The Endosome System and Membrane Trafficking

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Communication between cellular compartments frequently occurs by shuttle vesicles. The unique features of the endosomal system of Paramecium will be explored by the use of monoclonal antibodies, confocal fluorescence microscopy, replicas of quick-freeze freeze- dried cell fragments, immunogold labeling of cryosections and Lowicryl sections, and high resolution scanning electron microscopy of isolated organelles. The course of probes will be followed from coated pits to early endosomes and on to the next compartment, which may or many not be the acidosomes. Initial studies indicate that this latter transport is via 90 nm clathrin-coated shuttle vesicles arising from early endosomes. Selective labeling, antigen blocking and trypsinization techniques in conjunction with a monoclonal antibody will be used to follow the reappearance on the cell surface of a plasma membrane resident antigen, C6, which enters the early endosome compartment from coated pits via 190 nm preendosomal vesicles but does not enter the 90 nm early endosome- derived shuttle vesicles. Another mAb, specific for acidosomes, will be used to study the biogenesis of the acidosomal membranes and their relationship to the endosomal or contractile vacuole systems. Finally, quick-freeze, freeze-dry techniques and high resolution scanning electron microscopy will be used to visualize and study the true cytosolic surfaces of membranes associated with the endosomal, phagosome-lysosome, and contractile vacuole systems of this cell. %%% Virtually all eukaryotic cells contain an internal membrane system which creates a series of functionally and biochemically distinct intracellular compartments isolated from the "cytoplasm" by their membrane boundaries. Although these compartments are functionally and biochemically distinct, it is known that they "communicate" with and exchange contents with each other and with the extracellular space (isolated from the cytoplasm by the cell's plasma membrane) either directly or indirectly via membrane fusion events. This internal membrane system is the route by which cells take up materials from their environment and process them enzymatically, as well as secrete products to the environment. Understanding how this system functions, and how it is regulated, is a major goal in cell biology. In this project, the single- celled freeliving organism, Paramecium, is used as a readily manipulable model to study how cells communicate between selected compartments, how they package and return resident proteins to their home compartment or membrane, and how the topography of their true cytosolic membrane surface is related to the compartment's function.