FRAP-based analysis of Rho GTPase-dependent polar exocytosis in pollen tubes

Abstract

The regulation of exocytosis by Rho GTPases is conserved in eukaryotic kingdoms. Given the spatiotemporal dynamics of Rho GTPase activity, a method for visualizing exocytosis with high space and time resolution will be an important tool for the functional analysis of Rho GTPases. During tip growth of pollen tubes, both cell wall material and plasma membrane are renewed via rapid exocytosis. ROP1 GTPase, a plant Rho GTPase, is localized to the apical plasma membrane of pollen tubes and controls polar exocytosis via regulation of F-actin dynamics. Here, we describe a fluorescence recovery after photobleaching (FRAP) method for the analysis of exocytosis dynamics in living pollen tubes. This method has been shown to be a valuable tool for studying the effect of ROP1-dependent F-actin dynamics on polar exocytosis in pollen tubes and may also be applicable to other systems.