1. Actin Polymerization Mediated by AtFH5 Directs the Polarity Establishment and Vesicle Trafficking for Pollen Germination in Arabidopsis 
Chang Liu, Yi Zhang, Haiyun Ren 
Molecular Plant 
Volume 11, Issue 11, Pages (November 2018)
DOI: /j.molp
Copyright © 2018 The Author Terms and Conditions

2. Figure 1
The Actin Cytoskeleton Establishes the Polarity Prior to Pollen Germination.
(A) Representative time-lapse images (maximal projection of z sections of the whole cell) of actin filaments labeled with Lifeact-mEGFP during pollen germination. “0 s” was set at 1 h after the incubation of pollen grains on germination medium. The blue band indicates the region selected for kymograph analysis in (D). Scale bar, 4 μm.
(B) Relative fluorescent density of actin filaments at the prospective germination site (Site-1) and the opposite site (Site-2) in (A) during the rotation stage (0–1350 s).
(C) Frequency distribution of actin filament oscillation velocity in pollen grains (n = 20 grains).
(D) Kymograph analysis of actin filaments in the region indicated by the blue band in (A) starting from 1380 s. The actin bundle length is indicated and was used to calculate the average length.
(E) Top view of the actin filament structure at Site-1 in (A) at 1540 s. Scale bar, 4 μm.
(F) Three-dimensional heatmap renderings of the fluorescence intensity of actin filaments (rectangle in E).
(G) Relative fluorescent density of actin filaments at Site-1 and Site-2 in (A) during the collar-like actin-structure-construction stage (1350–4000 s).
(H) Single-layer images showing the elongation of actin filaments at the prospective germination site. Scale bar, 1 μm.
(I) Frequency distribution of actin filament elongation rates within the collar-like actin structures at the prospective germination sites (n = 22 from six grains).
(J) Representative time-lapse images (maximal projection of z sections of the whole cell) of actin filaments in atfh5-3 pollen grains. “0 s” was set at 1 h after the incubation of pollen grains on germination medium. Scale bar, 4 μm.
(K) Relative fluorescent density of actin filaments at Site-1 and Site-2 in (J).
(L) Analysis of the skewness of actin bundles in the wild-type control (WT) and atfh5-3 pollen grains. The values are the means ± SE (n = 20 grains for each genotype). **p < 0.01, as determined in a two-tailed Student's t-test. To correct for the decrease in fluorescent intensity resulting from fluorescent bleaching caused by intense imaging, we calculated the relative values in (B), (G), and (K) as the ratio of the fluorescent density in each site normalized by the fluorescent density in the entire cell.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

3. Figure 2
Spatiotemporal Interaction of AtFH5 and Actin Filaments during Pollen Germination.
(A) Co-localization analysis of AtFH5-mCherry with the vesicle marker protein YFP-RabA4b during the actin-filament-rotation stage. The representative images are single-layer images with the focal plane at the middle of the pollen grain. Scale bar, 4 μm.
(B) Co-localization analysis of AtFH5-mCherry and YFP-RabA4b during the collar-like actin-structure-construction stage. The representative images are single-layer images with the focal plane at the middle of the pollen grain. Scale bar, 4 μm.
(C) Representative time-lapse images (maximal projection of z sections of the whole cell) showing the rotational movement of AtFH5-mCherry and actin filaments in a pollen grain. “0 s” was set at 1 h after the incubation of pollen grains on germination medium. Scale bar, 4 μm.
(D) Relative fluorescent density of AtFH5-mCherry and actin filaments at Site-1 and Site-2 in (C). The relative values were calculated and are shown as the ratios of the fluorescent density in each site normalized by the fluorescent density in the entire cell.
(E) Frequency distribution of AtFH5-labeled vesicle velocity during rotational movement (n = 20 grains).
(F) Representative time-lapse images (maximal projection of z sections of the whole cell) showing that AtFH5-mCherry translocated to the plasma membrane and initiated actin assembly at the prospective germination site. “0 s” was set at the point that a portion of AtFH5-mCherry translocated to the plasma membrane (as indicated by the arrows). The blue band indicates the region selected for kymograph analysis in (G). Scale bar, 4 μm.
(G) Kymograph analysis of AtFH5-mCherry and actin filaments in the region indicated by the blue band in (F). Note that AtFH5-mCherry (red) accumulated at the plasma membrane prior to the actin filaments (green) and remained in front of actin filaments afterward.
(H) Top view of AtFH5-mCherry and the actin filament structure at the prospective germination site in (F) at 120 s. Scale bar, 4 μm.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

4. Figure 3
The Motion of AtFH5-Labeled Vesicles Depends on the Actin Cytoskeleton.
(A) Time-lapse images (maximal projection of z sections of the whole cell) of AtFH5-mCherry and actin filaments in the presence of LatB. “0 s” was set at 10 min after the incubation of pollen grains on germination medium with 10 nM LatB. Scale bar, 4 μm.
(B) Relative fluorescent density of AtFH5-mCherry and actin filaments at Site-1 and Site-2 in (A).
(C) Time-lapse imaging (maximal z projection of sections of the whole cell) of AtFH5-mCherry and actin filaments in the presence of BDM. “0 s” was set at 10 min after the incubation of pollen grains on germination medium with 30 mM BDM. Scale bar, 4 μm.
(D) Relative fluorescent density of AtFH5-mCherry and actin filaments at Site-1 and Site-2 in (C).
(E) Quantification of the oscillation velocity of AtFH5-mCherry and actin filaments in the presence of various doses of BDM (n = 5 grains for each treatment). *p < 0.05, **p < 0.01, as determined in a two-tailed Student's t-test; NS, not significant.
(F) Quantification of the lag time between AtFH5-mCherry and actin filaments in the presence of various doses of BDM.
Values are the means ± SE (n = 5 grains for each treatment). The relative values in (B) and (D) were calculated as the ratios of the fluorescent density in each site normalized by the fluorescent density in the whole cell.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

5. Figure 4
Interaction of AtFH5-Labeled Vesicles and Actin Filaments during the Recovery of Actin Polymerization after LatB Treatment.
Pollen grains were treated with 10 nM LatB for 15 min to depolymerize actin filaments and were then transferred to medium without inhibitors to recover from the treatment.
(A) Single-layer images showing that actin filaments were polymerized from AtFH5-labeled vesicles and that the assembly of actin filaments in turn pushed the separated piles of vesicles, causing them to merge together. Arrows indicate the outer edges of the AtFH5-labeled vesicle pools. “0 s” was set at the point where pollen grains were transferred to medium without LatB. Scale bar, 4 μm.
(B) Kymograph analysis of AtFH5-mCherry and actin filaments along the blue line in (A).
(C) AtFH5-labeled vesicles merged together and were surrounded by actin filaments and bundles 25 min after transferring pollen grains to medium without LatB. The rectangle indicates the region shown in (E) (30 s). Scale bar, 4 μm.
(D) Length of actin filaments and bundles in concave and convex regions (n = 15 from three grains for each group). **p < 0.01, as determined in a two-tailed Student's t-test.
(E) Higher-gain images of the region outlined by the rectangle in (C) at sequential time points. Arrows indicate the elongation of actin bundles. Note that AtFH5-labeled vesicles were pushed away from the region outlined by the rectangle when actin filaments elongated into this region. Scale bar, 1 μm.
(F) The fluorescence density in the region outlined by the rectangle in (E) was negatively correlated with the length of nearby actin filaments.
(G) Single-layer images showing the rotational movement of AtFH5-mCherry and actin filaments. An arrow indicates the buckling of actin filaments. “0 s” was set at 60 min after transferring pollen grains to germination medium without LatB. Scale bar, 4 μm.
(H) A representative image showing the “near” and “far” regions relative to AtFH5-labeled vesicles. The right panel was overexposed to show the actin filaments in the far region.
(I) Quantification of the convolutedness of actin filaments in the near and far regions defined in (H) (n = 65 from 10 grains for each group). **p < 0.01, as determined in a two-tailed Student's t-test.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

6. Figure 5
A Proposed Working Model Illustrating the Polarity Establishment of Actin Cytoskeleton and Vesicle Mobility during Pollen Germination.
(A) After hydration, actin filaments undergo an active rotational stage, referred to as the actin-filament-rotation stage, in pollen grains, where AtFH5 localizes at vesicles and rotates ahead of the actin filaments. The future positions of AtFH5-labeled vesicles and actin filaments are blurred to show the rotational movement.
(B) A portion of AtFH5 translocates to the plasma membrane at the prospective germination site and induces the formation of a collar-like actin structure prior to pollen germination. This stage is referred to as the collar-like actin-structure-construction stage.
(C) A probable model whereby actin polymerization provides the force for the mobility of vesicles in pollen grains. AtFH5 functions as a processive formin that localizes at the vesicle surface, binds to the barbed end of actin filaments, and promotes efficient actin polymerization and elongation at the end of the filament bound by AtFH5. The assembly of actin filaments, in turn, provides a propulsive force that drives the mobility of AtFH5-labeled vesicles in the same direction as the actin filaments elongate.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions