Synaptic membrane proteins form stable microdomains in early endosomes.

In the plasma membrane, membrane proteins are frequently organized in microdomains that are stabilized both by protein-protein and protein-lipid interactions, with the membrane lipid cholesterol being instrumental for microdomain stability. However, it is unclear whether such microdomains persist during endocytotic membrane trafficking. We used stimulated emission-depletion microscopy to investigate the domain structure of the endosomes. We developed a semiautomatic method for counting the individual domains, an approach that we have validated by immunoelectron microscopy. We found that in endosomes derived from neuroendocrine PC12 cells synaptophysin and several SNARE proteins are organized in microdomains. Cholesterol depletion by methyl-beta-cyclodextrin disintegrates most of the domains. Interestingly, no change in the frequency of microdomains was observed when endosomes were fused with protein-free liposomes of similar size (in what constitutes a novel approach in modifying acutely the lipid composition of organelles), regardless of whether the membrane lipid composition of the liposomes was similar or very different from that of the endosomes. Similarly, Rab depletion from the endosome membranes left the domain structure unaffected. Furthermore, labeled exogenous protein, introduced into endosomes by liposome fusion, equilibrated with the corresponding microdomains. We conclude that synaptic membrane proteins are organized in stable but dynamic clusters within endosomes, which are likely to persist during membrane recycling. Microsc. Res. Tech. 73:606-617, 2010. (C) 2009 Wiley-Liss, Inc

A fluorescence-based in vitro assay for investigating early endosome dynamics

early endosomes receive material from the plasma membrane by fusion with endocytotic vesicles. this material is sorted within endosomes and directed to subdomains at which carrier vesicles bud. these vesicles are then transported toward different cellular destinations. In this article, we describe a protocol for the cell-free reconstitution of endosome docking/fusion and sorting/budding, which is based on labeling of endosomes by endocytotic uptake with fluorescent cargoes. the protocol includes (i) the preparation of fluorescently labeled endosomes, (ii) assays for docking/fusion and for sorting/budding in vitro and (iii) imaging of the reaction mix by fluorescence microscopy to quantify docking, fusion, cargo sorting and budding using counting of single organelles. production of endosome stocks requires approximately 1 d. the in vitro reactions can then be performed separately (similar to 1 d) and are conveniently carried out with multiple samples in parallel. the assay can be adapted for studying the dynamics of organelles other than endosomes