Ferromagnetic isolation of endosomes: a novel method for subcellular fractionation of Xenopus oocytes

Abstract

A novel method has been developed using ferric particles to label endosomes, and to achieve magnetic sorting of the various endocytic compartments involved in lipoprotein uptake into cells. Ferric particles conjugated to a receptor-recognized ligand are bound to coated membrane pits and become internalized into the cytoplasm inside coated vesicles. After apparent fusion of the vesicles to tubular endosomes, the conjugates accumulate and finally discharge into multivesicular endosomes. Pulse-chase experiments elucidate the pathway of internalized conjugates and allow both early compartments (pinosomes and tubular endosomes) and late compartments (multivesicular endosomes and storage organelles) to be selectively labelled. After ferroloading of the various transport compartments, the cells are homogenized and subcellularly fractionated. Sorting of labelled endosomes is performed by a specially designed "free-flow" magnetic chamber. Prophase I-arrested oocytes of the toad Xenopus laevis are used as a model system for studying the transport pathway and the conversion of the yolk precursor vitellogenin. It is possible to follow the route of internalization of vitellogenin-iron conjugates via coated pits, coated vesicles, uncoated vesicles, tubular endosomes, multivesicular endosomes, and light primordial yolk platelets. These endosomes shuttle the ferric particles together with the vitellogenin from oolemma to performed heavy yolk organelles which are still growing. In addition, these various compartments can be isolated according to their function and subjected to electron microscopy and to gel electrophoresis for detailed characterization of their limiting membranes as well as their contents.