Clathrin-dependent and independent endocytic pathways in tobacco protoplasts revealed by labelling with charged nanogold

Abstract

Positively charged nanogold was used as a probe to trace the internalization of plasma membrane (PM) domains carrying negatively charged residues at an ultrastructural level. The probe revealed distinct endocytic pathways within tobacco protoplasts and allowed the morphology of the organelles involved in endocytosis to be characterized in great detail. Putative early endosomes with a tubulo-vesicular structure, similar to that observed in animal cells, are described and a new compartment, characterized by interconnected vesicles, was identified as a late endosome using the Arabidopsis anti-syntaxin family Syp-21 antibody. Endocytosis dissection using Brefeldin A (BFA), pulse chase, temperature- and energy-dependent experiments combined with quantitative analysis of nanogold particles in different compartments, suggested that recycling to the PM predominated with respect to degradation. Further experiments using ikarugamycin (IKA), an inhibitor of clathrin-dependent endocytosis, and negatively charged nanogold confirmed that distinct endocytic pathways coexist in tobacco protoplasts.

Salt-Induced Remodeling of Spatially Restricted Clathrin-Independent Endocytic Pathways in Arabidopsis Root

“…Several reports of clathrin-independent endocytic pathways taking up fluorescent nanobeads, glucose analogs, or gold nanoparticles in plant cells have appeared (Onelli et al, 2008;Bandmann et al, 2012). However, no detailed mechanistic insight into such pathways has been presented.…”

“…All transmembrane proteins studied so far have been shown to follow the clathrin-dependent uptake pathway, which operates in all cell layers of the Arabidopsis root (Robert et al, 2010;Kitakura et al, 2011), but information on other pathways is scarce. Clathrin-independent pathways have been implicated in studies of tobacco (Nicotiana tabacum) cells grown in suspension culture (Onelli et al, 2008) and in epidermal cells of Arabidopsis root (X. Li et al, 2011;R.…”

In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

“…Delivery of such probes through an intact plasma membrane in live cells, however, has not (yet) been shown to be feasible. Nanogold particles have nevertheless been shown to cross cellular membranes (44,45). Therefore, future development of a BG-nanogold probe that can reversibly cross membranes could considerably increase the applications of the SNAP labeling technique for cryo-ET studies of intact cells.…”

Interactions between engineered nanomaterials and agricultural crops: implications for food safety

“…It has been proposed that cell internalization of ENMs could occur by binding to carrier proteins, through aquaporins, ion channels, endocytosis, or by creating new pores (carbon nanotubes) (Rico et al, 2011). While experimental data are scarce and many proposed mechanisms are under intense debate, endocytosis has been demonstrated definitively through the use of temperature control and the addition of endocytosisinhibiting agents such as wortmannin (Onelli et al, 2008;Liu et al, 2009;Iversen et al, 2012;Miralles et al, 2012b). Enhanced expression of aquaporin proteins and up-regulation of water channel genes were found to support possible passive uptake mechanisms (Khodakovskaya et al, 2012).…”