Journal: The Journal of Cell Biology
Article Title: Rho-GTPase-dependent filamentous actin dynamics coordinate vesicle targeting and exocytosis during tip growth
Figure Lengend Snippet: The accumulation of YFP-RabA4d vesicles to the tip is associated with the apical F-actin dynamics. (A) F-actin is associated with YFP-RabA4d accumulation to the tip. YFP-RabA4d was transiently expressed alone (a) or coexpressed with RIC3 (b) in tobacco pollen tubes. For chemical treatment, control pollen tubes (YFP-RabA4d alone) or RIC3-overexpressing pollen tubes were treated with 5 nM LatB (c) or 100 μM LaCl 3 (d), respectively, for 1 h. Tip accumulation of YFP-RabA4d was completely disrupted by RIC3 overexpression (b) or LatB treatment (c). LaCl 3 treatment nearly restored normal accumulation of YFP-RabA4d in RIC3-overexpressing tubes (d). All images show a midplane section. (B) Accumulation of tip F-actin causes depolarized localization of YFP-RabA4d to the tip. YFP-RabA4d was transiently expressed alone (b and c) or coexpressed with the indicated RICs (RIC4 [a] and RIC3 + RIC4 [e]). 3 h after bombardment, control pollen tubes (YFP-RabA4d) were treated with 50 μM LaCl 3 for 1 h (b) or 100 nM jasplakinolide for 10 min (c) before imaging. RIC4 OX (a), LaCl 3 (b), or jasplakinolide treatment (c) induced increased accumulation of YFP-RabA4d near the cortex over an expanded region of the apex. Treatment with LatB (d) or coexpression with RIC3 (e) restored pollen tube growth and normal vesicle accumulation to the tip in RIC4-overxpressing tubes. Arrows indicate the depolarized localization of YFP-RabA4d. All images were taken from the midplane. Bars, 10 μm. (C) Quantitative comparison of YFP-RabA4d accumulation. Pixel values were measured along a central transect through the fluorescence image in wild-type, RIC3 OX, and RIC4 OX pollen tubes. Similar results were obtained from ∼20 individual pollen tubes.
Article Snippet: To determine the effect of jasplakinolide on exocytosis, a stock solution of 100 μM jasplakinolide (Invitrogen) was added to germinated pollen to a final concentration of 100 nM.
Techniques: Over Expression, Imaging, Fluorescence