title: Analyzing Endosomal Docking, Fusion, Sorting, and Budding Mechanisms in Isolated Organelles.
abstract: Due to their central role in the reception and sorting of newly internalized material, early endosomes undergo extensive membrane remodeling. They dock and fuse with endocytic carrier vesicles originating from the plasma membrane, sort the internalized material in internal microdomains, and allow the budding of new carrier vesicles from their membrane, destined to fuse with the plasma membrane (recycling) or other organelles. Early endosomal compartments might also be involved in the recycling of synaptic vesicles in nerve terminals. The present protocol describes a technique allowing to assess the mechanistic and molecular aspects of the membrane remodeling processes of docking, fusion, sorting, and budding in early endosomes of neuron-like (and other) cells. It involves the fluorescent labeling and isolation of endosomal organelles, the setup of assays allowing for docking/fusion or sorting/budding in vitro, and finally the assessment and quantification of the membrane remodeling events by fluorescent microscopy. The technique can be easily manipulated by the addition of inhibitors or activators, and can be combined with other techniques, such as immunostaining and high-resolution microscopy, expanding the experimental possibilities in the investigation of early endosomal characteristics.
authors.affiliation: Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Göttingen, Germany. s.barysch@zmbh.uni-heidelberg.de.|Zentrum für Molekulare Biologie Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany. s.barysch@zmbh.uni-heidelberg.de.
authors.forename: Sina V
authors.initials: SV
authors.lastname: Barysch
authors.affiliation: Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077, Göttingen, Germany.|Trilogy Writing & Consulting, Falkensteiner Str. 77, 60322, Frankfurt am Main, Germany.
authors.forename: Ioanna
authors.initials: I
authors.lastname: Bethani
mesh_terms: D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000378:metabolism; D002458:Cell Fractionation; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000378:metabolism; D015388:Organelles / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism
keywords: Budding; Endosomes; Fusion; In vitro; Post-nuclear supernatant (PNS); Rat brain cytosol