Endocytic pathways and membrane recycling involve the actin filaments in tobacco pollen tubes

The pollen tube is a tip growing cell that plays a fundamental role in the fertilization process of higher plants. Pollen tube growth is based on transport and accumulation of secretory vesicles. The incorporation of new segments of PM largely exceeds the quantity of membrane required for the extension, so the excess of PM must be removed by endocytosis. Recent studies showed internalization of subapical PM domains that were mainly recycled to exocytosis through the Golgi apparatus and a second mainly degradative pathway involving PM retrieval at the tip. The movements of the endomembrane system during the pollen tube growth depends on the concerted action and integrity of the cytoskeleton. In this study we disturb the system of cortical actin filaments using Latrunculin B to verify modifications in endocytosis, using specific FM dyes in time-lapse experiments and by ultrastructural experiments using charged Nanogold. Our results demonstrate that the system of cortical microfilaments determines a strong reduction of endocytosis in the subapical regions and consequently the alteration of recycling through the Golgi and the inhibition of the degradative pathways

Microtubule depolymerization affects endocytosis and exocytosis in the tip and influences endosome movement in tobacco pollen tubes

Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments (AFs) and microtubules (MTs). AFs convey post-Golgi secretory vesicles to the tip where some fuse with specific domains of the plasma membrane (PM). Secretory activity is balanced by PM retrieval that maintains cell membrane economy and regulates the polarized composition of the PM, by dividing lipids/proteins between the shank and the tip. Although AFs play a key role in PM internalization in the shank, the role of MTs in exoendocytosis needs to be characterized. The present results show that integrity of the MT cytoskeleton is necessary to control exoendocytosis events in the tip. MT polymerization plays a role in promoting PM invagination in the apex of tobacco pollen tubes since Nocodazole affected PM internalization in the tip and subsequent migration of endocytic vesicles from the apex for degradation. MT depolymerization in the apex and shank was associated with misallocation of a significantly greater amount of internalized PM to the Golgi apparatus and its early recycling to the secretory pathway. FRAP experiments also showed that MT depolymerization in the tip region influenced the rate of exocytosis in the central domain of the apical PM

Distinct endocytic pathways identified in tobacco pollen tubes using charged nanogold

In an attempt to dissect endocytosis in Nicotiana tabacum L. pollen tubes, two different probes \u2013 positively or negatively charged nanogold \u2013 were employed. The destiny of internalized plasma membrane domains, carrying negatively or positively charged residues, was followed at the ultrastructural level and revealed distinct endocytic pathways. Time-course experiments and electron microscopy showed internalization of subapical plasmamembrane domains that were mainly recycled to the secretory pathway through the Golgi apparatus and a second mainly degradative pathway involving plasma membrane retrieval at the tip. In vivo time-lapse experiments using FM4-64 combined with quantitative analysis confirmed the existence of distinct internalization regions. Ikarugamycin, an inhibitor of clathrin-dependent endocytosis, allowed us to further dissect the endocytic process: electron microscopy and time-lapse studies suggested that clathrin-dependent endocytosis occurs in the tip and subapical regions, because recycling of positively charged nanogold to the Golgi bodies and the consignment of negatively charged nanogold to vacuoles were affected. However, intact positively charged-nanogold transport to vacuoles supports the idea that an endocytic pathway that does not require clathrin is also present in pollen tubes

Pollen tube growth: a delicate equilibrium between secretory and endocytic pathways

Although pollen tube growth is a prerequisite for higher plant fertilization and seed production, the processes leading to pollen tube emission and elongation are crucial for understanding the basic mechanisms of tip growth. It was generally accepted that pollen tube elongation occurs by accumulation and fusion of Golgi-derived secretory vesicles (SVs) in the apical region, or clear zone, where they were thought to fuse with a restricted area of the apical plasma membrane (PM), defining the apical growth domain. Fusion of SVs at the tip reverses outside cell wall material and provides new segments of PM. However, electron microscopy studies have clearly shown that the PM incorporated at the tip greatly exceeds elongation and a mechanism of PM retrieval was already postulated in the mid-nineteenth century. Recent studies on endocytosis during pollen tube growth showed that different endocytic pathways occurred in distinct zones of the tube, including the apex, and led to a new hypothesis to explain vesicle accumulation at the tip; namely, that endocytic vesicles contribute substantially to V-shaped vesicle accumulation in addition to SVs and that exocytosis does not involve the entire apical domain. New insights suggested the intriguing hypothesis that modulation between exo- and endocytosis in the apex contributes to maintain PM polarity in terms of lipid/protein composition and showed distinct degradation pathways that could have different functions in the physiology of the cell. Pollen tube growth in vivo is closely regulated by interaction with style molecules. The study of endocytosis and membrane recycling in pollen tubes opens new perspectives to studying pollen tube-style interactions in vivo

Inhibition of actin polymerization by LatB affects endocytosis and secretion both in the apex and in the subapical regions of tobacco pollen tubes

The pollen tube is a tip growing cell that plays a fundamental role in the fertilization process of higher plants. Pollen tube growth is based on transport and accumulation of secretory vesicles. The incorporation of new segments of PM largely exceeds the quantity of membrane required for the extension, so the excess of PM must be removed by endocytosis. Recent studies showed internalization of subapical PM domains that were mainly recycled to exocytosis through the Golgi apparatus and a second mainly degradative pathway. The movements of the endomembrane system during the pollen tube growth depends on the concerted action and integrity of the cytoskeleton. In this study we disturb the system of cortical actin filaments using Latrunculin B to verify modifications in endocytosis, using specific FM dyes in time-lapse experiments and by ultrastructural experiments using charged Nanogold. Our results demonstrate that the system of cortical microfilaments determines a strong reduction of endocytosis and consequently the alteration of recycling through the Golgi, the inhibition of degradative pathways, and changes in the secretion pattern at the tip PM

Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes

Pollen tube growth depends on the integrity of the actin cytoskeleton that regulates cytoplasmic streaming and secretion. To clarify whether actin also plays a role in pollen tube endocytosis, Latrunculin B (LatB) was employed in internalisation experiments with tobacco pollen tubes, using the lipophilic dye FM4-64 and charged nanogold. Time-lapse analysis and dissection of endocytosis allowed us to identify internalisation pathways with different sensitivity to LatB. Co-localisation experiments and ultrastructural observations using positively charged nanogold revealed that LatB significantly inhibited endocytosis in the pollen tube shank, affecting internalisation of the plasma membrane (PM) recycled for secretion, as well as that conveyed to vacuoles. In contrast, endocytosis of negatively charged nanogold in the tip, which is also conveyed to vacuoles, was not influenced. Experiments of fluorescence recovery after photobleaching (FRAP) of the apical and subapical PM revealed domains with different rates of fluorescence recovery and showed that these differences depend on the actin cytoskeleton integrity. These results show the presence of distinct degradation pathways by demonstrating that actin-dependent and actin-indepedent endocytosis both operate in pollen tubes, internalising tracts of PM to be recycled and broken down. Intriguingly, although most studies concentrate on exocytosis and distension in the apex, the present paper shows that uncharacterised, actin-dependent secretory activity occurs in the shank of pollen tubes

Distinct endocytotic pathways identified in tobacco pollen tubes by using charged nanogold

By endocytosis eukaryotic cells can take up extracellular components and/or plasma membrane (PM) proteins for further delivering to endosomes. In pollen tube, endocytosis was supposed to be involved in regulating the membrane economy of the cell by retrieving the excess of PM incorporated at the tip during the cell wall secretion (Parton et al. 2001). Although in animal cells different endocytic pathways were identified based on the requirement of a clathrin coating for vesicle internalization (Nichols and Lippincott-Schwartz J. 2001, Kikham et al. 2005), endocytosis in plant cells still require to be fully characterized. In the attempt to dissect endocytosis in pollen tubes, two different probes, positively and negatively charged nanogold, were employed. The destiny of internalized PM domains, carrying negatively or positively charges residues, was followed at the ultrastructural level and revealed distinct endocytic pathways. Time course experiments and electron microscopy (EM) showed a massive internalization of sub apical PM domains which were mainly recycled to the secretory pathway through the Golgi apparatus and a second pathway involving PM retrieval at the very tip, which was mainly destined to the degradative pathway. In vivo time lapse experiments, by using FM4-64 combined with quantization analysis, confirmed the presence distinct internalization regions. The use of Ikarugamycin (Ika), an inhibitor of the clathrin-dependent endocytosis allowed us to further dissect the endocytic process: EM and time lapse studies suggested that clathrin-dependent endocytosis occurs both in the tip and in the subapical regions, since both the recycling of positively charged nanogold to the Golgi bodies and the delivery of negatively charged nanogold to vacuoles were affected. However, the intactness of the positively charged nanogold transport to vacuoles supports the idea that an endocytic pathway which does not require clathrin is present in pollen tube

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\uf06dM 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

Distinct endocytic pathways identified in tobacco pollen tubes using charged nanogold

In an attempt to dissect endocytosis in Nicotiana tabacum L. pollen tubes, two different probes positively or negatively charged nanogold were employed. The destiny of internalized plasma membrane domains, carrying negatively or positively charged residues, was followed at the ultrastructural level and revealed distinct endocytic pathways. Time-course experiments and electron microscopy showed internalization of subapical plasma-membrane domains that were mainly recycled to the secretory pathway through the Golgi apparatus and a second mainly degradative pathway involving plasma membrane retrieval at the tip. In vivo time-lapse experiments using FM4-64 combined with quantitative analysis confirmed the existence of distinct internalization regions. Ikarugamycin, an inhibitor of clathrin-dependent endocytosis, allowed us to further dissect the endocytic process: electron microscopy and time-lapse studies suggested that clathrin-dependent endocytosis occurs in the tip and subapical regions, because recycling of positively charged nanogold to the Golgi bodies and the consignment of negatively charged nanogold to vacuoles were affected. However, intact positively charged-nanogold transport to vacuoles supports the idea that an endocytic pathway that does not require clathrin is also present in pollen tube