Interdependence of endomembrane trafficking and actin dynamics during polarized growth of Arabidopsis pollen tubes

Abstract

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 (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 and the actin stabilization drug jasplakinolide 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.

Figure 1.

Optimization of pharmacological treatments for Arabidopsis pollen tubes. Pollen tube width was measured, or vacuoles were noted. A representative image for each concentration is shown above the graph. A, LatB treatment. B, Jas treatment. C, BFA treatment. D, Wortmannin treatment. Arrows indicate vacuoles. Scale bars = 50 μm.

Figure 2.

Fluorescent probes for transport vesicles, endosomes, and the actin cytoskeleton expressed in transgenic Arabidopsis pollen tubes. A, A pollen tube expressing YFP-RabA4b. B, A pollen tube expressing ARA6-YFP. C, A pollen tube expressing YFP-ARA7. D, A pollen tube expressing YFP-mTalin. Arrows in B and C indicate the apical clear zone, in which ARA6-labeled endosomes are excluded while ARA7-labeled endosomes are present. Asterisk in D marks the apical F-actin ring. Scale bar = 20 μm. [See online article for color version of this figure.]

Figure 3.

Time-lapse images of transgenic pollen tubes expressing YFP-RabA4b. A, Control. B, A pollen tube treated with BFA. Arrow indicates the RabA4b labeling below apex after BFA treatment, which resembles so-called BIA. Serial images were taken starting after 20 min incubation with BFA. Therefore time point 0 s is after 20 min treatment with BFA. C, A pollen tube treated with wortmannin. Serial images were taken starting after 60 min incubation with wortmannin. Therefore time point 0 s is after 60 min treatment with wortmannin. D, A representative pollen tube treated with BFA for 60 min. E, A representative pollen tube treated with wortmannin for 90 min. For D and E, left section, transmitted light; right section, YFP channel. Scale bars = 20 μm. [See online article for color version of this figure.]

Figure 4.

Endosomes respond differently to BFA and wortmannin. A, Pollen tubes expressing YFP-ARA7 treated with BFA. B, Pollen tubes expressing YFP-ARA7 treated with wortmannin. C, Pollen tubes expressing YFP-ARA6 treated with BFA. D, Pollen tubes expressing YFP-ARA6 treated with wortmannin. The images shown are representative, chosen from more than 30 pollen tubes at each time point (30, 60, and 90 min). Scale bars = 20 μm. [See online article for color version of this figure.]

Figure 5.

Time-lapse images of transgenic pollen tubes expressing YFP-RabA4b. A, A pollen tube treated with LatB for 15 min. Arrow indicates RabA4b labeling below apex, which resembles the so-called BIA. B, A pollen tube treated with Jas for 15 min. Asterisks indicate vacuoles. Scale bar = 20 μm. Serial images were taken starting after 15 min incubation with LatB (A) or Jas (B). Therefore time point 0 s is after 15 min treatment with the drugs. [See online article for color version of this figure.]

Figure 6.

Dynamic actin polymerization regulates ARA7- but not ARA6-labeled endosomes. A and B, Pollen tubes expressing YFP-ARA7. C and D, Pollen tubes expressing YFP-ARA6. A and C, LatB treatment for 30 min (top section) or 60 min (bottom section). B and D, Jas treatment for 30 min (top section) and 60 min (bottom section). The images shown are representative, chosen from more than 30 pollen tubes at each time point (30 and 60 min). Scale bar = 20 μm. [See online article for color version of this figure.]

Figure 7.

Apical F-actin, but not actin cables in the shank, is sensitive to disturbances of endomembrane trafficking. A, BFA treatment. B, Wortmannin treatment. The images shown are representative, chosen from more than 30 pollen tubes at each time point (for BFA: 20, 45, and 60 min; for wortmannin: 45, 60, and 90 min). Arrows indicate where the F-actin ring would be in control tubes. Scale bar = 20 μm. [See online article for color version of this figure.]