Microtubules affect endocytosis and membrane trafficking in the apical region in Nicotiana tabacum pollen tube

The pollen tube is a highly polarized cell that plays a fundamental role in the fertilization process of higher plants. Pollen tube growth depends on transport and accumulation of secretory vesicles (SVs) in the apical region where they fuse in a small area of the plasma membrane (PM) supplying material for the construction of new cell wall and providing, at the same time, new surface of PM. The incorporation of new PM area largely exceeds the quantity of membrane surface required for tube elongation, so the excess of PM must be removed through an active internalization process, known as endocytosis (Steer and Steer 1989). In vivo time-lapse experiments using FM4-64 combined with quantitative analysis allowed the observation that different endocytic pathways occurs in distinct zones of the tube, and that endocytic vesicles might substantially contribute to the V-shaped vesicle accumulation (Moscatelli et al. 2007; Zonia and Munnick 2008;). Pollen tube growth depends on the integrity of the cytoskeletal apparatus; is was shown that the actin cytoskeleton regulates cytoplasmic streaming and secretion and influence endocytosis in the shank of the tube (Moscatelli et al. 2012). However, the identification of kinesin-related polypeptides (Cai et al. 1993; Romagnoli et al. 2003) led to hypothesize that MTs could also have a role in membrane trafficking. In this study we disturb the system of MTs using 5M Nocodazole, to verify its effect on endocytosis and endosome transport. Time lapse experiments by using FM4-64 demonstrated that Nocodazole affects the PM internalization in the tip. Moreover, the use of FM4-64 in pollen tubes that transiently express GFP-RabA4b under the control of Lat52 pollen specific promoter, allowed us to show that MTs are involved in membrane sorting in the apex and/or in the progression of endocytic vesicles from the tip regions to vacuoles. Endocytosis dissection by using charged nanogold confirmed that Nocodazole has an effect in the internalization pathway occurring in the tip. In vitro binding assays showing that MTs are able to bind Syp-21 positive compartments confirmed a role of MTs in degradative pathways. In addition, FRAP experiments of the apical PM suggested that MTs are involved in maintaining the differential pattern of exocytosis, in different domains of the apical PM.