle and 254

Hepler, 1992), one can calculate that 𝑉v ~ 2.68 × 10−4 𝜇𝑚3 and 255

�̇�𝑤 ~ 0.051 𝜇𝑚3𝑠−1, and therefore, the vesicle fusion rate for a growing cell is 256

𝜙 ~ 189 𝑣𝑒𝑠𝑖𝑐𝑙𝑒𝑠/𝑠. 257

To relate observed vesicle diffusion to this calculated vesicle fusion rate, 258

we modeled the growing cell as a cylinder with an absorbable boundary at one 259

face of the cylinder. This model is equivalent in principle to latrunculin B treated 260

cells, in which diffusion alone must support growth. Here the cylinder is oriented 261

so that the tip is positioned at 𝑥 = 0 and the back end of the cylinder is at 𝑥 = 𝐿 262

(Fig. 4A and 4B). The cylinder contains a population of vesicles with a spatial 263

concentration that can be written as 𝑐(𝑥, 𝑡) . In wild type P.patens polarized 264

growth, we determined that large organelles such as chloroplasts and the 265

vacuole remain at a distance 𝐿 = 16 ± 1.4 𝜇𝑚 behind the growing tip. The 266

presence of these large organelles allows us to make two important 267

simplifications. First, the constant distance from these organelles to the tip makes 268

it reasonable to model the growing tip of the cell as a cylinder of constant size. 269

Second, we assume these organelles act as a reflective boundary (at 𝑥 = 𝐿) and 270

block the flux of vesicles through this edge, 𝑐′(𝐿, 𝑡) = 0. 271

At the opposite edge of the cylinder (at 𝑥 = 0), we assume perfect vesicle 272



10

absorbability (e.g. infinite on-rate), simulating a possible maximum limit for 273

growth rate. This assumption of infinite on-rate (or presumably unlimited supply 274

of receptors) uncouples vesicle diffusion limitations from receptor-mediated 275

vesicle-membrane fusion limitations by assuming that vesicles instantly fuse 276

upon contact with this face of the cylinder. This is represented by the vesicle 277

concentration at the extreme edge being zero, i.e. 𝑐(0, 𝑡) = 0. To model vesicle 278

production, we assume a spatially homogenous production rate 𝑄. We impose 279

this uniform production rate because of the almost uniform distribution of Golgi 280

bodies in the cell (Furt et al., 2012). We can then express the concentration of 281

vesicles 𝑐(𝑥, 𝑡) as a time-dependent diffusion equation with a constant production 282

rate 𝑄, i.e., 283

𝜕𝑐(𝑥,𝑡)

𝜕𝑡= 𝛻 ⋅ [𝐷(𝑥)𝛻𝑐(𝑥, 𝑡)] + 𝑄 . (3) 284

Here 𝛻𝑐(𝑥, 𝑡) represents the vesicle concentration gradient. At steady state 285

growth, we can take 𝜕𝑐(𝑥, 𝑡) 𝜕𝑡⁄ = 0 . Assuming homogeneous diffusion (𝐷 =286

𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡) , the radial symmetry of the cell lets us define the concentration 287

throughout the cell as dependent only on the linear position x. This produces a 288

general solution for c(x) of the form 289

𝑐(𝑥) = − (𝑄

2𝐷) 𝑥2 + 𝐾1𝑥 + 𝐾2 , (4) 290

where 𝐾1 and 𝐾2 are constants determined by the boundary conditions. Solving 291

for the two boundary conditions ( 𝑐(0) = 0 and 𝑐′(𝐿) = 0 ) produces a profile 292

dependent on the vesicle production rate 𝑄 such that 293

𝑐(𝑥) = − (𝑄

2𝐷) 𝑥2 + (

𝑄𝐿

𝐷) 𝑥 . (5) 294

There is an additional constraint that fixes the value of 𝑄—this steady state 295

condition requires that the concentration of vesicles at the cell shank 𝑐(𝐿) is 296

equal to a predetermined concentration 𝑐𝐿 . This constraint produces an 297

expression for 𝑄, namely, 298



11

𝑄 = 2𝐷𝑐𝐿/𝐿2. (6) 299

During steady state growth, the vesicle production rate 𝑄 must match the number 300

of vesicle fusion events 𝜙 such that, 301

𝜙 = 𝑄𝐿𝜋𝑅2 . (7) 302

Here, 𝑅 is the radius of the growing cylinder. This expression allows us to 303

determine the number of vesicle fusion events for any given 𝑐𝐿, 𝐷, 𝑅 and 𝐿, and 304

the values used for these four parameters are listed in Table I. Diffusion 305

coefficient, 𝐷, was measured as described in the previous section with FRAP 306

analysis (also see attached supporting manuscript) . To determine 𝑐𝐿, we used 307

our confocal particle-based simulation (see attached supporting manuscript 308

companion manuscript) to produce a range of potential concentrations, and 309

found that resolving individual particles becomes impossible at concentrations 310

above 10 𝑣𝑒𝑠𝑖𝑐𝑙𝑒𝑠/𝜇𝑚3 (Fig. 4C). Since we cannot resolve individual particles 311

experimentally, we let this be the lower bound estimate for 𝑐𝐿. Finally, we can 312

relate the vesicle fusion rate predicted by our analytical model in Eq. (7) to the 313

observed vesicle fusion rate calculated using Eq. (2). 314

For the range of possible values of cL, the model predicts two consistent 315

effects that influence cell growth. First, there is a parabolic reduction in vesicle 316

concentration from the shank out toward the growing tip (Fig. 4D). Additionally, 317

we find a linear relationship between cell growth rates and cL. At the lower bound 318

vesicle concentration 𝑐𝐿 = 10 𝑣𝑒𝑠𝑖𝑐𝑙𝑒𝑠/𝜇𝑚 3, the predicted vesicle fusion rate 319

is 𝜙 = 41 ± 20 𝑣𝑒𝑠𝑖𝑐𝑙𝑒𝑠/𝑠 and the corresponding cell growth rate is 1.2 ±320

0.6 𝑛𝑚/𝑠 (Fig. 4E and 4F). At the upper bound vesicle concentration 𝑐𝐿 =321

100 𝑣𝑒𝑠𝑖𝑐𝑙𝑒𝑠/𝜇𝑚3, 𝜙 = 410 ± 201 𝑣𝑒𝑠𝑖𝑐𝑙𝑒𝑠/𝑠 and the growth rate is 12 ± 6 𝑛𝑚/𝑠. 322

The confidence bounds for these estimates are the result of lower and upper 323

bound estimates for the vesicle diffusion coefficient (see Materials and Methods). 324

This growth rate indicates that diffusion cannot sustain cell growth at our 325

estimated lower bound vesicle concentrations (Fig. 4E) but, can easily support 326

growth at our upper bound estimate (Fig. 4F). 327



12

This means that diffusion limitations at the growing cell edge may not 328

serve as the only reason why these cells stop growing. Interestingly this implies 329

that under idealized vesicle fusion and vesicle concentrations, diffusion could 330

support cell growth. Additional factors such as membrane-vesicle fusion kinetics, 331

small active exocytic regions, and the three-dimensional cell geometry may also 332

prevent latrunculin B treated cells from growing. 333

334 Vesicle Concentrations Yield an Estimate of Vesicle Fusion Kinetics 335 336 To explore the additional factors that may limit cell growth and better determine 337

the concentration of VAMP72 labeled vesicles during latrunculin B treatment, we 338

used Numbers and Brightness (N&B) analysis (Digman et al., 2008). In N&B 339

analysis, the mean and variance of a fluorophore’s intensity fluctuations are used 340

to determine the concentration of molecules in solution. The foundation of this 341

analysis is the assumption that molecule number fluctuations in a given volume 342

are Poisson distributed—which holds true for a freely diffusing population of 343

vesicles (Digman et al., 2008). Since the mean and variance of a Poisson 344

distribution are equal, it is possible to algebraically solve for the brightness of a 345

fluorophore even in the presence of detector shot noise (Digman et al., 2008). 346

Using this analysis, we found that the vesicle concentrations of latrunculin B 347

treated cells were 39 ± 6 and 38 ± 2 𝑣𝑒𝑠𝑖𝑐𝑙𝑒𝑠/𝜇𝑚3 at the tip and shank, 348

respectively (See Materials and Methods for more details). No statistically 349

significant difference in concentrations were found at the tip and shank (𝑝 −350

𝑣𝑎𝑙𝑢𝑒 = 0.8349, 𝑛 = 6 ). At these vesicle concentrations the analytical model 351

predicts growth at 5 ± 2.9 𝑛𝑚/𝑠, similar to normal growth. 352

Since diffusion-based growth with ideal vesicle fusion kinetics is enough to 353

support cell growth, we sought to estimate the true kinetics of vesicle membrane 354

fusion during exocytosis. Docking and fusion of exocytic vesicles is a multistep 355

process mediated by protein complexes such as the Exocyst (Kulich et al., 2010; 356

Bloch et al., 2016) and the SNARE complex (Lipka et al., 2007; El Kasmi et al., 357

2013); which we simplify by assuming VAMP72-vesicles interact with one type of 358

receptor on the plasma membrane at the cell tip. We also assume that this type 359



13

of receptor has one reaction rate, and facilitates the integration of vesicles into 360

the plasma membrane. This allows us to write the flux equation through the 361

plasma membrane as follows (Phillips, 2013), 362

363

𝜙 = 𝑚𝐾𝑜𝑛𝑐(0) . (8) 364

365

Here m is the total number of receptors on the plasma membrane at the cell tip, 366

Kon is the binding reaction rate between vesicles and the receptors, and 𝜙 is the 367

measured number of vesicle fusion events per second (as previously defined 368

with Eq. (2)). As a first approximation, and for the most parsimonious case, we 369

assume that 𝑚𝐾𝑜𝑛 is constant during growth. Since the intensity of vesicles at 370

the very cell tip, 𝑐(0), for growing cells is roughly two-fold higher than 𝑐(0) for 371

latrunculin B treated cells (Supplementary Data Fig. S2), we can solve Eq. (8) to 372

get 𝑚𝐾𝑜𝑛 ~ 2.5 𝑠−1𝜇𝑚3. 373

374 Refined Diffusion Model Illustrates the Requirement for F-actin in Polarized 375 Growth 376 377 With relevant vesicle concentrations and fusion kinetics known, we then built a 378

more comprehensive model to determine the physiological conditions under 379

which diffusion could or could not sustain cell growth, and developed an insight 380

into the limiting factors in polarized growth from a vesicle trafficking perspective. 381

To this end, we created a diffusion-based growth model without the actin 382

cytoskeleton. To incorporate a more realistic geometry of the growing moss cell 383

tip, we used the finite element analysis modeling software Comsol Multiphysics 384

(Comsol Inc, Stockholm, Sweden) to solve Eq. (3) within the moss geometry for 385

biologically relevant boundary conditions. We used impermeable (reflective) 386

boundary conditions for most of the plasma membrane, except for the active 387

region of exocytosis at the cell tip, where the 𝑚 receptors were concentrated. In 388

this region we used Eq. (8) as a boundary condition to simulate diffusion-389

mediated exocytosis. Since the size of the zone of vesicle exocytosis is 390

unknown, we simulated four different discrete exocytic zones, centered at the cell 391



14

apex, with diameters Ω = 1, 2, 4, and 10 𝜇𝑚 (Fig. 5A). To satisfy Eq. (8), each of 392

the four simulated exocytic zones maintained the same total number of 393

receptors, 𝑚, but at different densities. Although it has been previously shown 394

that vesicle exocytic zones may have a monotonically increasing 𝑚𝐾𝑜𝑛 focused 395

at the tip (Campàs and Mahadevan, 2009), we simulate Ω with a constant 𝑚𝐾𝑜𝑛 396

to establish the effective length scales for the exocytic zone. 397

To achieve growth rates similar to a wild type cell, our model predicts that 398

a cell growing by diffusion would have to maintain vesicle concentrations at the 399

shank much greater than those observed experimentally. Specifically, these 400

concentrations range between ~125 and ~477 𝑣𝑒𝑠𝑖𝑐𝑙𝑒𝑠/𝜇𝑚3 at the cell shank, 𝑐𝐿 , 401

while our experimentally measured shank concentration is 38 ± 2 𝑣𝑒𝑠𝑖𝑐𝑙𝑒𝑠/𝜇𝑚3. 402

Based on the size of the exocytosis zone we found that these concentrations 403

exhibited different gradients as they approached the cell tip. Large exocytic 404

zones produced concentration gradients that fell slowly as they approached the 405

cell tip, while smaller zones produced concentration gradients that rapidly fell as 406

they approached the cell tip (Fig. 5B and 5C). We also found that larger exocytic 407

zones could support cell growth at lower vesicle concentrations than smaller 408

exocytic zones. This means that a cell with larger exocytic zones could grow 409

more quickly than a cell with a smaller exocytic zone, if both cells had the same 410

steady state vesicle concentrations. For large exocytic regions and 411

experimentally measured vesicle concentrations, diffusion alone could lead to 412

cell growth rates slightly slower than 2 𝑛𝑚/𝑠 (Fig. 5E). Smaller exocytic regions 413

at experimentally relevant vesicle concentrations produced growth rates slower 414

than 1 𝑛𝑚/𝑠 (Fig. 5E). Although actin could serve other functions associated with 415

growth, our results demonstrate that there is a specific requirement for active 416

transport to sustain cell growth, and that this need is dependent of the size of the 417

exocytic zone. 418

419 Wall Extensibility is Weakest at the Cell Tip 420 421 In order to constrain our diffusion model with an experimentally relevant secretion 422

size, we examined the material properties of the cell wall. To probe these 423



15

properties during polarized growth, we used the enzyme cocktail driselase, which 424

enzymatically degrades the wall. When exposed to driselase, we found that the 425

wall of the tip-growing cells would begin to extrude and eventually fail (Fig. 6A). 426

Following treatment extrusion and failure was observed within minutes. We found 427

that the average size of the extruded region was approximately 5.8 ± 0.5 µ𝑚 in 428

diameter (Fig. 6B), and its center was within 3 µ𝑚 from the center of the cell (Fig. 429

6C). Assuming degradation happens uniformly, this indicates that the cell wall is 430

most extensible at the cell tip. Due to the fact that exocytosis mediates wall 431

extensibility, we inferred that exocytosis likely takes place within a region smaller 432

than 5.8 µ𝑚 in diameter. This upper limit illustrates that without F-actin, diffusion-433

based cell growth could never achieve growth rates greater than 2 𝑛𝑚/𝑠 (Fig 6D 434

and 6E), which is significantly less than 5.5 ± 0.4 𝑛𝑚/𝑠 observed 435

experimentally. 436

437

Discussion and Conclusion 438 439 This work demonstrates that in moss protonemata actin drives vesicle transport, 440

and shows quantitatively that this transport is necessary to sustain normal cell 441

growth. Specifically, the experimentally measured vesicle diffusion coefficient, 442

vesicle concentrations, membrane reaction kinetics, and size of the exocytosis 443

region collectively impose enough limitations on diffusion based growth to require 444

an active transport system in polarized secretion, Fig 7. 445

While it is well-known that filamentous actin is necessary for polarized 446

growth, here we showed that latrunculin B treatments halt tip growth within 2 447

minutes, indicating an immediate reliance on actin. We then showed with FRAP 448

that VAMP72 labeled vesicles are transported cortically to the cell apex in an 449

actin-dependent manner. Nevertheless, this FRAP analysis is limited in that it 450

does not have the spatiotemporal resolution to infer additional modes of vesicle 451

dynamics, such as transport to the plasma membrane and/or endocytic vesicle 452

resupply. 453

Depolymerizing the actin cytoskeleton with latrunculin B, reduced the 454

number of modes of vesicles dynamics, and allowed us to use FRAP to measure 455



16

the vesicle diffusion coefficient (~ 0.29 𝜇𝑚2𝑠−1 ). While we assumed that the 456

VAMP72 fluorescent marker exclusively labeled secretory vesicles, it remains a 457

possibility that the marker could have labeled a small fraction of the Golgi or ER. 458

However, our Numbers and Brightness (N&B) analysis shows that the vast 459

majority of pixels in the images have a brightness distribution consistent with the 460

marker labeling a single species (Supplement Data Fig. S6). In addition, 461

fluorescence labeling of the ER and Golgi (Furt et al., 2012) did not match the 462

fluorescence intensity distribution found with VAMP72. Furthermore, the vesicle 463

diffusion coefficient we measured is in agreement with previously measured 464

pollen tube vesicle diffusion coefficients (Bove et al., 2008; Kroeger et al., 2009), 465

and measurements of the viscosity of the moss cytoplasm (Kingsley, Bibeau et 466

al., see attached supporting manuscript). 467

Our quantitative estimate of the vesicle diffusion coefficient, the number of 468

vesicles (through N&B analysis of VAMP72), and the kinetics of vesicle fusion 469

(𝑚𝐾𝑜𝑛 ) allowed us to build a model to describe diffusion-based growth with 470

relevant parameters from moss. While the variability in number of VAMP72 471

molecules on a vesicle can contribute to the observed intensity fluctuations, we 472

considered this effect to be negligible since our measured concentration of 473

vesicles is in agreement with EM estimates (Mccauley and Hepler, 1992). We 474

used this concentration to solve the flux equation at the cell tip, and determined 475

an estimate for the kinetics of vesicle fusion, 𝑚𝐾𝑜𝑛. To explore how the reaction 476

kinetics observed during normal cell growth limit diffusion based cell growth, we 477

assumed 𝑚𝐾𝑜𝑛 is constant and independent of the presence of F-actin. With 478

these parameters used as inputs, we built and solved a comprehensive model of 479

diffusion-based cell growth in a realistic moss geometry. It is important to note 480

that this model is a theoretical proof used to illustrate limitations that highlight the 481

importance of F-actin, and is not expected to match the physiological conditions 482

in latrunculin B treated cells. Nevertheless, our results predict that diffusion-483

based transport is slower than normal cell growth and heavily dependent on the 484

size of the active region of exocytosis. 485



17

Finally with driselase treatments, we found that the effective length scale 486

for the region of greatest extensibility (maximum secretion) was on the order of a 487

few microns and located at the cell tip. This analysis holds as long the cell wall 488

extruded because of an increased extensibility, and not because of increased 489

stresses resulting from the intrinsic geometry of the cell. Based on the shape of 490

the cell wall, and the assumption that the cell wall is a thin sheet, one can show 491

that the maximum stresses on the cell wall are not at the regions of rupture 492

(Campàs and Mahadevan, 2009). For thin walled shells under uniform pressure, 493

it has been shown that the curvature of the cell wall is inversely proportional to 494

stress (Campàs and Mahadevan, 2009). Since the shank of the cell is less 495

curved than the tip, greater stresses are predicted at the shank; therefore the 496

observed cell rupture is most likely a result of increased extensibility at the tip. 497

Additionally, these measurements for the size of the exocytic zone are consistent 498

with the localization of myosin XI at the cell tip (Vidali et al., 2010). 499

Although we built a tip-growth model that quantitatively highlights the 500

necessity for an active transport mechanism in polarized growth, we cannot rule 501

out the possibility that other factors could limit polarized growth. For example, 502

the F-actin could be necessary for maintaining the active region of exocytosis. 503

Specifically, F-actin could facilitate the transport and polarization of the receptors 504

required in vesicle fusion. However, elucidating such potential limitations is left 505

for future work. Importantly, we have shown and quantified specific reasons why 506

active transport is necessary in polarized growth. These analyses and modeling 507

approaches are likely to apply to other tip growth systems in plants such as root 508

hairs and pollen tubes. 509

510

Materials and Methods 511 512 Measuring Moss Growth Rates 513 514 The moss cell line used for measuring growth rates was 3mEGFP-VAMP72, and 515

was transformed as done in (Furt et al., 2013) the gene used corresponds to 516

accession number Pp3c4_13580V3.1 (Phytozome 12). Moss samples were 517

cultured using the protocol described previously (Vidali et al., 2007). Microscope 518



18

samples were prepared as described previously (Furt et al., 2013). To stop 519

growth, liquid medium (PpNO3) with 200μL of 5 μM of latrunculin B was pipetted 520

directly on top of the growing cells. Samples were imaged on a Leica TCS SP5 521

confocal microscope with a 63X objective and a numerical aperture of 1.4. The 522

argon laser was set to 25% power and the 488 𝑛𝑚 laser line was set to 10% 523

power. The emission bandwidth was set between 499 − 546 𝑛𝑚, and a hybrid 524

detector was used to acquire images. Kymographs and growth rates were 525

constructed using ImageJ. 526

Experimental FRAP Acquisition, Processing, and Analysis 527 528 The moss cell line used for FRAP was a 3mCherry-VAMP72 3mEGFP-myosin 529

XIa double line previously published by (Furt et al., 2013). Moss samples were 530

cultured using the protocol described previously (Kingsley, Bibeau et al., see 531

attached supporting manuscript). Samples were prepared in QR-43C chambers 532

(Warner Instruments) and perfused with 5 μM latrunculin B. Samples were 533

imaged on a Leica TCS SP5 scanning confocal microscope using the Leica 534

FRAP wizard. A 63𝑋 objective with a 1.4 numerical aperture was used. The 535

pinhole was set to 2 airy disks and the camera zoom was 9. To visualize the 536

3mCherry-VAMP72 cell line developed in (Furt et al., 2013), the DPSS 537

561 𝑛𝑚 laser was turned on and the 561 𝑛𝑚 laser line was set to 10%. The laser 538

scanning speed was set to 1400 𝐻𝑧 with the bi-directional scanning. The 539

emission bandwidth for 3mCherry was set between 574 and 646 𝑛𝑚 . Images 540

were acquired at 256 × 256 pixel resolution, with a bit depth of 12 bits. 541

Following image acquisition, images were subjected to several processing 542

steps. First experimental TIFF stacks were analyzed with an ImageJ macro that 543

tracked the photobleaching Region Of Interest (ROI), performed background 544

subtraction, and normalized the mean ROI to pre-bleach ROI intensities. Image 545

acquisition photobleaching was measured by tracking fluorescence intensity over 546

time while imaging cells at the same sampling rate used during FRAP 547

experiments. This fluorescence time trace was fit to a piecewise exponential. To 548

correct for acquisition photobleaching and reversible photobleaching, normalized 549



19

FRAP experimental ROI intensities were divided by this piecewise exponential 550

(Supplementary Data Fig. S1). All untreated curves were normalized to the mean 551

pre-bleach shank intensity to illustrate changes in fluorescence intensity relative 552

to the shank vesicle concentration. Recovery curves from latrunculin B treated 553

cells, at the tip and shank, were normalized to their mean pre-bleach intensities 554

at their respective locations. 555

For spatial analysis of fluorescence recovery, experimental images were 556

analyzed as follows. First an image cropping macro was used to extract FRAP 557

ROIs. Cropped ROIs were then normalized to the mean of the pre-bleach ROIs 558

and averaged across experimental replicates. To correct for the limited 559

accessible volume at the cell tip, we fit an exponential decay to a line scan 560

through the cropped prebleach ROIs. We then divided each ROI during 561

fluorescence recovery by this exponential decay (Supplementary Data Fig. S5). 562

For more details on experimental FRAP acquisition and processing see, 563

(Kingsley, Bibeau et al., see attached supporting manuscript). 564

After ROI processing, we spatially sub-sampled the ROIs with an angular 565

sector that was rotated 360 degrees about the ROI (Fig. 2B and 3B). To reduce 566

noise, we averaged about the horizontal axis of the ROI; this produced an 567

intensity profile from 0 − 180 degrees. 568

569 Measuring Confocal Imaging and FRAP Parameters 570 571 Since our particle-based FRAP simulation considers the imaging and 572

photobleaching properties of the confocal microscope, we measured the Point 573

Spread Function (PSF) of our imaging system, and the photobleaching properties 574

of 3mCherry-VAMP72 (Kingsley, Bibeau et al., see attached supporting 575

manuscript). for more details on the simulation inputs and parameter estimates). 576

To determine the PSF of our imaging system, we used the PS-Speck Microscope 577

Point Source Kit P7220 (Invirtogen). Bead images (Supplementary Data Fig. 578

S3A and S3B) were deconvolved with a box car function equal to the width of the 579

bead. This ensured that the experimental images represented a true point 580

source. The deconvolved PSF was then fit to a three-dimensional squared 581



20

Gaussian beam (Supplementary Data Fig. S3A and S3B) and used for imaging in 582

the simulation (Kingsley, Bibeau et al., see attached supporting manuscript). . 583

Based on these fits, we found that the minimum beam width, 𝑤0 , and the 584

Rayleigh range, 𝑧𝑅, were 520 and 500 𝑛𝑚, respectively. 585

To measure the photobleaching properties of the beam, we fixed cells 586

expressing 3mCherry-VAMP72 with formaldehyde (Fritzsche and Charras, 587

2015). For fixation, a solution of 100 𝑚𝑀 HEPES with 2% formaldehyde at pH 7, 588

was perfused into QR-43C chambers (Warner Instruments) for 30 minutes (Vidali 589

et al., 2007). Fixed cells were subjected to a 1 × 6 𝜇𝑚 rectangular 590

photobleaching region of interest (Supplementary Data Fig. S1C, solid white 591

rectangle) and imaged in the same plane as the photobleach. A rectangular ROI 592

was chosen because it is composed of confocal scans of equal length; which 593

permits fluorescence averaging along the long axis of the rectangle. A line scan 594

was created by collapsing a 4 × 8 𝜇𝑚 rectangular region including the 595

photobleached ROI (Supplementary Data Fig. S1B, dashed yellow rectangle) into 596

a one-dimensional profile. After photobleaching, we fit the photobleaching 597

function described by (Kingsley, Bibeau et al., see attached supporting 598

manuscript) to the line scan. This function characterizes the horizontal line scan 599

of a Gaussian beam as it photobleaches across a fixed distance. As shown 600

previously (Braeckmans et al., 2006), we found that the photobleaching 601

Gaussian beam waist was two-fold larger than the imaging beam waist at 602

920 𝑛𝑚. This fit also yielded a photobleaching proportionality constant, which 603

was used as a starting point in fluorescence recovery fitting routines to measure 604

VAMP-72 vesicle diffusion. For more details on simulating confocal 605

photobleaching see, (Kingsley, Bibeau et al., see attached supporting 606

manuscript) and for general use we developed a Digital Confocal Microscopy 607

Suite (DCMS), which can be found at 608

http://tuzelgroup.wpi.edu/desktop/research/software/dcms/. 609

610

FRAP Parameter Minimization 611 612 To determine the simulation parameters that best characterize the recovery 613



21

curves for VAMP72-vesicles in latrunculin B treated cells, a minimization routine 614

was used similar to the one by (Kingsley, Bibeau et al., see attached supporting 615

manuscript). Averaged experimental recovery curves 𝑅𝑒𝑥𝑝(𝑡) at the tip and shank 616

were characterized into classes 𝑅𝑇𝑒𝑥𝑝

(𝑡) and 𝑅𝑆𝑒𝑥𝑝(𝑡), respectively. Averaged 617

simulation recovery curves were characterized in the same way to produce 618

𝑅𝑇𝑠𝑖𝑚(𝑡) and 𝑅𝑆

𝑠𝑖𝑚(𝑡) . These two classes of simulation recovery curves were 619

generated in a two-dimensional parameter space for the parameters 𝐷 and 𝐾, 620

where a range of each parameter was simulated ( 𝐷 = 0.1 − 0.5 𝜇𝑚2𝑠−1 and 621

𝐾 = 0.01 − 0.03 ). Here 𝐷 is the diffusion coefficient and 𝐾 is the bleaching 622

proportionality coefficient. The bleaching proportionality coefficient 𝐾 measured 623

during fixation was used only as a reference in generating the range for possible 624

values of 𝐾. This was done because formaldehyde treatment was found to affect 625

fluorophore bleaching properties at the concentrations used. Unlike the approach 626

from Kingsley, Bibeau et al., (see attached supporting manuscript) only two 627

parameters were used here because the photobleaching width, w0, was 628

measured with formaldehyde treated cells. The recovery curve for each 629

parameter pair was stored in a Matlab (MathWorks, Natick, MA) data structure. 630

Best fit simulation parameters for the averaged experimental recovery profiles at 631

the tip, 𝑅𝑇𝑒𝑥𝑝

(𝑡), and shank, 𝑅𝑆𝑒𝑥𝑝(𝑡),, were found by reducing the sum of squared 632

differences between the curves. Averaged tip and shank recovery curves can be 633

represented as 𝑅𝑇𝑠𝑖𝑚(𝑡)𝐷,𝐾,𝑤0

and 𝑅𝑆𝑠𝑖𝑚(𝑡)𝐷,𝐾,𝑤0

, respectively. 634

To account for reversible bleaching we imposed a correction on all 635

simulated recovery curves (Sinnecker et al., 2005; Mueller et al., 2012; Morisaki 636

and McNally, 2014) (Kingsley, Bibeau et al., see attached supporting 637

manuscript). During intentional photobleaching, some fraction of the bleached 638

molecules, 𝛼, convert back to an unbleached state at a given rate, 𝑅(𝑡). We used 639

𝛼 and 𝑅(𝑡) that were previously measured for mCherry. Then using the 640

relationship previously devised,𝐼𝑀(𝑡) = 𝐼𝐹𝑅𝐴𝑃(𝑡) + 𝛼𝑅(𝑡)𝐼𝐹𝐿𝐴𝑃(𝑡), we corrected the 641

simulated recovery curves (Mueller et al., 2012). Here 𝐼𝑀(𝑡) is the resultant 642

fluorescence recovery that incorporates reversible bleaching, 𝐼𝐹𝑅𝐴𝑃(𝑡) represents 643

the fraction of the fluorescent recovery due to movement of unbleached 644



22

fluorophores and 𝐼𝐹𝐿𝐴𝑃(𝑡) represents the motion of the reversibly photobleached 645

molecules. After correction the averaged simulation tip and shank recoveries 646

become, 𝑅𝑇𝑠𝑖𝑚(𝑡)𝐷,𝐾,𝑤0

𝑟𝑒𝑣 and 𝑅𝑆𝑠𝑖𝑚(𝑡)𝐷,𝐾,𝑤0

𝑟𝑒𝑣 , respectively, where the superscript 𝑟𝑒𝑣 647

indicates reversible bleaching correction. Following this correction, best fit 648

diffusion coefficients for VAMP72-vesicle experiments, were found with the 649

following argument minimization, 650

651

𝑎𝑟𝑔𝑚𝑖𝑛𝐷𝑣,𝐾𝑡,𝐾𝑠[∑ (𝑅𝑇

𝑒𝑥𝑝(𝑡) −𝑡>0 𝑅𝑇𝑠𝑖𝑚(𝑡)𝐷𝑣,𝐾𝑡

𝑟𝑒𝑣 )2 + ∑ (𝑅𝑆𝑒𝑥𝑝(𝑡) − 𝑅𝑆

𝑠𝑖𝑚(𝑡)𝐷𝑣,𝐾𝑠

𝑟𝑒𝑣 )2𝑡>0 ] (9) 652

653

In this minimization, experimental recoveries at the cell tip and shank were 654

compared to their corresponding simulations to find the parameters 𝐷𝑣, 𝐾𝑡, and 655

𝐾𝑠. Here, the subscript 𝑣 denotes VAMP72-vesicles. As shown in Eq. (9), the 656

diffusion coefficient 𝐷𝑣 was shared between the tip and shank. To improve the 657

argument minimization, two unshared bleaching proportionality coefficients were 658

used at the tip 𝐾𝑡 and shank 𝐾𝑠 (Kingsley, Bibeau et al., see attached supporting 659

manuscript). Although using two unshared bleaching probabilities improves the 660

argument minimization, a shared bleached probability for the tip and shank did 661

not appreciably change the mean value of our measured diffusion coefficient, 662

𝐷 = 0.45 ± 0.04 𝜇𝑚2𝑠−1 . This measurement has confidence intervals that 663

overlap with the intervals of our VAMP72-vesicles diffusion coefficient ( 𝐷 =664

0.29 ± 0.14 𝜇𝑚2𝑠−1) and does not change our conclusions. Similarly, we found 665

that the ignoring reversible photobleaching would have had little impact on our 666

measured diffusion coefficient (𝐷 = 0.32 ± 0.18 𝜇𝑚2𝑠−1). 667

To determine the error associated with the measured parameters, we 668

used the Monte Carlo simulation as described in detail in (Kingsley, Bibeau et al., 669

see attached supporting manuscript)(Motulsky and Christopoulos, 2004). In brief, 670

we generated new experimental and simulated recovery curves via Monte Carlo 671

simulation by sampling from a normal distribution for each condition. The mean 672

and standard deviations of this normal distribution were taken from the mean and 673

standard deviations of each point in the corresponding recovery curve. We then 674

performed the argument minimization described in Eq. (9) on the newly 675



23

generated curves to find new values for the parameters, 𝐷𝑣, 𝐾𝑡, and 𝐾𝑠. We then 676

repeated this process to produce a distribution of these parameters. We used 677

two standard deviations from each distribution to represent the error of our 678

measured parameters such that, 𝐷𝑣 = 0.29 ± 0.24 𝜇𝑚2𝑠−1 , 𝐾𝑡 = 0.02 ±679

0.004,𝐾𝑠 = 0.0125 ± 0.004. 680

681 Comsol Cell Growth Model 682 683 Cell growth models were generated using the Comsol Multiphysics (Comsol Inc, 684

Stockholm, Sweden) modeling software using the “three-dimensional transport of 685

dilute species” interface with one species. To achieve the steady state solution of 686

our growth model, the “stationary study” option was selected. We model the cell 687

as a cylinder of length 10 𝜇𝑚 and radius 6 𝜇𝑚 , capped by a hemisphere. To 688

simulate the active region of exocytosis, we induced a flux, consistent with Eq. 689

(8), through the cell membrane at the cell tip. To simulate the uniform production 690

of vesicles, we imposed a homogenous reaction rate. All simulations were run 691

with a mesh setting of “extremely fine” in the software. To determine the 692

concentration profiles required to maintain wild type growth, for the various 693

secretion sizes, we set the production term to match the desired growth rate. We 694

then modified this production term to match the experimentally measured shank 695

concentrations and reported the growth rate. Vesicle concentration profiles were 696

then exported as text files, and visualized in Matlab. 697

Brightness and Numbers Analysis 698 699 Moss cultures and microscope samples were prepared as described in 700

measuring moss growth rates. Samples were imaged on a Leica TCS SP5 701

confocal microscope with a 63 × objective with a numerical aperture of 1.4. The 702

argon laser was set to 25% power and the 488 𝑛𝑚 laser line was set to 20% 703

power. The emission bandwidth was set between 499 − 546 𝑛𝑚 and a hybrid 704

detector in photon counting mode was used. Images were scanned at 700 𝐻𝑧. 705

Six latrunculin B-treated cells were imaged at both the shank and the cell tip 706

within a 6 × 6 𝜇𝑚 area, and a pixel resolution of 512 × 512. Image stacks were 707



24

analyzed in Matlab (MathWorks, Natick, MA) as previously described (Digman et 708

al., 2008). 709

To conduct our analysis, we collected confocal image time series at the tip 710

and shank of latrunculin B treated cells expressing 3mEGFP-VAMP72 711

(Supplemental Data Fig. S5A). We assumed that all the VAMP72s on a vesicle 712

equally contribute to the brightness of a vesicle because the size of a vesicle is 713

consistent with a diffraction limited spot (Lancelle and Hepler, 1992). To ensure 714

that detector shot noise and VAMP72-vesicle number fluctuations were the 715

primary contributors to intensity fluctuations, all images were acquired with a 716

hybrid detector in photon counting mode (Dalal et al., 2008; Digman et al., 2008). 717

The intensity fluctuations over time for each pixel were calculated and used to 718

determine the brightness and number of molecules within each pixel 719

(Supplemental Data Fig. S5B). We then manually filtered the pixels by their 720

apparent brightness and intensity. This was done to remove background pixels 721

and anomalously bright pixels (with brightness value 𝐵 > 2) (Supplemental Data 722

Fig. S5C). To convert the number of molecules within a given pixel to a 723

concentration, the number of molecules must be divided by the volume of the 724

Point Spread Function (PSF). To obtain the apparent volume of the PSF, we 725

used our particle-based confocal simulation (Kingsley, Bibeau et al., see 726

attached supporting manuscript). with our measured PSF beam width and 727

Rayleigh range as input parameters. These parameters were measured in the 728

same way as 3mCherry(Supplementary Data Fig. S3). With the particle-based 729

confocal simulation we then generated an array of vesicle concentrations, and 730

used a calibration curve to convert the number of molecules per pixel to the 731

known concentrations (Supplemental Data Fig. S5D). From the calibration curve, 732

we were able to determine the volume of our PSF given our measured 733

experimental parameters. This PSF volume was within 20% error of theoretical 734

PSF volumes (Moens et al., 2011). 735

736



25

Driselase Treatment on the Cell Wall 737

Moss cultures were prepared as described in measuring moss growth rates. 738

Microscope preparations were made on the bottom of 60X15 mm petri dishes. 10 739

ml of PPN03 with 1% agar was pipetted into the dish. Six 1 ml pipette tips were 740

placed face up onto the dish. Once solidified, the pipette tips were removed and 741

50 𝜇𝐿 of PPN03 with 1% agar was pipetted into the newly made holes. A single 742

moss colony was then placed into one of the holes. Then a 200 𝜇𝐿 solution of 8% 743

mannitol and 2% driselase was pipetted into the hole. The cells were imaged 744

using a Zeiss Axio Observer inverted microscope with a 20 × objective. Images 745

were acquired with a Photometrics Cool Snap Camera with the Pixelfly software. 746

Images were taken every 0.1 seconds. 747

Images were analyzed with Matlab (MathWorks, Natick, MA). Cell edges 748

were detected using the canny edge detection algorithm with the function edge. 749

Rupture points and positions were found manually by selecting points on the 750

image. Polynomial fitting was used to estimate relevant arc lengths. 751

752

Supplemental Figure 1. Image acquisition photobleaching/reversible 753

photoswitching and correction. 754

Supplemental Figure 2. Latrunculin B abolishes tip localization of VAMP72-755

vesciles. 756

Supplemental Figure 3. Point Spread Function (PSF) for imaging and 757

photobleaching. 758

Supplemental Figure 4. VAMP72-vesicle accessible volume correction. 759

Supplemental Figure 5. Spatial fluorescence recovery of VAMP72-vesicles at 760

the cell shank. 761

Supplemental Figure 6. Brightness and numbers analysis reveals vesicle 762

concentrations. 763



26

Supplemental Movie 1. Analysis of spatial recovery of VAMP72-vesicles at the 764

cell tip. 765

Supplemental Movie 2. Analysis of spatial recovery of VAMP72-veislces at the 766

cell tip of latrunculin B treated cell. 767

768

769

Supplemental Movie 1. Analysis of spatial recovery of VAMP72-vesicles at the 770

cell tip. Cropped and frame averaged photobleaching ROI at the cell tip for 771

3mCherry-VAMP72-vesicles( left). Angular intensity profile of VAMP72-vesicles 772

at the cell tip (right). n = 10; ROI is 4 μm in diameter. Mean subtracted image 773

intensity denoted with rainbow lookup table. 774

Supplemental Movie 2. Analysis of spatial recovery of VAMP72-veislces at the 775

cell tip of latrunculin B treated cell. Cropped and frame averaged photobleaching 776

ROI at the cell tip for latrunculin B treated (left) 3mCherrry-VAMP72-vesicles. 777

Angular intensity profile of VAMP72-vesicles at the cell tip following latrunculin B 778

treatment (right). n = 8; ROI is 4 μm in diameter. Mean subtracted image intensity 779

denoted with rainbow lookup table. 780

781 Acknowledgements 782

We thank Leica Microsystems GmbH for technical guidance, and all members of 783

the Vidali and Tüzel labs for helpful discussions, especially Iman Mousavi. This 784

work was supported by National Science Foundation CBET 1309933 to ET, and 785

NSF-MCB 1253444 to LV. ET and LV also acknowledge support from Worcester 786

Polytechnic Institute Startup Funds. JK acknowledges support from the WPI 787

Alden Fellowship. 788

789

790

791



27

792

Table 1 Analytical Model Parameters 793 794 Parameter Value Source

Diffusion Coefficient, 𝐷 0.29 ± 0.14 𝜇𝑚2𝑠−1 Measured with FRAP

Cylinder length, 𝐿 16 ± 1.4 𝜇𝑚 Measured from microscope images (n=8)

Cylinder Radius, 𝑅 6 ± 0.2 𝜇𝑚 Measured from microscope images (n=8)

Concentration at 𝑐(𝐿), 𝑐𝐿 10 − 100 𝑣𝑒𝑠/𝜇𝑚3 Estimated from Figure 1B

795 796 797



28

Figure Legends 798 799 Figure 1. Latruculin B stops growth. A) Representative kymographic analysis of 800

caulonemal cells expressing 3mEGFP-VAMP72 vesicles before (top) and after 801

(bottom) vehicle (left) or 5 𝜇𝑀 latrunculin B treatment (right). Black vertical arrow 802

indicates vertical time axis. Horizontal arrow denotes treatment time. White 803

space between top and bottom figures represents the approximate time 804

necessary to apply the treatment to the culture. Dotted white box indicates region 805

for line scan. B) Representative cells before (top) and after (bottom) latrunculin B 806

treatment. Scale bar is 5 𝜇𝑚. C) Measured intensity gradient for latrunculin B 807

treated cells. Measurement was taken from the white dotted box in (B) for each 808

measured cell. (error bars indicate standard error, and 𝑛 = 4) 809

810

Figure 2. VAMP72-vesicle dynamics during polarized growth. A) Fluorescence 811

recovery of 3mCherry-VAMP72-vesicles at the cell shank (green circles) and cell 812

tip (black squares). 𝑛 = 8 and 10, respectively (error bars indicate standard 813

deviation). B) Cropped and frame averaged photobleaching ROI at the cell tip for 814

3mCherry-VAMP72-vesicles (top) and simulation (bottom) n = 8 and 50, 815

respectively. Image intensity denoted with rainbow lookup table; ∆𝑡1 = 0.7 −816

1.6 𝑠 and ∆𝑡2 = 20 − 40 𝑠. White arrows mark direction of recovery(left). White 817

circle and arrow denotes how the perimeter of the ROI was measured (right). C-818

D) Intensity profiles of 3mcherry-VAMP72 along the ROI perimeter at the cell tip 819

during the time intervals ∆𝑡1 = 0.7 − 1.6 𝑠 (C) and ∆𝑡2 = 20 − 40 𝑠 (D) 820

(𝑛 = 10, error bars indicate standard error). 821

822

Figure 3. Latrunculin B treated VAMP72-vesicle dynamics. A) Fluorescence 823

recovery of latrunculin B treated 3mCherry-VAMP72-vesicles at the cell shank 824

(green circles) and cell tip (black squares) with corresponding best fit simulation 825

results (red). 𝑛 = 6 and 8, respectively (error bars indicate standard deviation). 826

B) Cropped and frame averaged photobleaching ROI at the cell tip for latrunculin 827

B treated 3mCherry-VAMP72-vesicles (top) and simulation (bottom) 𝑛 =828

8 𝑎𝑛𝑑 50, respectively. Image intensity denoted with rainbow lookup table; 829



29

∆𝑡1 = 0.8 − 2.5 𝑠 and ∆𝑡2 = 20 − 40 𝑠. White arrows mark direction of recovery 830

(left). White circle and arrow denote how the perimeter of the ROI was measured 831

(right). C-F) Intensity profiles of latrunculin B treated VAMP72 along the ROI 832

perimeter at the cell tip during the time intervals ∆𝑡1 = 0.8 − 2.5 𝑠 (C) and 833

∆𝑡2 = 20 − 40 𝑠 (D) with corresponding simulations in red (E and F) (𝑛 = 8, 834

error bars indicate standard error). 835

836

Figure 4. Differential equation model of diffusive cell growth with idealized 837

geometry. A) Diagram of the cylindrical approximation of the moss geometry. 838

Green circles represent vesicles. Red boundaries are reflective boundaries and 839

green boundary is perfectly absorbable. B) Representative bright field image of 840

growing cell with cylindrical approximation of analytical model in red. Scale bar is 841

5 𝜇𝑚. C) Simulated 3mEGFP-VAMP72-vesicles with 𝑐𝐿 = 10 𝑣𝑒𝑠𝑖𝑐𝑙𝑒𝑠/𝜇𝑚3. 842

Scale bar is 4 𝜇𝑚. D) Normalized concentration profile for the solution of the 843

analytical model, Eqs. (3)-(7). E+F) Growth rates for the analytical model, Eqs. 844

(3)-(7), solved for 𝑐𝐿 = 10 𝑣𝑒𝑠𝑖𝑐𝑙𝑒𝑠/𝜇𝑚3 (E) and 𝑐𝐿 = 100 𝑣𝑒𝑠𝑖𝑐𝑙𝑒𝑠/𝜇𝑚3 (F). 845

Model error bars represent predicted growth rates within 95% confidence 846

intervals, with 𝐷 = 0.15 𝜇𝑚2𝑠−1 and 𝐷 = 0.43 𝜇𝑚2𝑠−1, respectively. Wild type 847

error bars represent 95% confidence intervals, 𝑛 = 4. 848

849 Figure 5. Differential equation model of diffusive growth with moss cell geometry. 850

A) Plasma membrane with the active area of exocytosis marked in red. Black line 851

indicates reflective boundary. Dashed line indicates the exocytosis region 852

diameter 𝛺. B) Model predicted steady-state vesicle concentration profile 853

necessary to sustain wild type cell growth. Color table indicates concentration 854

gradients with high concentrations as warm colors and low concentrations as 855

cool colors. C+D) Normalized (C) and unormalized(D) concentration profiles from 856

(B) necessary to sustain wild type cell growth. (B). E) Comsol model predicted 857

growth rates for experimentally measured shank concentrations for 𝛺 = 10,4,2, 858

and 1 𝜇𝑚 from left to right. Model error bars represent predicted growth rates for 859



30

𝐷 = 0.15 and 𝐷 = 0.43 𝜇𝑚2𝑠−1, respectively. Wild type error bars represent 860

standard error of the mean, 𝑛 = 4. 861

862

Figure 6. Driselase reveals cell wall extensibility. A) Representative time series 863

of cell wall rupture following exposure to driselase. Cell perimeter is highlighted in 864

blue. Green circles mark the end points of the rupture arc length. Black circles 865

mark the cell tip. Red circle shows maximally deflected rupture potion. Red star 866

shows the projected rupture position. Scale bar is 5𝜇𝑚. Dashed line indicates the 867

rupture diameter 𝛺. B) Cumulative frequency of rupture diameter 𝛺. Insert 868

displays a box plot of the rupture diameter. Median is marked in red, the quartiles 869

are marked in blue, and the minimum and maximums are in black. Red dot is the 870

mean. C) Location of cell rupture along the edge. Blue dots indicate the cell 871

boundary. Red stars indicate region of rupture. (𝑛 = 17). D) Comsol model 872

predicted concentration profile, necessary to sustain wild type cell growth, for 873

𝛺 = 5.8 𝜇𝑚. E) Comsol model predicted growth rate for experimentally measured 874

shank concentrations and 𝛺 = 5.8 𝜇𝑚 , compared to wild type growth rate. Model 875

error bars represent predicted growth rates for 𝐷 = 0.15 and 𝐷 = 0.43 𝜇𝑚2𝑠−1, 876

respectively. Wild type error bars represent standard error of the mean, 𝑛 = 4. 877

878

Figure 7. F-actin must overcome the limiting factors in polarized cell growth. A) 879

Illustration of limiting factors in cell growth. Without F-actin, our measured 880

vesicle diffusion, vesicle concentrations, reaction kinetics (𝑚𝐾𝑜𝑛), and the active 881

secretion zone size would result in a significantly slower polarized growth. B) 882

With F-actin the cell can overcome these measured limitations and sustain cell 883

growth. 884

885



31

References 886

887

Bloch D, Pleskot R, Pejchar P, Potocky M, Trpkosova P, Cwiklik L, 888

Vukasinovic N, Sternberg H, Yalovsky S, Zarsky V (2016) Exocyst 889

SEC3 and phosphoinositides define sites of exocytosis in pollen tube 890

initiation and growth. Plant Physiology 172: 980-1002 891

Bove J, Vaillancourt B, Kroeger J, Hepler PK, Wiseman PW, Geitmann A 892

(2008) Magnitude and direction of vesicle dynamics in growing pollen 893

tubes using spatiotemporal image correlation spectroscopy and 894

fluorescence recovery after photobleaching. Plant Physiology 147: 1646-895

1658 896

Braeckmans K, Stubbe BG, Remaut K, Demeester J, De Smedt SC (2006) 897

Anomalous photobleaching in fluorescence recovery after photobleaching 898

measurements due to excitation saturation--a case study for fluorescein. J 899

Biomed Opt 11: 044013 900

Burkart GM, Baskin TI, Bezanilla M (2015) A family of ROP proteins that 901

suppresses actin dynamics, and is essential for polarized growth and cell 902

adhesion. Journal of Cell Science 128: 2553-2564 903

Campàs O, Mahadevan L (2009) Shape and dynamics of tip-growing cells. 904

Current Biology 19: 2102-2107 905

Cardenas L, Lovy-Wheeler A, Kunkel JG, Hepler PK (2008) Pollen tube 906

growth oscillations and intracellular calcium levels are reversibly 907

modulated by actin polymerization. Plant Physiol 146: 1611-1621 908

Dalal RB, Digman MA, Horwitz AF, Vetri V, Gratton E (2008) Determination of 909

particle number and brightness using a laser scanning confocal 910

microscope operating in the analog mode. Microscopy Research and 911

Technique 71: 69-81 912

Derksen J, Rutten T, Vanamstel T, Dewin A, Doris F, Steer M (1995) 913

Regulation of Pollen-Tube Growth. Acta Botanica Neerlandica 44: 93-119 914



32

Digman MA, Dalal R, Horwitz AF, Gratton E (2008) Mapping the number of 915

molecules and brightness in the laser scanning microscope. Biophysical 916

Journal 94: 2320-2332 917

El Kasmi F, Krause C, Hiller U, Stierhof YD, Mayer U, Conner L, Kong L, 918

Reichardt I, Sanderfoot AA, Jurgens G (2013) SNARE complexes of 919

different composition jointly mediate membrane fusion in Arabidopsis 920

cytokinesis. Mol Biol Cell 24: 1593-1601 921

Fritzsche M, Charras G (2015) Dissecting protein reaction dynamics in living 922

cells by fluorescence recovery after photobleaching. Nature Protocols 10: 923

660-680 924

Furt F, Lemoi K, Tüzel E, Vidali L (2012) Quantitative analysis of organelle 925

distribution and dynamics in Physcomitrella patens protonemal cells BMC 926

Plant Biology 12: 70 927

Furt F, Liu YC, Bibeau JP, Tüzel E, Vidali L (2013) Apical myosin XI 928

anticipates F-actin during polarized growth of Physcomitrella patens cells. 929

Plant J 73: 417-428 930

Hepler PK, Rounds CM, Winship LJ (2013) Control of cell wall extensibility 931

during pollen tube growth. Molecular Plant 6: 998-1017 932

Hepler PK, Vidali L, Cheung AY (2001) Polarized cell growth in higher plants. 933

Annu Rev Cell Dev Biol 17: 159-187 934

Kroeger JH, Daher FB, Grant M, Geitmann A (2009) Microfilament orientation 935

constrains vesicle flow and spatial distribution in growing pollen tubes. 936

Biophys J 97: 1822-1831 937

Kulich I, Cole R, Drdova E, Cvrckova F, Soukup A, Fowler J, Zarsky V (2010) 938

Arabidopsis exocyst subunits SEC8 and EXO70A1 and exocyst interactor 939

ROH1 are involved in the localized deposition of seed coat pectin. New 940

Phytologist 188: 615-625 941

Lancelle SA, Hepler PK (1992) Ultrastructure of freeze-substituted pollen tubes 942

of lilium-longiflorum. Protoplasma 167: 215-230 943

Lee YJ, Yang ZB (2008) Tip growth: signaling in the apical dome. Current 944

Opinion in Plant Biology 11: 662-671 945



33

Lipka V, Kwon C, Panstruga R (2007) SNARE-ware: the role of SNARE-946

domain proteins in plant biology. Annu Rev Cell Dev Biol 23: 147-174 947

Loren N, Hagman J, Jonasson JK, Deschout H, Bernin D, Cella-Zanacchi F, 948

Diaspro A, McNally JG, Ameloot M, Smisdom N, Nyden M, 949

Hermansson AM, Rudemo M, Braeckmans K (2015) Fluorescence 950

recovery after photobleaching in material and life sciences: putting theory 951

into practice. Quarterly Reviews of Biophysics 48: 323-387 952

Lovy-Wheeler A, Wilsen KL, Baskin TI, Hepler PK (2005) Enhanced fixation 953

reveals the apical cortical fringe of actin filaments as a consistent feature 954

of the pollen tube. Planta 221: 95-104 955

Madison SL, Buchanan ML, Glass JD, McClain TF, Park E, Nebenfuhr A 956

(2015) Class XI myosins move specific organelles in pollen tubes and are 957

required for normal fertility and pollen tube growth in Arabidopsis. Plant 958

Physiology 169: 1946-1960 959

Madison SL, Nebenfuhr A (2013) Understanding myosin functions in plants: are 960

we there yet? Curr Opin Plant Biol 16: 710-717 961

Martin A, Lang D, Hanke ST, Mueller SJX, Sarnighausen E, Vervliet-962

Scheebaum M, Reski R (2009) Targeted gene knockouts reveal 963

overlapping functions of the five Physcomitrella patens FtsZ isoforms in 964

chloroplast division, chloroplast shaping, cellpatterning, plant 965

development, and gravity sensing. Molecular Plant 2: 1359-1372 966

Mccauley MM, Hepler PK (1992) Cortical Ultrastructure of Freeze-Substituted 967

Protonemata of the Moss Funaria-Hygrometrica. Protoplasma 169: 168-968

178 969

McKenna ST, Kunkel JG, Bosch M, Rounds CM, Vidali L, Winship LJ, 970

Hepler PK (2009) Exocytosis precedes and predicts the increase in 971

growth in oscillating pollen tubes. Plant Cell 21: 3026-3040 972

McNally JG (2008) Quantitative FRAP in analysis of molecular binding dynamics 973

in vivo. Methods Cell Biol 85: 329-351 974



34

Menand B, Calder G, Dolan L (2007) Both chloronemal and caulonemal cells 975

expand by tip growth in the moss Physcomitrella patens. Journal of 976

Experimental Botany 58: 1843-1849 977

Moens PDJ, Gratton E, Salvemini IL (2011) Fluorescence correlation 978

spectroscopy, Raster image correlation spectroscopy, and Number & 979

Brightness on a commercial confocal laser scanning microscope with 980

analog detectors (Nikon C1). Microscopy Research and Technique 74: 981

377-388 982

Morisaki T, McNally JG (2014) Photoswitching-free FRAP analysis with a 983

genetically encoded fluorescent tag. PLoS One 9: e107730 984

Moscatelli A, Idilli AI, Rodighiero S, Caccianiga M (2012) Inhibition of actin 985

polymerisation by low concentration Latrunculin B affects endocytosis and 986

alters exocytosis in shank and tip of tobacco pollen tubes. Plant Biol 987

(Stuttg) 14: 770-782 988

Motulsky H, Christopoulos A (2004) Fitting models to biological data using 989

linear and nonlinear regression : a practical guide to curve fitting. Oxford 990

University Press, Oxford ; New York 991

Mueller F, Morisaki T, Mazza D, McNally JG (2012) Minimizing the impact of 992

photoswitching of fluorescent proteins on FRAP analysis. Biophys J 102: 993

1656-1665 994

Phillips R (2013) Physical biology of the cell, Ed Second edition /. Garland 995

Science, London New York, NY 996

Rojas ER, Hotton S, Dumais J (2011) Chemically mediated mechanical 997

expansion of the pollen tube cell wall. Biophys J 101: 1844-1853 998

Rounds CM, Bezanilla M (2013) Growth mechanisms in tip-growing plant cells. 999

Annual Review of Plant Biology, Vol 64 64: 243-265 1000

Rounds CM, Hepler PK, Winship LJ (2014) The apical actin fringe contributes 1001

to localized cell walldeposition and polarized growth in the lily pollen tube. 1002

Plant Physiology 166: 139-151 1003



35

Sanati Nezhad A, Packirisamy M, Geitmann A (2014) Dynamic, high precision 1004

targeting of growth modulating agents is able to trigger pollen tube growth 1005

reorientation. Plant J 80: 185-195 1006

Sanderfoot A (2007) Increases in the number of SNARE genes parallels the rise 1007

of multicellularity among the green plants. Plant Physiol 144: 6-17 1008

Shimmen T (2007) The sliding theory of cytoplasmic streaming: fifty years of 1009

progress. Journal of Plant Research 120: 31-43 1010

Sinnecker D, Voigt P, Hellwig N, Schaefer M (2005) Reversible photobleaching 1011

of enhanced green fluorescent proteins. Biochemistry 44: 7085-7094 1012

Vidali L, Augustine RC, Kleinman KP, Bezanilla M (2007) Profilin is essential 1013

for tip growth in the moss Physcomitrella patens. Plant Cell 19: 3705-3722 1014

Vidali L, Bezanilla M (2012) Physcomitrella patens: a model for tip cell growth 1015

and differentiation. Curr Opin Plant Biol 15: 625-631 1016

Vidali L, Burkart GM, Augustine RC, Kerdavid E, Tüzel E, Bezanilla M (2010) 1017

Myosin XI is essential for tip growth in Physcomitrella patens. Plant Cell 1018

22: 1868-1882 1019

Vidali L, McKenna ST, Hepler PK (2001) Actin polymerization is essential for 1020

pollen tube growth. Molecular Biology of the Cell 12: 2534-2545 1021

Vidali L, Rounds CM, Hepler PK, Bezanilla M (2009) Lifeact-mEGFP reveals a 1022

dynamic apical F-actin network in tip growing plant cells. Plos One 4: 1023

e5744 1024

Winship LJ, Obermeyer G, Geitmann A, Hepler PK (2010) Under pressure, 1025

cell walls set the pace. Trends in Plant Science 15: 363-369 1026

Zonia L, Munnik T (2008) Vesicle trafficking dynamics and visualization of zones 1027

of exocytosis and endocytosis in tobacco pollen tubes. Journal of 1028

Experimental Botany 59: 861-873 1029



4 μm"

2 m

in"

Tim

e"

Latrunculin B "Vehicle"

Before Treatment" Before Treatment"A)

B) C)

Befo

re"

LatB"

Figure 1. Latruculin B stops growth. A) Representative kymographic analysis of caulonemal cells expressing3mEGFP-VAMP72 vesicles before (top) and after (bottom) vehicle (left) or 5 μM latrunculin B treatment(right). Black vertical arrow indicates vertical time axis. Horizontal arrow denotes treatment time.White spacebetween top and bottom figures represents the approximate time necessary to apply the treatment to theculture. Dottedwhite box indicates region for line scan. B) Representative cells before (top) andafter (bottom)latrunculin B treatment. Scale bar is 5 μm. C) Measured intensity gradient for latrunculin B treated cells.Measurementwas taken from thewhite dottedbox in (B) for eachmeasured cell. (error bars indicate standarderror, and n = 4)

Figure 2. VAMP72-vesicle dynamics during polarized growth. A) Fluorescence recovery of 3mCherry-VAMP72-vesicles at the cell shank (green circles) and cell tip (black squares). n = 8 and 10, respectively(error bars indicate standard deviation). B) Cropped and frame averaged photobleaching ROI at thecell tip for 3mCherry-VAMP72-vesicles (top) and simulation (bottom) n = 8 and 50, respectively. Imageintensity denoted with rainbow lookup table; ∆t1 = 0.7−1.6 s and ∆t2 = 20−40 s. White arrows markdirection of recovery(left). White circle and arrow denotes how the perimeter of the ROI wasmeasured (right). C-D) Intensity profiles of 3mcherry-VAMP72 along the ROI perimeter at the cell tipduring the time intervals ∆t1 = 0.7 − 1.6 s (C) and ∆t2 = 20 − 40 s (D) (n = 10, error bars indicatestandarderror).

Tip!

0.7-1.6s! 20-40s!

VAM

P !

C)! D)!

Δt1! Δt2!

Δt1! Δt2!

A)! B)!shank! tip!

Figure 3. Latrunculin B treated VAMP72-‐vesicle dynamics. A) Fluorescence recovery of latrunculin B treated 3mCherry-‐VAMP72-‐vesicles at the cell shank (green circles) and cell Dp (black squares) with corresponding best fit simulaDon results (red). n = 6 and 8, respecDvely (error bars indicate standard deviaDon). B) Cropped and frame averaged photobleaching ROI at the cell Dp for latrunculin B treated 3mCherry-‐VAMP72-‐vesicles (top) and simulaDon (boQom) n = 8 and 50, respecDvely. Image intensity denoted with rainbow lookup table; ∆t1 = 0.8−2.5 s and ∆t2 = 20−40 s. White arrows mark direcDon of recovery (leZ). White circle and arrow denote how the perimeter of the ROI was measured (right). C-‐F) Intensity profiles of latrunculin B treated VAMP72 along the ROI perimeter at the cell Dp during the Dme intervals ∆t1 = 0.8 − 2.5 s (C) and ∆t2 = 20 − 40 s (D) with corresponding simulaDons in red (E and F) (n = 8, error bars indicate standard error).

0 10 20 30 40Time (sec)

0.2

0.6

1

1.4

Inte

nsity VA

MP !

SIM!

Tip LatB!

Δt1! Δt2!

A)! B)!

C)! D)!

E)! F)!

Δt1! Δt2!

Δt1! Δt2!

Model wt0

1

2

3

4

5

6

Gro

wth

Rat

e (n

m/s

)

φ

10 ves/µm3

A)

C)

B)C(x)

0 x D)

E) F)

10#ves/μm3#

A)

C)

B)

D)

E) F)

Figure 4. Differen)al equa)on model of diffusive cell growth with idealized geometry. A) Diagram of our cylindrical approxima)on of the moss geometry. Green circles represent vesicles. Red boundaries are reflec)ve boundaries and green boundary is perfectly absorbable. B) Representa)ve bright field image of growing cell with cylindrical approxima)on of analy)cal model in red. Scale bar is 5 μm. C) Simulated 3mEGFP-‐VAMP72-‐vesicles with cL = 10 vesicles/μm3. Scale bar is 4 μm. D) Normalized concentra)on profile for solu)on of the analy)cal model, Eqs. (3)-‐(7). E+F) Growth rates for analy)cal model, Eqs. (3)-‐(7), solved for cL = 10 vesicles/μm3 (E) and cL = 100 vesicles/μm3 (F). Model error bars represent predicted growth rates within 95% confidence intervals, with D = 0.15 μm2s−1 and D = 0.43 μm2s−1, respec)vely. Wild type error bars represent 95% confidence intervals, n=4.

Model wt0

5

10

15

20G

row

th R

ate

(nm

/s)

Ω=10μm Ω=4μm Ω=2μm Ω=1μm

A)

Figure 5. Differen'al equa'on model of diffusive growth with moss cell geometry. A) Plasma membrane with the ac've area of exocytosis marked in red. Black line indicates reflec've boundary. Dashed line indicates the exocytosis diameter Ω. B) Model predicted steady-‐state vesicle concentra'on profile necessary to sustain wild type cell growth. Color table indicates concentra'on gradient with high concentra'ons as warm colors and low concentra'ons as cool colors. C+D) Normalized (C) and unormalized(D) concentra'on profiles from (B) necessary to sustain wild type cell growth. (B). E) Comsol model predicted growth rates for experimentally measured shank concentra'ons for Ω=10,4,2, and 1 μm from leT to right. Model error bars represent predicted growth rates for D = 0.14 and D = 0.43μm2s−1, respec'vely. Wild type error bars represent standard error of the mean, n=4.

C)

D)

E)

B)

−10 −5 0 5 100

5

10

15

20

(µm)

(µm)

2 4 6 8 10

Rupture Diameter ( m)0

0.2

0.4

0.6

0.8

1

Rel

ativ

e Fr

eque

ncy Rupture

Positions

A)

B)

Figure 6. Driselase reveals cell wall extensibility. A) Representative time series of cell wall rupture followingexposure to driselase. Cell perimeter is highlighted in blue. Green circlesmark the end points of the rupturearc length. Black circles mark the cell tip. Red circle shows maximally defl ected rupture potion. Red starshows the projected rupture position. Scale bar is 5 μm. Dashed line indicates the rupture diameter Ω.B)Cumulative frequency of rupture diameter Ω. Insert displays a box plot of the rupture diameter. Median ismarked in red, the quartiles are marked in blue, and the minimum and maximums are in black. Red dot isthemean. C) Location of cell rupture along the edge. Bluedots indicate the cell boundary. Red stars indicateregion of rupture. (n=17). D) Comsol model predicted concentration profile, necessary to sustain wild typecell growth, for Ω = 5.8 μm . E) Comsol model predicted growth rate for experimentally measured shankconcentrations and Ω= 5.8 μm compared to wild type growth rate. Model error bars represent predictedgrowth rates for D = 0.14 and D =0.43μm2s−1, respectively. Wild type error bars represent standard error ofthe mean, n=4.

72.5sec 72.6sec71.4sec0.0sec

DriselaseTreatm

ent

1

4

6

8

Rup

ture

Dia

met

er

(m

)

D) E)

C)

A)

Inac(ve Membrane

Ac(ve Membrane

F-‐Ac(n

Vesicle Before Fusion

Fusing Vesicle Growth Rate

B)

Figure 7. F-‐ac(n must overcome the limi(ng factors in polarized cell growth. A) Illustra(on of limi(ng factors in cell growth. Without F-‐ac(n, our measured vesicle diffusion, vesicle concentra(ons, reac(on kine(cs (mKon), and the ac(ve secre(on zone size would result in a significantly slower polarized growth. B) With F-‐ac(n the cell can overcome these measured limita(ons and drive cell growth.

Parsed CitationsBloch D, Pleskot R, Pejchar P, Potocky M, Trpkosova P, Cwiklik L, Vukasinovic N, Sternberg H, Yalovsky S, Zarsky V (2016) ExocystSEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth. Plant Physiology 172: 980-1002

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bove J, Vaillancourt B, Kroeger J, Hepler PK, Wiseman PW, Geitmann A (2008) Magnitude and direction of vesicle dynamics in growingpollen tubes using spatiotemporal image correlation spectroscopy and fluorescence recovery after photobleaching. Plant Physiology147: 1646-1658


Braeckmans K, Stubbe BG, Remaut K, Demeester J, De Smedt SC (2006) Anomalous photobleaching in fluorescence recovery afterphotobleaching measurements due to excitation saturation--a case study for fluorescein. J Biomed Opt 11: 044013


Burkart GM, Baskin TI, Bezanilla M (2015) A family of ROP proteins that suppresses actin dynamics, and is essential for polarizedgrowth and cell adhesion. Journal of Cell Science 128: 2553-2564


Campàs O, Mahadevan L (2009) Shape and dynamics of tip-growing cells. Current Biology 19: 2102-2107Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Cardenas L, Lovy-Wheeler A, Kunkel JG, Hepler PK (2008) Pollen tube growth oscillations and intracellular calcium levels arereversibly modulated by actin polymerization. Plant Physiol 146: 1611-1621


Dalal RB, Digman MA, Horwitz AF, Vetri V, Gratton E (2008) Determination of particle number and brightness using a laser scanningconfocal microscope operating in the analog mode. Microscopy Research and Technique 71: 69-81


Derksen J, Rutten T, Vanamstel T, Dewin A, Doris F, Steer M (1995) Regulation of Pollen-Tube Growth. Acta Botanica Neerlandica 44:93-119


Digman MA, Dalal R, Horwitz AF, Gratton E (2008) Mapping the number of molecules and brightness in the laser scanning microscope.Biophysical Journal 94: 2320-2332


El Kasmi F, Krause C, Hiller U, Stierhof YD, Mayer U, Conner L, Kong L, Reichardt I, Sanderfoot AA, Jurgens G (2013) SNAREcomplexes of different composition jointly mediate membrane fusion in Arabidopsis cytokinesis. Mol Biol Cell 24: 1593-1601


Fritzsche M, Charras G (2015) Dissecting protein reaction dynamics in living cells by fluorescence recovery after photobleaching.Nature Protocols 10: 660-680


Furt F, Lemoi K, Tüzel E, Vidali L (2012) Quantitative analysis of organelle distribution and dynamics in Physcomitrella patensprotonemal cells BMC Plant Biology 12: 70

Furt F, Liu YC, Bibeau JP, Tüzel E, Vidali L (2013) Apical myosin XI anticipates F-actin during polarized growth of Physcomitrella patenscells. Plant J 73: 417-428

Pubmed: Author and Title

http://www.ncbi.nlm.nih.gov/pubmed?term=Bloch D%2C Pleskot R%2C Pejchar P%2C Potocky M%2C Trpkosova P%2C Cwiklik L%2C Vukasinovic N%2C Sternberg H%2C Yalovsky S%2C Zarsky V %282016%29 Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth%2E Plant Physiology 172%3A 980%2D1002&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bloch D, Pleskot R, Pejchar P, Potocky M, Trpkosova P, Cwiklik L, Vukasinovic N, Sternberg H, Yalovsky S, Zarsky V (2016) Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth. Plant Physiology 172: 980-1002

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bloch&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bloch&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Bove J%2C Vaillancourt B%2C Kroeger J%2C Hepler PK%2C Wiseman PW%2C Geitmann A %282008%29 Magnitude and direction of vesicle dynamics in growing pollen tubes using spatiotemporal image correlation spectroscopy and fluorescence recovery after photobleaching%2E Plant Physiology 147%3A 1646%2D1658&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bove J, Vaillancourt B, Kroeger J, Hepler PK, Wiseman PW, Geitmann A (2008) Magnitude and direction of vesicle dynamics in growing pollen tubes using spatiotemporal image correlation spectroscopy and fluorescence recovery after photobleaching. Plant Physiology 147: 1646-1658

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bove&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Magnitude and direction of vesicle dynamics in growing pollen tubes using spatiotemporal image correlation spectroscopy and fluorescence recovery after photobleaching&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Magnitude and direction of vesicle dynamics in growing pollen tubes using spatiotemporal image correlation spectroscopy and fluorescence recovery after photobleaching&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bove&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Braeckmans K%2C Stubbe BG%2C Remaut K%2C Demeester J%2C De Smedt SC %282006%29 Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation%2D%2Da case study for fluorescein%2E J Biomed Opt 11%3A 044013&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Braeckmans K, Stubbe BG, Remaut K, Demeester J, De Smedt SC (2006) Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein. J Biomed Opt 11: 044013

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Braeckmans&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Braeckmans&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Burkart GM%2C Baskin TI%2C Bezanilla M %282015%29 A family of ROP proteins that suppresses actin dynamics%2C and is essential for polarized growth and cell adhesion%2E Journal of Cell Science 128%3A 2553%2D2564&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Burkart GM, Baskin TI, Bezanilla M (2015) A family of ROP proteins that suppresses actin dynamics, and is essential for polarized growth and cell adhesion. Journal of Cell Science 128: 2553-2564

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Burkart&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=A family of ROP proteins that suppresses actin dynamics, and is essential for polarized growth and cell adhesion&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=A family of ROP proteins that suppresses actin dynamics, and is essential for polarized growth and cell adhesion&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Burkart&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Camp�s O%2C Mahadevan L %282009%29 Shape and dynamics of tip%2Dgrowing cells%2E Current Biology 19%3A 2102%2D2107&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Camp�s O, Mahadevan L (2009) Shape and dynamics of tip-growing cells. Current Biology 19: 2102-2107

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Campàs&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Shape and dynamics of tip-growing cells&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Shape and dynamics of tip-growing cells&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Campàs&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Cardenas L%2C Lovy%2DWheeler A%2C Kunkel JG%2C Hepler PK %282008%29 Pollen tube growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization%2E Plant Physiol 146%3A 1611%2D1621&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Cardenas L, Lovy-Wheeler A, Kunkel JG, Hepler PK (2008) Pollen tube growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization. Plant Physiol 146: 1611-1621

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Cardenas&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Pollen tube growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Pollen tube growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Cardenas&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Dalal RB%2C Digman MA%2C Horwitz AF%2C Vetri V%2C Gratton E %282008%29 Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode%2E Microscopy Research and Technique 71%3A 69%2D81&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Dalal RB, Digman MA, Horwitz AF, Vetri V, Gratton E (2008) Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode. Microscopy Research and Technique 71: 69-81

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Dalal&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Dalal&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Derksen J%2C Rutten T%2C Vanamstel T%2C Dewin A%2C Doris F%2C Steer M %281995%29 Regulation of Pollen%2DTube Growth%2E Acta Botanica Neerlandica 44%3A 93%2D119&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Derksen J, Rutten T, Vanamstel T, Dewin A, Doris F, Steer M (1995) Regulation of Pollen-Tube Growth. Acta Botanica Neerlandica 44: 93-119

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Derksen&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Regulation of Pollen-Tube Growth&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Regulation of Pollen-Tube Growth&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Derksen&as_ylo=1995&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Digman MA%2C Dalal R%2C Horwitz AF%2C Gratton E %282008%29 Mapping the number of molecules and brightness in the laser scanning microscope%2E Biophysical Journal 94%3A 2320%2D2332&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Digman MA, Dalal R, Horwitz AF, Gratton E (2008) Mapping the number of molecules and brightness in the laser scanning microscope. Biophysical Journal 94: 2320-2332

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Digman&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Mapping the number of molecules and brightness in the laser scanning microscope&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Mapping the number of molecules and brightness in the laser scanning microscope&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Digman&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=El Kasmi F%2C Krause C%2C Hiller U%2C Stierhof YD%2C Mayer U%2C Conner L%2C Kong L%2C Reichardt I%2C Sanderfoot AA%2C Jurgens G %282013%29 SNARE complexes of different composition jointly mediate membrane fusion in Arabidopsis cytokinesis%2E Mol Biol Cell 24%3A 1593%2D1601&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=El Kasmi F, Krause C, Hiller U, Stierhof YD, Mayer U, Conner L, Kong L, Reichardt I, Sanderfoot AA, Jurgens G (2013) SNARE complexes of different composition jointly mediate membrane fusion in Arabidopsis cytokinesis. Mol Biol Cell 24: 1593-1601

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=El&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=SNARE complexes of different composition jointly mediate membrane fusion in Arabidopsis cytokinesis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=SNARE complexes of different composition jointly mediate membrane fusion in Arabidopsis cytokinesis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=El&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Fritzsche M%2C Charras G %282015%29 Dissecting protein reaction dynamics in living cells by fluorescence recovery after photobleaching%2E Nature Protocols 10%3A 660%2D680&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Fritzsche M, Charras G (2015) Dissecting protein reaction dynamics in living cells by fluorescence recovery after photobleaching. Nature Protocols 10: 660-680

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Fritzsche&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Dissecting protein reaction dynamics in living cells by fluorescence recovery after photobleaching&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Dissecting protein reaction dynamics in living cells by fluorescence recovery after photobleaching&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Fritzsche&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Furt F%2C Liu YC%2C Bibeau JP%2C T�zel E%2C Vidali L %282013%29 Apical myosin XI anticipates F%2Dactin during polarized growth of Physcomitrella patens cells%2E Plant J 73%3A 417%2D428&dopt=abstract

CrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hepler PK, Rounds CM, Winship LJ (2013) Control of cell wall extensibility during pollen tube growth. Molecular Plant 6: 998-1017Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hepler PK, Vidali L, Cheung AY (2001) Polarized cell growth in higher plants. Annu Rev Cell Dev Biol 17: 159-187Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kroeger JH, Daher FB, Grant M, Geitmann A (2009) Microfilament orientation constrains vesicle flow and spatial distribution in growingpollen tubes. Biophys J 97: 1822-1831


Kulich I, Cole R, Drdova E, Cvrckova F, Soukup A, Fowler J, Zarsky V (2010) Arabidopsis exocyst subunits SEC8 and EXO70A1 andexocyst interactor ROH1 are involved in the localized deposition of seed coat pectin. New Phytologist 188: 615-625


Lancelle SA, Hepler PK (1992) Ultrastructure of freeze-substituted pollen tubes of lilium-longiflorum. Protoplasma 167: 215-230Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lee YJ, Yang ZB (2008) Tip growth: signaling in the apical dome. Current Opinion in Plant Biology 11: 662-671Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lipka V, Kwon C, Panstruga R (2007) SNARE-ware: the role of SNARE-domain proteins in plant biology. Annu Rev Cell Dev Biol 23: 147-174


Loren N, Hagman J, Jonasson JK, Deschout H, Bernin D, Cella-Zanacchi F, Diaspro A, McNally JG, Ameloot M, Smisdom N, Nyden M,Hermansson AM, Rudemo M, Braeckmans K (2015) Fluorescence recovery after photobleaching in material and life sciences: puttingtheory into practice. Quarterly Reviews of Biophysics 48: 323-387


Lovy-Wheeler A, Wilsen KL, Baskin TI, Hepler PK (2005) Enhanced fixation reveals the apical cortical fringe of actin filaments as aconsistent feature of the pollen tube. Planta 221: 95-104


Madison SL, Buchanan ML, Glass JD, McClain TF, Park E, Nebenfuhr A (2015) Class XI myosins move specific organelles in pollentubes and are required for normal fertility and pollen tube growth in Arabidopsis. Plant Physiology 169: 1946-1960


Madison SL, Nebenfuhr A (2013) Understanding myosin functions in plants: are we there yet? Curr Opin Plant Biol 16: 710-717Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Martin A, Lang D, Hanke ST, Mueller SJX, Sarnighausen E, Vervliet-Scheebaum M, Reski R (2009) Targeted gene knockouts revealoverlapping functions of the five Physcomitrella patens FtsZ isoforms in chloroplast division, chloroplast shaping, cellpatterning, plantdevelopment, and gravity sensing. Molecular Plant 2: 1359-1372


Mccauley MM, Hepler PK (1992) Cortical Ultrastructure of Freeze-Substituted Protonemata of the Moss Funaria-Hygrometrica.Protoplasma 169: 168-178

Pubmed: Author and TitleCrossRef: Author and Title

http://search.crossref.org/?page=1&rows=2&q=Furt F, Liu YC, Bibeau JP, T�zel E, Vidali L (2013) Apical myosin XI anticipates F-actin during polarized growth of Physcomitrella patens cells. Plant J 73: 417-428

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Furt&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Apical myosin XI anticipates F-actin during polarized growth of Physcomitrella patens cells&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Apical myosin XI anticipates F-actin during polarized growth of Physcomitrella patens cells&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Furt&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Hepler PK%2C Rounds CM%2C Winship LJ %282013%29 Control of cell wall extensibility during pollen tube growth%2E Molecular Plant 6%3A 998%2D1017&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Hepler PK, Rounds CM, Winship LJ (2013) Control of cell wall extensibility during pollen tube growth. Molecular Plant 6: 998-1017

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hepler&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Control of cell wall extensibility during pollen tube growth&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Control of cell wall extensibility during pollen tube growth&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hepler&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Hepler PK%2C Vidali L%2C Cheung AY %282001%29 Polarized cell growth in higher plants%2E Annu Rev Cell Dev Biol 17%3A 159%2D187&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Hepler PK, Vidali L, Cheung AY (2001) Polarized cell growth in higher plants. Annu Rev Cell Dev Biol 17: 159-187

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hepler&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Polarized cell growth in higher plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Polarized cell growth in higher plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hepler&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Kroeger JH%2C Daher FB%2C Grant M%2C Geitmann A %282009%29 Microfilament orientation constrains vesicle flow and spatial distribution in growing pollen tubes%2E Biophys J 97%3A 1822%2D1831&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kroeger JH, Daher FB, Grant M, Geitmann A (2009) Microfilament orientation constrains vesicle flow and spatial distribution in growing pollen tubes. Biophys J 97: 1822-1831

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kroeger&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Microfilament orientation constrains vesicle flow and spatial distribution in growing pollen tubes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Microfilament orientation constrains vesicle flow and spatial distribution in growing pollen tubes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kroeger&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Kulich I%2C Cole R%2C Drdova E%2C Cvrckova F%2C Soukup A%2C Fowler J%2C Zarsky V %282010%29 Arabidopsis exocyst subunits SEC8 and EXO70A1 and exocyst interactor ROH1 are involved in the localized deposition of seed coat pectin%2E New Phytologist 188%3A 615%2D625&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Kulich I, Cole R, Drdova E, Cvrckova F, Soukup A, Fowler J, Zarsky V (2010) Arabidopsis exocyst subunits SEC8 and EXO70A1 and exocyst interactor ROH1 are involved in the localized deposition of seed coat pectin. New Phytologist 188: 615-625

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kulich&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Arabidopsis exocyst subunits SEC8 and EXO70A1 and exocyst interactor ROH1 are involved in the localized deposition of seed coat pectin&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Arabidopsis exocyst subunits SEC8 and EXO70A1 and exocyst interactor ROH1 are involved in the localized deposition of seed coat pectin&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kulich&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Lancelle SA%2C Hepler PK %281992%29 Ultrastructure of freeze%2Dsubstituted pollen tubes of lilium%2Dlongiflorum%2E Protoplasma 167%3A 215%2D230&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Lancelle SA, Hepler PK (1992) Ultrastructure of freeze-substituted pollen tubes of lilium-longiflorum. Protoplasma 167: 215-230

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lancelle&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Ultrastructure of freeze-substituted pollen tubes of lilium-longiflorum&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Ultrastructure of freeze-substituted pollen tubes of lilium-longiflorum&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lancelle&as_ylo=1992&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Lee YJ%2C Yang ZB %282008%29 Tip growth%3A signaling in the apical dome%2E Current Opinion in Plant Biology 11%3A 662%2D671&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Lee YJ, Yang ZB (2008) Tip growth: signaling in the apical dome. Current Opinion in Plant Biology 11: 662-671

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lee&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Tip growth: signaling in the apical dome&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Tip growth: signaling in the apical dome&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lee&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Lipka V%2C Kwon C%2C Panstruga R %282007%29 SNARE%2Dware%3A the role of SNARE%2Ddomain proteins in plant biology%2E Annu Rev Cell Dev Biol 23%3A 147%2D174&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Lipka V, Kwon C, Panstruga R (2007) SNARE-ware: the role of SNARE-domain proteins in plant biology. Annu Rev Cell Dev Biol 23: 147-174

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lipka&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=SNARE-ware: the role of SNARE-domain proteins in plant biology&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=SNARE-ware: the role of SNARE-domain proteins in plant biology&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lipka&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Loren N%2C Hagman J%2C Jonasson JK%2C Deschout H%2C Bernin D%2C Cella%2DZanacchi F%2C Diaspro A%2C McNally JG%2C Ameloot M%2C Smisdom N%2C Nyden M%2C Hermansson AM%2C Rudemo M%2C Braeckmans K %282015%29 Fluorescence recovery after photobleaching in material and life sciences%3A putting theory into practice%2E Quarterly Reviews of Biophysics 48%3A 323%2D387&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Loren N, Hagman J, Jonasson JK, Deschout H, Bernin D, Cella-Zanacchi F, Diaspro A, McNally JG, Ameloot M, Smisdom N, Nyden M, Hermansson AM, Rudemo M, Braeckmans K (2015) Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice. Quarterly Reviews of Biophysics 48: 323-387

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Loren&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Loren&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Lovy%2DWheeler A%2C Wilsen KL%2C Baskin TI%2C Hepler PK %282005%29 Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube%2E Planta 221%3A 95%2D104&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Lovy-Wheeler A, Wilsen KL, Baskin TI, Hepler PK (2005) Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube. Planta 221: 95-104

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lovy-Wheeler&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lovy-Wheeler&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Madison SL%2C Buchanan ML%2C Glass JD%2C McClain TF%2C Park E%2C Nebenfuhr A %282015%29 Class XI myosins move specific organelles in pollen tubes and are required for normal fertility and pollen tube growth in Arabidopsis%2E Plant Physiology 169%3A 1946%2D1960&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Madison SL, Buchanan ML, Glass JD, McClain TF, Park E, Nebenfuhr A (2015) Class XI myosins move specific organelles in pollen tubes and are required for normal fertility and pollen tube growth in Arabidopsis. Plant Physiology 169: 1946-1960

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Madison&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Class XI myosins move specific organelles in pollen tubes and are required for normal fertility and pollen tube growth in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Class XI myosins move specific organelles in pollen tubes and are required for normal fertility and pollen tube growth in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Madison&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Madison SL%2C Nebenfuhr A %282013%29 Understanding myosin functions in plants%3A are we there yet%3F Curr Opin Plant Biol 16%3A 710%2D717&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Madison SL, Nebenfuhr A (2013) Understanding myosin functions in plants: are we there yet? Curr Opin Plant Biol 16: 710-717

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Madison&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Understanding myosin functions in plants: are we there yet&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Understanding myosin functions in plants: are we there yet&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Madison&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Martin A%2C Lang D%2C Hanke ST%2C Mueller SJX%2C Sarnighausen E%2C Vervliet%2DScheebaum M%2C Reski R %282009%29 Targeted gene knockouts reveal overlapping functions of the five Physcomitrella patens FtsZ isoforms in chloroplast division%2C chloroplast shaping%2C cellpatterning%2C plant development%2C and gravity sensing%2E Molecular Plant 2%3A 1359%2D1372&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Martin A, Lang D, Hanke ST, Mueller SJX, Sarnighausen E, Vervliet-Scheebaum M, Reski R (2009) Targeted gene knockouts reveal overlapping functions of the five Physcomitrella patens FtsZ isoforms in chloroplast division, chloroplast shaping, cellpatterning, plant development, and gravity sensing. Molecular Plant 2: 1359-1372

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Martin&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Targeted gene knockouts reveal overlapping functions of the five Physcomitrella patens FtsZ isoforms in chloroplast division, chloroplast shaping, cellpatterning, plant development, and gravity sensing&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Targeted gene knockouts reveal overlapping functions of the five Physcomitrella patens FtsZ isoforms in chloroplast division, chloroplast shaping, cellpatterning, plant development, and gravity sensing&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Martin&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Mccauley MM%2C Hepler PK %281992%29 Cortical Ultrastructure of Freeze%2DSubstituted Protonemata of the Moss Funaria%2DHygrometrica%2E Protoplasma 169%3A 168%2D178&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Mccauley MM, Hepler PK (1992) Cortical Ultrastructure of Freeze-Substituted Protonemata of the Moss Funaria-Hygrometrica. Protoplasma 169: 168-178

Google Scholar: Author Only Title Only Author and Title

McKenna ST, Kunkel JG, Bosch M, Rounds CM, Vidali L, Winship LJ, Hepler PK (2009) Exocytosis precedes and predicts the increasein growth in oscillating pollen tubes. Plant Cell 21: 3026-3040


McNally JG (2008) Quantitative FRAP in analysis of molecular binding dynamics in vivo. Methods Cell Biol 85: 329-351Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Menand B, Calder G, Dolan L (2007) Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens.Journal of Experimental Botany 58: 1843-1849


Moens PDJ, Gratton E, Salvemini IL (2011) Fluorescence correlation spectroscopy, Raster image correlation spectroscopy, andNumber & Brightness on a commercial confocal laser scanning microscope with analog detectors (Nikon C1). Microscopy Researchand Technique 74: 377-388


Morisaki T, McNally JG (2014) Photoswitching-free FRAP analysis with a genetically encoded fluorescent tag. PLoS One 9: e107730Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Moscatelli A, Idilli AI, Rodighiero S, Caccianiga M (2012) Inhibition of actin polymerisation by low concentration Latrunculin B affectsendocytosis and alters exocytosis in shank and tip of tobacco pollen tubes. Plant Biol (Stuttg) 14: 770-782


Motulsky H, Christopoulos A (2004) Fitting models to biological data using linear and nonlinear regression : a practical guide to curvefitting. Oxford University Press, Oxford ; New York


Mueller F, Morisaki T, Mazza D, McNally JG (2012) Minimizing the impact of photoswitching of fluorescent proteins on FRAP analysis.Biophys J 102: 1656-1665


Phillips R (2013) Physical biology of the cell, Ed Second edition /. Garland Science, London New York, NYPubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Rojas ER, Hotton S, Dumais J (2011) Chemically mediated mechanical expansion of the pollen tube cell wall. Biophys J 101: 1844-1853Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Rounds CM, Bezanilla M (2013) Growth mechanisms in tip-growing plant cells. Annual Review of Plant Biology, Vol 64 64: 243-265Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Rounds CM, Hepler PK, Winship LJ (2014) The apical actin fringe contributes to localized cell walldeposition and polarized growth inthe lily pollen tube. Plant Physiology 166: 139-151


Sanati Nezhad A, Packirisamy M, Geitmann A (2014) Dynamic, high precision targeting of growth modulating agents is able to triggerpollen tube growth reorientation. Plant J 80: 185-195


http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Mccauley&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Cortical Ultrastructure of Freeze-Substituted Protonemata of the Moss Funaria-Hygrometrica&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Cortical Ultrastructure of Freeze-Substituted Protonemata of the Moss Funaria-Hygrometrica&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Mccauley&as_ylo=1992&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=McKenna ST%2C Kunkel JG%2C Bosch M%2C Rounds CM%2C Vidali L%2C Winship LJ%2C Hepler PK %282009%29 Exocytosis precedes and predicts the increase in growth in oscillating pollen tubes%2E Plant Cell 21%3A 3026%2D3040&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=McKenna ST, Kunkel JG, Bosch M, Rounds CM, Vidali L, Winship LJ, Hepler PK (2009) Exocytosis precedes and predicts the increase in growth in oscillating pollen tubes. Plant Cell 21: 3026-3040

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=McKenna&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Exocytosis precedes and predicts the increase in growth in oscillating pollen tubes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Exocytosis precedes and predicts the increase in growth in oscillating pollen tubes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=McKenna&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=McNally JG %282008%29 Quantitative FRAP in analysis of molecular binding dynamics in vivo%2E Methods Cell Biol 85%3A 329%2D351&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=McNally JG (2008) Quantitative FRAP in analysis of molecular binding dynamics in vivo. Methods Cell Biol 85: 329-351

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=McNally&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Quantitative FRAP in analysis of molecular binding dynamics in vivo&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Quantitative FRAP in analysis of molecular binding dynamics in vivo&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=McNally&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Menand B%2C Calder G%2C Dolan L %282007%29 Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens%2E Journal of Experimental Botany 58%3A 1843%2D1849&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Menand B, Calder G, Dolan L (2007) Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens. Journal of Experimental Botany 58: 1843-1849

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Menand&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Menand&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Moens PDJ%2C Gratton E%2C Salvemini IL %282011%29 Fluorescence correlation spectroscopy%2C Raster image correlation spectroscopy%2C and Number %26 Brightness on a commercial confocal laser scanning microscope with analog detectors %28Nikon C1%29%2E Microscopy Research and Technique 74%3A 377%2D388&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Moens PDJ, Gratton E, Salvemini IL (2011) Fluorescence correlation spectroscopy, Raster image correlation spectroscopy, and Number & Brightness on a commercial confocal laser scanning microscope with analog detectors (Nikon C1). Microscopy Research and Technique 74: 377-388

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Moens&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Fluorescence correlation spectroscopy, Raster image correlation spectroscopy, and Number & Brightness on a commercial confocal laser scanning microscope with analog detectors (Nikon C1)&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Fluorescence correlation spectroscopy, Raster image correlation spectroscopy, and Number & Brightness on a commercial confocal laser scanning microscope with analog detectors (Nikon C1)&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Moens&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Morisaki T%2C McNally JG %282014%29 Photoswitching%2Dfree FRAP analysis with a genetically encoded fluorescent tag%2E PLoS One 9%3A e107730&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Morisaki T, McNally JG (2014) Photoswitching-free FRAP analysis with a genetically encoded fluorescent tag. PLoS One 9: e107730

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Morisaki&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Photoswitching-free FRAP analysis with a genetically encoded fluorescent tag.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Photoswitching-free FRAP analysis with a genetically encoded fluorescent tag.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Morisaki&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Moscatelli A%2C Idilli AI%2C Rodighiero S%2C Caccianiga M %282012%29 Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes%2E Plant Biol %28Stuttg%29 14%3A 770%2D782&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Moscatelli A, Idilli AI, Rodighiero S, Caccianiga M (2012) Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes. Plant Biol (Stuttg) 14: 770-782

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Moscatelli&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Moscatelli&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Motulsky H%2C Christopoulos A %282004%29 Fitting models to biological data using linear and nonlinear regression %3A a practical guide to curve fitting%2E Oxford University Press%2C Oxford %3B New York&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Motulsky H, Christopoulos A (2004) Fitting models to biological data using linear and nonlinear regression : a practical guide to curve fitting. Oxford University Press, Oxford ; New York

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Motulsky&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Fitting models to biological data using linear and nonlinear regression : a practical guide to curve fitting.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Fitting models to biological data using linear and nonlinear regression : a practical guide to curve fitting.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Motulsky&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Mueller F%2C Morisaki T%2C Mazza D%2C McNally JG %282012%29 Minimizing the impact of photoswitching of fluorescent proteins on FRAP analysis%2E Biophys J 102%3A 1656%2D1665&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Mueller F, Morisaki T, Mazza D, McNally JG (2012) Minimizing the impact of photoswitching of fluorescent proteins on FRAP analysis. Biophys J 102: 1656-1665

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Mueller&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Minimizing the impact of photoswitching of fluorescent proteins on FRAP analysis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Minimizing the impact of photoswitching of fluorescent proteins on FRAP analysis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Mueller&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Phillips R %282013%29 Physical biology of the cell%2C Ed Second edition %2F%2E Garland Science%2C London New York%2C NY&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Phillips R (2013) Physical biology of the cell, Ed Second edition /. Garland Science, London New York, NY

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Phillips&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Physical biology of the cell, Ed Second edition /.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Physical biology of the cell, Ed Second edition /.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Phillips&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Rojas ER%2C Hotton S%2C Dumais J %282011%29 Chemically mediated mechanical expansion of the pollen tube cell wall%2E Biophys J 101%3A 1844%2D1853&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Rojas ER, Hotton S, Dumais J (2011) Chemically mediated mechanical expansion of the pollen tube cell wall. Biophys J 101: 1844-1853

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Rojas&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Chemically mediated mechanical expansion of the pollen tube cell wall&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Chemically mediated mechanical expansion of the pollen tube cell wall&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Rojas&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Rounds CM%2C Bezanilla M %282013%29 Growth mechanisms in tip%2Dgrowing plant cells%2E Annual Review of Plant Biology%2C Vol 64 64%3A 243%2D265&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Rounds CM, Bezanilla M (2013) Growth mechanisms in tip-growing plant cells. Annual Review of Plant Biology, Vol 64 64: 243-265

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Rounds&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Growth mechanisms in tip-growing plant cells&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Growth mechanisms in tip-growing plant cells&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Rounds&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Rounds CM%2C Hepler PK%2C Winship LJ %282014%29 The apical actin fringe contributes to localized cell walldeposition and polarized growth in the lily pollen tube%2E Plant Physiology 166%3A 139%2D151&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Rounds CM, Hepler PK, Winship LJ (2014) The apical actin fringe contributes to localized cell walldeposition and polarized growth in the lily pollen tube. Plant Physiology 166: 139-151

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Rounds&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The apical actin fringe contributes to localized cell walldeposition and polarized growth in the lily pollen tube&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The apical actin fringe contributes to localized cell walldeposition and polarized growth in the lily pollen tube&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Rounds&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Sanati Nezhad A%2C Packirisamy M%2C Geitmann A %282014%29 Dynamic%2C high precision targeting of growth modulating agents is able to trigger pollen tube growth reorientation%2E Plant J 80%3A 185%2D195&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Sanati Nezhad A, Packirisamy M, Geitmann A (2014) Dynamic, high precision targeting of growth modulating agents is able to trigger pollen tube growth reorientation. Plant J 80: 185-195

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Sanati&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Dynamic, high precision targeting of growth modulating agents is able to trigger pollen tube growth reorientation&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Dynamic, high precision targeting of growth modulating agents is able to trigger pollen tube growth reorientation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sanati&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

Sanderfoot A (2007) Increases in the number of SNARE genes parallels the rise of multicellularity among the green plants. PlantPhysiol 144: 6-17


Shimmen T (2007) The sliding theory of cytoplasmic streaming: fifty years of progress. Journal of Plant Research 120: 31-43Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sinnecker D, Voigt P, Hellwig N, Schaefer M (2005) Reversible photobleaching of enhanced green fluorescent proteins. Biochemistry44: 7085-7094


Vidali L, Augustine RC, Kleinman KP, Bezanilla M (2007) Profilin is essential for tip growth in the moss Physcomitrella patens. PlantCell 19: 3705-3722


Vidali L, Bezanilla M (2012) Physcomitrella patens: a model for tip cell growth and differentiation. Curr Opin Plant Biol 15: 625-631Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Vidali L, Burkart GM, Augustine RC, Kerdavid E, Tüzel E, Bezanilla M (2010) Myosin XI is essential for tip growth in Physcomitrellapatens. Plant Cell 22: 1868-1882


Vidali L, McKenna ST, Hepler PK (2001) Actin polymerization is essential for pollen tube growth. Molecular Biology of the Cell 12: 2534-2545


Vidali L, Rounds CM, Hepler PK, Bezanilla M (2009) Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells.Plos One 4: e5744


Winship LJ, Obermeyer G, Geitmann A, Hepler PK (2010) Under pressure, cell walls set the pace. Trends in Plant Science 15: 363-369Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zonia L, Munnik T (2008) Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes.Journal of Experimental Botany 59: 861-873


http://www.ncbi.nlm.nih.gov/pubmed?term=Sanderfoot A %282007%29 Increases in the number of SNARE genes parallels the rise of multicellularity among the green plants%2E Plant Physiol 144%3A 6%2D17&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Sanderfoot A (2007) Increases in the number of SNARE genes parallels the rise of multicellularity among the green plants. Plant Physiol 144: 6-17

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Sanderfoot&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Increases in the number of SNARE genes parallels the rise of multicellularity among the green plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Increases in the number of SNARE genes parallels the rise of multicellularity among the green plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sanderfoot&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Shimmen T %282007%29 The sliding theory of cytoplasmic streaming%3A fifty years of progress%2E Journal of Plant Research 120%3A 31%2D43&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Shimmen T (2007) The sliding theory of cytoplasmic streaming: fifty years of progress. Journal of Plant Research 120: 31-43

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Shimmen&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The sliding theory of cytoplasmic streaming: fifty years of progress&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The sliding theory of cytoplasmic streaming: fifty years of progress&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Shimmen&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Sinnecker D%2C Voigt P%2C Hellwig N%2C Schaefer M %282005%29 Reversible photobleaching of enhanced green fluorescent proteins%2E Biochemistry 44%3A 7085%2D7094&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Sinnecker D, Voigt P, Hellwig N, Schaefer M (2005) Reversible photobleaching of enhanced green fluorescent proteins. Biochemistry 44: 7085-7094

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Sinnecker&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Reversible photobleaching of enhanced green fluorescent proteins&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Reversible photobleaching of enhanced green fluorescent proteins&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Sinnecker&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Vidali L%2C Augustine RC%2C Kleinman KP%2C Bezanilla M %282007%29 Profilin is essential for tip growth in the moss Physcomitrella patens%2E Plant Cell 19%3A 3705%2D3722&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Vidali L, Augustine RC, Kleinman KP, Bezanilla M (2007) Profilin is essential for tip growth in the moss Physcomitrella patens. Plant Cell 19: 3705-3722

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Vidali&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Profilin is essential for tip growth in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Profilin is essential for tip growth in the moss Physcomitrella patens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vidali&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Vidali L%2C Bezanilla M %282012%29 Physcomitrella patens%3A a model for tip cell growth and differentiation%2E Curr Opin Plant Biol 15%3A 625%2D631&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Vidali L, Bezanilla M (2012) Physcomitrella patens: a model for tip cell growth and differentiation. Curr Opin Plant Biol 15: 625-631


http://scholar.google.com/scholar?as_q=Physcomitrella patens: a model for tip cell growth and differentiation&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Physcomitrella patens: a model for tip cell growth and differentiation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vidali&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Vidali L%2C Burkart GM%2C Augustine RC%2C Kerdavid E%2C T�zel E%2C Bezanilla M %282010%29 Myosin XI is essential for tip growth in Physcomitrella patens%2E Plant Cell 22%3A 1868%2D1882&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Vidali L, Burkart GM, Augustine RC, Kerdavid E, T�zel E, Bezanilla M (2010) Myosin XI is essential for tip growth in Physcomitrella patens. Plant Cell 22: 1868-1882


http://scholar.google.com/scholar?as_q=Myosin XI is essential for tip growth in Physcomitrella patens&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Myosin XI is essential for tip growth in Physcomitrella patens&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vidali&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Vidali L%2C McKenna ST%2C Hepler PK %282001%29 Actin polymerization is essential for pollen tube growth%2E Molecular Biology of the Cell 12%3A 2534%2D2545&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Vidali L, McKenna ST, Hepler PK (2001) Actin polymerization is essential for pollen tube growth. Molecular Biology of the Cell 12: 2534-2545


http://scholar.google.com/scholar?as_q=Actin polymerization is essential for pollen tube growth&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Actin polymerization is essential for pollen tube growth&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vidali&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Vidali L%2C Rounds CM%2C Hepler PK%2C Bezanilla M %282009%29 Lifeact%2DmEGFP reveals a dynamic apical F%2Dactin network in tip growing plant cells%2E Plos One 4%3A e5744&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Vidali L, Rounds CM, Hepler PK, Bezanilla M (2009) Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells. Plos One 4: e5744


http://scholar.google.com/scholar?as_q=Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vidali&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Winship LJ%2C Obermeyer G%2C Geitmann A%2C Hepler PK %282010%29 Under pressure%2C cell walls set the pace%2E Trends in Plant Science 15%3A 363%2D369&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Winship LJ, Obermeyer G, Geitmann A, Hepler PK (2010) Under pressure, cell walls set the pace. Trends in Plant Science 15: 363-369

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Winship&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Under pressure, cell walls set the pace&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Under pressure, cell walls set the pace&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Winship&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Zonia L%2C Munnik T %282008%29 Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes%2E Journal of Experimental Botany 59%3A 861%2D873&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Zonia L, Munnik T (2008) Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes. Journal of Experimental Botany 59: 861-873

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zonia&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zonia&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

Documents

F-Actin Meditated Focusing of Vesicles at the Cell …...74 have heavily implicated the cytoskeleton as a key player in exocytosis. Evidence 75 suggests that myosin XI transports vesicle