Interdependence of Endomembrane Trafficking and Actin Dynamics during Polarized Growth of Arabidopsis Pollen Tubes

During polarized growth of pollen tubes, endomembrane trafficking and actin polymerization are two critical processes that establish membrane/wall homeostasis and maintain growth polarity. Fine-tuned interactions between these two processes are therefore necessary but poorly understood. To better understand such cross-talk in the model plant Arabidopsis thaliana , we first established optimized concentrations of drugs that interfere with either endomembrane trafficking or the actin cytoskeleton, then examined pollen tube growth using fluorescent protein markers that label transport vesicles, endosomes, or the actin cytoskeleton. Both Brefeldin A (BFA) and Wortmannin disturbed the motility and structural integrity of ARA7- but not ARA6-labeled endosomes, suggesting heterogeneity of the endosomal populations. Disrupting endomembrane trafficking by BFA or Wortmannin perturbed actin polymerization at the apical region but not in the longitudinal actin cables in the shank. The interference of BFA/Wortmannin with actin polymerization was progressive rather than rapid, suggesting an indirect effect, possibly due to perturbed endomembrane trafficking of certain membrane-localized signaling proteins. Both the actin depolymerization drug Latrunculin B (LatB) and the actin stabilization drug Jasplakinolide (Jas) rapidly disrupted transport of secretory vesicles, but each drug caused distinct responses on different endosomal populations labeled by ARA6 or ARA7, indicating that a dynamic actin cytoskeleton was critical for some steps in endomembrane trafficking. Our results provide evidence of cross-talk between endomembrane trafficking and the actin cytoskeleton in pollen tubes.


INTRODUCTION
Pollen tubes of flowering plants are specialized cells that deliver immotile sperm to the proximity of female gametes for successful reproduction (Johnson and Preuss, 2002).
The growth of pollen tubes is both polar and directional ; many cellular activities contribute to such growth, the most important being the dynamics of the actin cytoskeleton system, targeted exocytosis, and endocytosis .
Pollen tubes contain longitudinal actin cables along the shank, which are important for providing structural support and acting as tracks for the movement of large organelles (Staiger et al., 1994). The apical area of pollen tubes instead contains dynamic filamentous actin (F-actin), as shown by fluorescently-labeled actin-binding proteins (Kost et al., 1999;Fu et al., 2001;Chen et al., 2002;Wilsen et al., 2006). The dynamics of F-actin are critical for the polarized growth of pollen tubes. Genetically manipulating the activities of the small GTPases ROP (Kost et al., 1999;Fu et al., 2001;Cheung et al., 2008) and Rab (de Graaf et al., 2005), or of actin-binding proteins such as profilin and formin (Staiger et al., 1994;Chen et al., 2002;Cheung and Wu, 2004), disrupted F-actin dynamics and inhibited tube growth and caused apical bulges. Application of drugs such as Latrunculin B (LatB) and Jasplakinolide (Jas) showed similar effects (Gibbon et al., 1999;Cardenas et al., 2005;Hörmanseder et al., 2005;Chen et al., 2007).
Targeted exocytosis delivers building materials for cell membranes and cell walls and therefore is critical for maintaining growth polarity and directionality of growing pollen tubes . Because targeted exocytosis brings more membrane and wall materials than needed to the apex of a pollen tube, an active endocytic system exists to retrieve excess secreted materials. In addition to this non-selective bulk membrane retrieval, pollen tubes may have selective and regulated endocytic trafficking pathways.
For example, experiments using charged gold particles indicated the existence of two distinct endocytic pathways in tobacco pollen tubes (Moscatelli et al., 2007), and other studies showed that pollen tubes are able to take in materials from the extracellular matrix (Lind et al., 1996;Goldraij et al., 2006). The axis of targeted exocytosis correlated with the direction of tube growth and it asymmetrically changed toward the new apex during tube re-orientation (Camacho and Malho, 2003;de Graaf et al., 2005). Disruption of membrane trafficking altered growth trajectories (de Graaf et al., 2005). Both suggest that membrane trafficking is a critical part of polarity maintenance and re-orientation.
As two important cellular processes in pollen tube growth, membrane trafficking and actin polymerization are conceivably dependent on each other. For example, several studies demonstrated that dynamic actin polymerization was essential for membrane trafficking (Hörmanseder et al., 2005;Wang et al., 2005;Chen et al., 2007;Lee et al., 2008), while others explored whether membrane trafficking affected actin polymerization (de Graaf et al., 2005;Hörmanseder et al., 2005). These studies, however, were mostly done with rapidly growing pollen tubes from tobacco or lily. For the model plant Arabidopsis thaliana, whose pollen tubes grow slower, little is known in this regard.
Given a robust protocol for Arabidopsis pollen germination (Boavida and McCormick, 2007), it is now possible to investigate the interactions between these two cellular activities.
In this study, we analyzed the effects of drug treatments on Arabidopsis pollen tubes expressing fluorescent protein probes for transport vesicles, endosomes, or the actin cytoskeleton. We show that perturbing actin dynamics by LatB or Jas treatments disrupted the V-shaped distribution of transport vesicles, caused aggregation and finally dissipation of a subpopulation of endosomes, indicating that actin dynamics are critical at some steps of endomembrane trafficking. On the other hand, disturbing endomembrane trafficking with Brefeldin A (BFA) or Wortmannin abolished the F-actin structure at the apical region without affecting the longitudinal actin cables at the shank. These results provide evidence that endomembrane trafficking and actin dynamics interact at certain steps during polarized growth of Arabidopsis pollen tubes.

Optimization of Pharmacological Treatments on Arabidopsis Pollen Tube Growth
Inhibitors perturb cellular processes and thereby are useful for understanding their mechanisms. BFA is a fungal toxin that interferes with the activity of Arf guanine nucleotide exchange factors (ArfGEFs) and thereby interferes with endomembrane trafficking (Nebenfuhr et al., 2002). Wortmannin inhibits the activity of Phosphatidylinositol 3-phosphate kinase (PI3K) and therefore disturbs Phosphatidylinositol 3-phosphate (PtdIns3P) production and possibly its distribution, which is critical for endomembrane trafficking (van Leeuwen et al., 2004). Wortmannin at high concentrations (above 30 μ M) also inhibited PI4P and PI(4,5)P 2 production by inhibiting type III PI4 Kinase (Krinke et al., 2007). Because PI4P and PI(4,5)P 2 regulate both vesicle trafficking and the actin cytoskeleton in animal cells (Balla and Balla, 2006), we were concerned that Wortmannin treatment might not be specific. Therefore we used a concentration well below 30 μ M, to minimize interference with the actin cytoskeleton or with PI4P and PI(4,5)P 2 production. Studies with tobacco BY2 cells as well as with Arabidopsis seedlings showed that Wortmannin treatment below 30 μ M did not obviously affect the PI4P level, as shown by a fluorescent-tagged PI4P probe, PH FAPP1 (Vermeer et al., 2009). LatB depolymerizes the actin cytoskeleton (Gibbon et al., 1999) while Jas stabilizes it (Cardenas et al., 2005). Except for Wortmannin, these other inhibitors have been used in pollen tubes of various species such as pea, lily and tobacco, where they resulted in growth arrest and tube bulges (Gibbon et al., 1999;Cardenas et al., 2005;de Graaf et al., 2005;Wang et al., 2005;Wilsen et al., 2006;Chen et al., 2007;Lovy-Wheeler et al., 2007). Because pollen tubes from different plant species may have distinct sensitivities toward pharmacological drugs, optimized concentrations for Arabidopsis had to be determined. To examine the dynamic changes of endomembrane trafficking as well as actin polymerization, we used inhibitor concentrations that disturb these cellular activities without causing cell death. At such concentrations, the effects of inhibitor treatment, as assessed by reductions in growth rate and increases in tube width, could be visualized over time.
We applied serial dilutions of different inhibitors based on concentrations in previous reports (Gibbon et al., 1999;Cardenas et al., 2005;de Graaf et al., 2005;Wang et al., 2005;Wilsen et al., 2006;Chen et al., 2007;Lovy-Wheeler et al., 2007). Because inhibitors were dissolved in DMSO, we also confirmed that adding equivalent volumes of DMSO to control tubes had no phenotypic consequences over the time course of the experiments. Inhibitors were added 4 hr after pollen germination in liquid germination medium (PGM). Several concentrations of BFA were used: there was hardly any effect on tube growth rate or width at 0.7 µM or 1 µM ( Figure 1C), however, at 1.4 µM inhibitory effects, i.e. reduced growth rate and increased tube width, became obvious ( Figure 1C). Tube width and growth rate were not more affected at higher concentrations (i.e., 3 µM), suggesting that there is a threshold for inhibitory effects of BFA. Threshold concentrations were also identified for LatB and Jas. Treatment with LatB and Jas both resulted in tube bulging and growth arrest at high concentrations ( Figure 1A-B). LatB at 1 to 2 nM and Jas at 0.1 to 0.2 μ M showed a steeply increasing effect on pollen tube width ( Figure 1A-B). The increased tube width upon application of BFA, LatB and Jas suggested that these drugs compromised tube polarity. In contrast, the most prominent phenotypic effect upon Wortmannin treatment was increased vacuolation.
After treatment with 0.

Fluorescent Protein Markers
To study the dynamics of endomembrane trafficking and actin polymerization in Arabidopsis pollen tubes, we generated constructs expressing Yellow Fluorescent Protein (YFP)-fused markers. Arabidopsis RabA4b was used as the marker for transport vesicles.
RabA4b is a Rab GTPase specifically localized at an inverted cone-shaped area in the apical region of both tobacco pollen tubes (Lee et al., 2008) and Arabidopsis root hairs (Preuss et al., 2004). Two other Rab GTPases, ARA6 and ARA7, were used as markers for endosomes, although neither has been used before in pollen tubes. Although it is unequivocal that both ARA6 and ARA7 label endosomes, different groups differed as to the type of endosomes they label, either early endosomes, later endosomes or prevacuolar compartments (Ueda et al., 2001;Tse et al., 2004;Ueda et al., 2004).
We used YFP-fused mouse Talin as the marker for the actin cytoskeleton. Although studies have shown that mTalin bundles actin and therefore affects organization of the actin cytoskeleton (Ketelaar et al., 2004;Wilsen et al., 2006), it has been used in pollen tubes to demonstrate the dynamics of F-actin at the apical clear zone (Kost et al., 1999;Fu et al., 2001). We found that strong expression of mTalin indeed bundled actin microfilaments and caused tubes to bulge (data not shown). However, moderate expression of YFP-mTalin did not cause growth inhibition or changes of tube morphology ( Figure 2).
We first tested these fluorescent markers in tobacco pollen using particle bombardment.
As expected, YFP-RabA4b formed an inverted cone shape in the apical cytoplasm and a longitudinal tail in the center of pollen tubes (Supplemental Figure 1A). This localization resembled that of tobacco Rab11 (de Graaf et al., 2005) and thus indicates where transport vesicles are. ARA6 and ARA7 both labeled punctate, highly motile vesicular structures (Supplemental Figure 1B, C) characteristic of endosomes (Cheung and Wu, 2007). However, ARA7-labeled endosomes were present not only in the tube shank but also in the apical clear zone (Supplemental Figure 1C), whereas ARA6-labeled endosomes were excluded from the apical clear zone (Supplemental Figure 1B). Such distinct localizations suggested that ARA6 and ARA7 label different populations of endosomes in pollen tubes. Recently two distinct endocytic pathways were proposed to occur in pollen tubes, one at the very apex and the other at the subapical region (Zonia and Munnik, 2009). The distinct distribution of ARA6-and ARA7-labeled endosomes may reflect such a spatial separation of endocytic pathways. YFP-mTalin (Supplemental Figure 1D) labeled longitudinal or slightly helical cables along the tube shank as well as an F-actin collar/ring at the junction between the apical and subapical region, as previously described (Kost et al., 1999;Fu et al., 2001).
Having established the validity of these fluorescent markers, we then generated stable transgenic Arabidopsis plants in the quartet1 background. For each construct we obtained more than 50 individual T1 lines and selected 10 lines having single T-DNA insertions, based on the criterion that 2 of the 4 grains in a given quartet showed fluorescence.
Homozygous T3 plants were used for subsequent analyses.
The subcellular localization of RabA4b ( Figure 2A) and mTalin ( Figure 2D) in Arabidopsis pollen tubes was the same as in tobacco pollen tubes. The distinction between ARA6-and ARA7-labeled endosomes regarding whether they were present in the clear zone was less obvious but still distinguishable ( Figure 2B, C). In addition to endosome localization, ARA6 was detected at the plasma membrane of pollen tubes and on the invaginated vegetative cell plasma membrane surrounding the sperm cells in transgenic lines that had high expression levels (data not shown). This plasma membrane localization of ARA6 may result from its reported N-myristoylation and palmitoylation (Ueda et al., 2001).
To verify that the expression of these fusion proteins did not perturb tube growth and morphology, we measured pollen tube width and growth rates during in vitro pollen germination assays. There was no significant difference between pollen tubes expressing these fusion proteins and wild-type pollen tubes, either in tube width or in growth rate (Supplemental Figure 2), indicating that the expression of these fusion proteins did not disturb normal cellular activities in pollen tubes.

Treatment
In control pollen tubes, transport vesicles labeled by RabA4b accumulated in an inverted cone-shaped region in the apical cytoplasm ( Figure 2A). The axis of such inverted cones changed to a new growth direction during tube re-orientation ( Figure 3A, Supplemental Movie 1), as did that of NtRab11 (de Graaf et al., 2005), suggesting that the directional movement of transport vesicles is part of the re-orientation process during tube growth.
Often, longitudinal RabA4b-labeling could be seen in the center of pollen tubes ( Figure   3A), which may indicate the path of cytoplasmic streaming.
After 5 min incubation with BFA, pollen tubes continued growth and RabA4b localization resembled the pattern in control tubes (data not shown). At around 20 min, tube growth slowed down, and this was accompanied by increased fluorescence intensity in the apical cytoplasm ( Figure 3B). BFA inhibits exocytosis but may increase endocytosis (Wang et al., 2005), which explains the increased signal intensity after a short incubation with BFA. The fluorescence intensity increase appeared to oscillate, although pollen tubes had almost stopped growth ( Figure 3B, Supplemental Movie 2).
The YFP accumulation zone was shifted from the very apical region to the sub-apical area ( Figure 3B) and the YFP label formed aggregates resembling the so-called BFAinduced aggregates (BIA) that were previously described in tobacco pollen tubes (Parton et al., 2001;Parton et al., 2003). Prolonged BFA treatment (60-90 min) caused apical bulges and growth arrest, with vacuoles invading the apical region and an absence of the typical V-shaped RabA4b labeling ( Figure 3D). The fluorescence was evenly distributed in the cytoplasm ( Figure 3D), indicating that directional vesicle transport had stopped.
Wortmannin did not affect the V-shaped localization of RabA4b within the first 45 min of treatment (data not shown), but after 60 min the RabA4b labeling shifted backwards to the junction between the apical and sub-apical region ( Figure 3C), and this was correlated with slowed growth. RabA4b labeling was in distinct motile patches in the apical and sub-apical area ( Figure 3C, Supplemental Movie 3). Unlike treatment with BFA, Wortmannin-treated tubes did not show an oscillating fluorescence pattern ( Figure   3C). Further incubation resulted in vacuolation at the apical region and caused redistribution of the RabA4b signal to the cytoplasm ( Figure 3E).
To confirm these results, we also applied FM4-64, a lipophilic dye that labels vesicles via endocytosis, to RabA4b-labeled tubes treated with either BFA (Supplemental Figure   3) or Wortmannin (Supplemental Figure 4). BFA treatment increased FM4-64 uptake into pollen tubes. Treatment with BFA disrupted the distribution pattern of both RabA4blabeled and FM4-64-labeled transport vesicles and formed BFA-induced aggregates (Supplemental Figure 3). In contrast, although Wortmannin also disrupted the distribution pattern of transport vesicles labeled by both RabA4b and FM4-64, pre-treatment with Wortmannin inhibited FM4-64 uptake (Supplemental Figure 4). The RabA4b-signal overlapped with the FM4-64 signal at the apical region but not at the pollen tube shank (Supplemental Figure 4).
ARA7-labeled endosomes were fast-moving punctuate vesicles detected throughout pollen tubes and including the apical clear zone ( Figure 2C). A 30 min incubation with BFA caused aggregation of ARA7-labeled endosomes ( Figure 4A), which were distinct from the ring-shaped compartments that ARA7-labeled endosomes formed upon BFA treatment or in protoplasts prepared from the Arabidopsis gnom mutant (Geldner et al., 2003). This may indicate a cell-specific endosomal response to BFA. ARA7-labeling dissipated into the cytoplasm after prolonged (60-90 min) BFA incubation ( Figure 4A), when growth was arrested.
Incubation with Wortmannin did not affect the localization pattern and motility of ARA7-labeled endosomes within the first 30 min (data not shown), consistent with the normal tube morphology and growth rate. However, ARA7-labeled endosomes started to form large aggregates after 45 min incubation with Wortmannin (Supplemental Movie 4); 90 min incubation resulted in vacuolation at the apex and growth arrest, when a few large aggregates were labeled by ARA7 ( Figure 4B).
Surprisingly, ARA6-labeled endosomes showed responses distinct from those of ARA7-labeled endosomes, with both BFA and Wortmannin treatments. The ARA6 signal was seen at punctate motile vesicles in the cytoplasm ( Figure 2B, Supplemental Movie 5).
BFA treatment depleted ARA6 from the apical plasma membrane (Figure 4C), likely a result of inhibited exocytosis, which is a well-documented effect of BFA treatment (Nebenfuhr et al., 2002;Geldner, 2004). However, ARA6-labeled endosomes did not aggregate, even after prolonged BFA treatment ( Figure 4C). ARA6-labeled endosomes penetrated the apical clear zone upon BFA treatment ( Figure 4C, Supplemental Movie 5) where they were usually excluded, indicating disrupted cellular structures due to BFA treatment. ARA6-labeled endosomes were less numerous after prolonged BFA treatment ( Figure 4C). Previous studies in Arabidopsis protoplasts were contradictory regarding the sensitivity of ARA6-labeled endosomes to BFA (Grebe et al., 2003;Ueda et al., 2004).
That ARA6-labeled endosomes were BFA-insensitive was previously indicated, in that they were not affected in the gnom mutant, in which a BFA-sensitive ArfGEF is defective (Geldner et al., 2003). Our data showed that in pollen tubes ARA6-labeled endosomes are BFA-insensitive.
Wortmannin treatment also did not affect the morphology or motility of ARA6labeled endosomes ( Figure 4D). Only when pollen tubes were incubated with Wortmannin for more than 60 min did ARA6-labeled endosomes start to aggregate ( Figure 4D), although to a much lesser extent than for ARA7-labeled aggregates ( Figure   4B). In tubes treated with Wortmannin for more than 80 min, when tubes were highly vacuolated, ARA6 occasionally accumulated at ring-shaped compartments ( Figure 4D).
That ARA7-and ARA6-labeled endosomes showed different responses to BFA and Wortmannin suggests that they are distinct subpopulations of endosomes.
Because both BFA and Wortmannin treatments disrupted the FM4-64 distribution pattern, we labeled ARA7-or ARA6-expressing tubes with FM4-64 to verify the treatments with these two drugs. Indeed, disturbance of the FM4-64-labeling pattern upon BFA or Wortmannin treatments correlated with the aggregation of ARA7-labeled endosomes but not of ARA6-labeled endosomes (Supplemental Figures 5 and 6).
Furthermore, we generated transgenic plants co-expressing ARA6-CFP and YFP-ARA7 and treated these transgenic pollen tubes with either BFA or Wortmannin. As expected,

ARA7-and ARA6-labeled endosomes showed different responses to BFA and
Wortmannin (Supplemental Figure 7), strongly supporting the heterogeneity of endosomes in pollen tubes.

The Effects of LatB and Jas Treatments on Transport Vesicles and Endosomes in Arabidopsis Pollen Tubes
The effects of LatB treatment on the dynamics of the actin cytoskeleton have been extensively studied with pollen tubes of several plant species (Fu et al., 2001;Chen et al., 2002;Chen et al., 2007). We obtained similar results with Arabidopsis pollen tubes. A 30 min incubation with LatB disrupted the apical F-actin ring structure (Supplemental Figure 8A), and after 60 min ectopic actin cables penetrated the apical region (Supplemental Figure 8B). Application of Jas disrupted actin cables along the tube shank (Supplemental Figure 8C) within 10 min, and resulted in enrichment of short thick actin patches beneath the plasma membrane (Supplemental Figure 8D), as reported for lily pollen tubes (Cardenas et al., 2005).
To find out how the dynamics of the actin cytoskeleton affected the dynamics of transport vesicles in Arabidopsis pollen tubes, we applied LatB or Jas to tubes expressing

RabA4b-YFP. LatB treatment caused rapid relocation of the inverted cone labeled by
RabA4b backward from the very apex, forming a patch at the subapical region ( Figure   5A). Further incubation with LatB caused dissipation of the RabA4b signal, resulting in an even cytoplasmic distribution and growth arrest (Supplemental Movie 6). These results are consistent with the dissipation of the RabA4b signal to the cytoplasm and with the cessation of root hair growth seen upon treatment with 200 nM LatB (Preuss et al., 2004). The RabA4b signal was fragmented upon treatment with Jas ( Figure 5B). Because Jas treatment caused fragmentation of actin cables at the shank and aggregation of actin microfilaments at the apical region (Supplemental Figure 8C, D), the fragmented RabA4b signal upon Jas treatment indicates that transport vesicles in pollen tubes require an intact actin cytoskeleton for directional movement. Further incubation with Jas re-distributed the RabA4b signal evenly in the cytoplasm (data not shown). These results support the idea that a dynamic actin cytoskeleton is critical for the trafficking of transport vesicles.
Endosomes labeled by ARA6 or ARA7 showed distinct responses to LatB and Jas.

ARA7-labeled endosomes started to form large aggregates after 10 min incubation with
LatB (data not shown), which were more obvious after 30 min incubation ( Figure 6A).
However, such aggregates were few, reduced in motility and persisted in tubes that had been treated with LatB for 60 min, when growth was already arrested ( Figure 6A). Jas treatment had similar aggregation effects on ARA7-labeled endosomes ( Figure 6B).
However, prolonged (60 min) Jas treatment caused dissipation of the ARA7 signal from these aggregates to an even cytoplasmic distribution ( Figure 6B). In contrast, ARA6labeled endosomes were insensitive to both Jas and LatB treatments and no obvious aggregation was seen ( Figure 6C, D), although they penetrated to the apical region ( Figure 6C, D) where they were excluded in control tubes ( Figure 2B). These results indicated that a dynamic actin cytoskeleton is critical for certain steps during endocytic routes that affect a subpopulation of endosomes.

Cytoskeleton
In contrast to the regulation of endomembrane trafficking by the actin cytoskeleton, few studies have been done to explore whether and how endomembrane trafficking affects actin dynamics during tube growth (de Graaf et al., 2005;Hörmanseder et al., 2005).
Expression of either a dominant negative or constitutively active version of NtRab11b abolished the apical actin ring structure without disrupting actin cables in the shank (de Graaf et al., 2005), suggesting that transport vesicles influence actin dynamics. In addition, BFA treatment of lily pollen tubes caused association of a so-called "actin basket" with BIA, on which organelles move between the plasma membrane and BIA (Hörmanseder et al., 2005).
To find out how perturbing endomembrane trafficking affected the actin cytoskeleton, we applied either BFA or Wortmannin to tubes expressing mTalin. Because we established the effects of BFA or Wortmannin on endomembrane trafficking, the dynamics of the actin cytoskeleton after these drug treatments should reflect its response to perturbed endomembrane trafficking. Incubation with BFA for 5 min did not change the actin cables in the shank or the F-actin ring at the apex (data not shown). After 20 min, when BFA treatment affected tube growth as expected (i.e. an increased apical dome and a disrupted clear zone was seen), the apical F-actin ring disappeared while actin cables at the tube shank were still intact ( Figure 7A). Tangled actin cables penetrated to the apical region and surrounded small vacuoles in the apex ( Figure 7A). However, these actin cable structures disappeared after prolonged (60 min) incubation with BFA, when a large vacuole occupied the apex and growth was arrested ( Figure 7A).
The effect of Wortmannin on the actin cytoskeleton was slower, in that 45 min incubation did not change either the F-actin ring at the apex or the actin cables in the shank ( Figure 7B). After 60 min, however, the actin ring disappeared ( Figure 7B).
Instead, a strong fluorescent labeling of tangled actin cable structures was detected below the apex ( Figure 7B), similar to the previously reported BIA-associated actin structures (Hörmanseder et al., 2005). Prolonged (90 min) treatment with Wortmannin caused tube vacuolation at the apex and the actin cables penetrated the apex, surrounded the vacuole and were also tangled beneath the apical plasma membrane ( Figure 7B). Both drugs caused disappearance of the F-actin ring at the apex, suggesting that this F-actin ring is sensitive to perturbations of endomembrane trafficking.

Interdependency of Actin Dynamics and Endomembrane Trafficking in Pollen
Tubes A dynamic actin cytoskeleton and regulated endomembrane trafficking are two critical cellular activities leading to polarized growth of pollen tubes . We show here using pharmacological drugs that these two cellular activities are interdependent in Arabidopsis pollen tubes.
A dynamic actin cytoskeleton at the apex, as reflected by the presence of the F-actin ring, was critical for the directional movement of transport vesicles to the tip, based on the effects of LatB and Jas on spatiotemporal RabA4b distribution ( Figure 5). A dynamic actin cytoskeleton is essential for endomembrane trafficking involving ARA7-labeled endosomes but not ARA6-labeled endosomes, in that ARA7-but not ARA6-labeled endosomes quickly became immotile and aggregated in the presence of LatB or Jas ( Figure 6). ARA7 most likely labels an early endosome population (Ueda et al., 2004) and/or vesicles immediately exiting the trans-Golgi network (TGN), which were also considered early endosomes (Samaj et al., 2006). Indeed, it was reported that early endosomes, but not late endosomes or prevacuolar compartments (PVCs), are sensitive to BFA (Tse et al., 2004;Ueda et al., 2004). On the other hand, disturbing endomembrane trafficking by BFA or Wortmannin disrupted the apical F-actin ring at the apex without affecting actin cables in the shank.
Such an effect was delayed and progressive and therefore might have resulted from an altered membrane presence of signaling proteins. Plausible candidates for such signaling proteins are ROP GTPases. ROP GTPases regulate dynamic actin polymerization in pollen tubes (Fu et al., 2001), and both in pollen tubes and in sporophytic cells they also regulate membrane trafficking through actin dynamics and/or their effectors (Lavy et al., 2007;Lee et al., 2008). ROP-GTP is spatiotemporally restricted to the apical plasma membrane  by the coordinated function of a receptor kinase (PRK2a) and a ROP guanine nucleotide exchange factor (ROPGEF) (Zhang and McCormick, 2007), suggesting that ROP-mediated signaling is a convergence point for actin dynamics and endomembrane trafficking in pollen tubes.

Generation of Stable Transgenic Protein Fusion Marker Lines
The expression construct for mTalin (Zhang et al., 2009)  and ARA7 were generated by TOPO ligation (Invitrogen) and verified by sequencing.
The YFP and CFP fusion expression constructs were generated by LR reactions with previously described pollen-specific Gateway (Invitrogen) destination vectors (Zhang and McCormick, 2007). The Arabidopsis quartet1-2 (qrt1) mutant in the Col-0 ecotype was used as wild type for stable transformation by the floral dipping method (Clough and Bent, 1998

Transient Expression in Tobacco Pollen Tubes by Particle Bombardment
Transient expression assays in tobacco pollen were conducted as described (Zhang and McCormick, 2007). Images were captured from 4-8 hrs after germination.

Arabidopsis in vitro Pollen Germination
Arabidopsis in vitro pollen germination was conducted at 22.5°C, as described (Boavida and McCormick, 2007

Microscopy
An inverted Axiophot microscope (Zeiss) with either bright field or epifluorescence optics was used. Images were captured using a Spot digital camera (Diagnostic Instruments), exported using AxioVision (Zeiss), and processed using Adobe Photoshop 7.0 (Adobe). All movies were generated using the 6D acquisition option, with 30 images taken at 30 sec intervals.

ACKNOWLEDGMENTS
We thank Leonor Boavida for helpful discussions, and Weihua Tang and Leonor Boavida for comments on the manuscript. We are grateful to UC Berkeley undergraduates Sarah Liu, James Pao and Carolyn Jin for excellent technical assistance. We thank Shaul Yalovsky for a YFP-mTalin construct.