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
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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
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
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
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)
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Inte
nsity VA
MP !
SIM!
Tip LatB!
Δt1! Δt2!
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Model wt0
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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
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th R
ate
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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)
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Positions
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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
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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.
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