AtARO1 localization in the tube tip is BFA dependent but LatB insensitive

To further verify the participation of AtARO1 in establishment of the actin cytoskeleton, the effect of the actin nucleation inhibitor Latrunculin B (LatB) from the red sea sponge Latrunculia magnifica on AtARO1-GFP distribution was investigated. Furthermore, the accumulation of AtARO1-GFP in spot-like structures (Fig. 3.23G) suggested another role of the protein in exo- and/or endocytotic processes.

The transport of Golgi vesicles, filled with cell wall material, to sites of exocytosis was shown to be highly dependent on the actin cytoskeleton (Hepler et al., 2001). Brefeldin A (BFA), which is known to inhibit exocytosis and enhance endocytosis in pollen tubes (Wang et al., 2005), was used as another drug to analyze possible effects on AtARO1-GFP localization. Pollen were germinated in vitro for three hours and subsequently treated with LatB or BFA in liquid germination medium for two hours respectively. Control pollen tubes were incubated in liquid germination medium containing only the dissolvent dimethylsulfoxide (DMSO) or Methanol (MeOH), respectively. The control pollen tubes showed normal AtARO1-GFP distribution in the cytoplasm and vegetative nucleus of the pollen tube,

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with the tip-high accumulation described above (Fig. 3.26A, C; compare to 3.23G). Intriguingly, BFA treatment led to a loss of accumulation of AtARO1-GFP in the pollen tube tip (Fig.3.26B) and the fusion protein was evenly distributed throughout the cytoplasm. Morphological changes of the pollen, like increased tube diameter, tip swelling or accumulation of vacuoles were observed. These effects of BFA treatment were also observed elsewhere (Wang et al, 2005) and therefore not regarded to be specific for altered AtARO1-GFP distribution but a consequence of inhibited exocytosis and enhanced endocytosis. LatB, in contrast, did not abolish the tip accumulation of AtARO1-GFP (Fig. 3.26D), although it was often weaker than in the controls (Fig.3.26C). A high number of bright fluorescent spots could be detected in pollen tubes treated with BFA and to a lesser extent, in pollen tubes treated with LatB (Fig. 3.26B and D). A few bright fluorescent spots were also observed in the control experiments or in non-treated pollen tubes (arrows in Fig. 3.26C, Fig.3.23G).

Fig.3.26. Localization of AtARO1-GFP in pollen tubes treated with Brefeldin A (BFA) or Latrunculin B (LatB), respectively. Control pollen tubes were treated each with the corresponding solvent. (A) Typical phenotype of a control pollen tube incubated with MeOH for two hours (single optical section; 0.50 µm). No alterations compared to non-treated pollen tubes were detected. AtARO1-GFP accumulates in the pollen tube tip (arrowhead), the two sperm cells, free of fluorescence, are visible (arrows). (B) BFA treatment leads to reduced growth rate as well as altered pollen tube morphology. The tip-high accumulation of AtARO1-GFP disappears (arrowhead) while bright fluorescent spots can be detected throughout the pollen tube and grain (arrows, 52 0.30 µm optical sections;

15.32µm). (C) Control pollen tube treated with DMSO (36 optical sections; 17.63 µm). Growth rate, pollen tube morphology and distribution of AtARO1-GFP were comparable to non-treated pollen tubes. The tip-high accumulation of AtARO1-GFP (arrowhead) and spots of lower fluorescence intensity are visible in the cytoplasm (arrows). (D) Treatment of pollen tubes with LatB (3D stack of 59 optical sections; 17.42 µm). Pollen tube morphology and the distribution of AtARO1-GFP fusion protein did not show strong alterations after LatB treatment. However, punctiform fluorescence (arrows) in the cytoplasm is brighter, while the amount of AtARO1-GFP in the tip seems to decrease (arrowhead). Scale bars: 10 µm

In a second set of experiments, the actin cytoskeleton was fixed and stained with rhodamine-phalloidin after treatments with BFA or LatB. While the control experiments showed the same co-localization of AtARO1-GFP and actin as non-treated cells (not shown), BFA treatment revealed even more severe changes in AtARO1-GFP distribution after fixation (Fig. 3.27A-C). The accumulation in the tip vanished as in unfixed BFA-treated cells, additionally the filamentous AtARO1-GFP distribution changed into a punctiform fluorescence of varying size and shape (on average 0.5 µm diameter). These accumulations of AtARO1-GFP mostly did not seem to be associated with thick actin bundles, as can

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also be seen as an L-shaped scatterplot of red (actin) and green (AtARO1-GFP) fluorescence intensities in Fig. 3.27D. Instead, the fluorescent spots were often found to be distributed along fine actin filaments like pearls on a string (dashed arrow in Fig. 3.27A-C).

After the complete disruption of the actin cytoskeleton by LatB, labeling of remnants of F-actin by rhodamine-phalloidin was very weak (Fig. 3.27F). The disruption of actin filaments was accompanied by the disappearance of filament-like GFP signals in the shank of pollen tubes and AtARO1-GFP was dispersed through the cytoplasm (Fig. 3.27E). Again, several brighter AtARO1-AtARO1-GFP spots were detected throughout the cytoplasm, of which few co-localized with red fluorescent spots of actin.

Co-localization was still present at the pollen tube tip, where both, AtARO1-GFP and actin accumulated to a weaker extent compared to non treated cells (Fig. 3.27G). This reduced co-localization is also reflected in a broader distribution of pixels in the scatterplot (compare Fig. 3.27H with Fig. 3.24H). Therefore, AtARO1-GFP accumulation in the pollen tube tip seems not or only to a low extent depend on an intact cytoskeleton.

Fig. 3.27. localization of AtARO1-GFP and actin after treatment with BFA and LatB, respectively. (A-D) Co-localization of AtARO1-GFP and actin after BFA treatment. (A) The distribution of the fusion protein is strongly altered compared to fixed pollen tubes without BFA treatment as well as compared to unfixed but BFA treated pollen tubes. AtARO1-GFP strongly accumulates as bright fluorescent structures of approximately 0.5 µm diameter (arrows) that are dispersed throughout the cytoplasm. (B) Thick actin cables (arrowheads) and finer filaments (dashed arrow) are visible in the red channel. (C) The merged image reveals that AtARO1-GFP seems to associate with finer actin filaments (dashed arrow). However, most AtARO1-GFP accumulations do not co-localize with actin bundles (arrowheads). (D) The scatterplot reveals only little correlation between red and green pixels, which are distributed in an L-shaped fashion. (E-H) Co-localization of AtARO1-GFP and the actin cytoskeleton after treatment with LatB. (E) The filamentous fluorescence of AtARO1-GFP which is found after fixation of untreated pollen tubes is lost as actin bundles are disrupted due to LatB treatment (F). Some bright fluorescent AtARO1-GFP spots are dispersed throughout the cytoplasm (arrows) but do not coincide with spots of actin accumulations (dashed arrow). (G) AtARO1-GFP and actin still co-localize, to a certain extent, in the pollen tube tip (arrowhead). (H) The reduced co-localization of actin and AtARO1-GFP is reflected in a broader distribution of pixels in the scatterplot.

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Due to the presence of vesicle-like, fluorescent structures throughout the pollen tube shank and the accumulation of AtARO1-GFP in the vesicle-enriched tip region of the pollen tubes, a possible association of AtARO1 with transport vesicles was examined using the fluorescent membrane dye FM4-64 (Bolte et al., 2004; Ovečka et al., 2005) in combination with BFA. So called “BFA compartments” were reported to form from endosomes and Golgi derived endomembrane systems (Baluska et al., 2002) after BFA treatment and a localization of AtARO1-GFP to these compartments could point to an involvement of AtARO1 in vesicle trafficking. Before fixation, BFA treated and control pollen tubes were incubated for half an hour with the fixable derivative FM^®4-64FX to visualize endomembrane compartments. In BFA-free control experiments, AtARO1-GFP co-localized with the actin cytoskeleton and bright fluorescent spots were visible, but these did not coincide with small endocytic vesicles stained by FM4-64 (Fig. 3.28A-D). Moreover, no co-localization was detected in BFA treated pollen tubes, where BFA induced compartments became visible as enlarged FM4-64 stained membrane compartments (Fig. 3.28E-H).

Fig. 3.28 Co-localization of AtARO1-GFP, endomembrane compartments and actin. AtARO1-GFP does not co-localize with endocytotic vesicles. (A) AtARO1-GFP co-co-localizes with actin bundles (B) in the pollen tube shank (arrowheads). Punctiform fluorescence of AtARO1-GFP (arrow) does not co-localize with endocytotic vesicles labeled by FM^®4-64FX (dashed arrow, C, D). (E-H) BFA treated pollen tube. AtARO1-GFP no longer decorates thick actin bundles (arrowheads) but appears as fluorescent bodies (arrow). AtARO1-GFP does not co-localize with FM^®4-64FX labeled BFA-induced membrane compartments (dashed arrow). Scale bars: 5 µm

Recapitulatory, the co-localization experiments clearly demonstrate that AtARO1-GFP is definitely associated with the actin cytoskeleton in the shank of the pollen tube but not with endocytotic vesicles, although it shares a common spatial distribution with endo- and exocytotic vesicles.

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3.5.3 Distribution of AtARO1-GFP and actin in the female gametophyte before and after