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

Abstract

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