Immunocytochemical analysis of secretion mutants of Tetrahymena using a mucocyst-specific monoclonal antibody.
Dense-core granules represent an adaptation of specialized secretory cells to facilitate stimulus-regulated release of stored proteins. Such granules are a prominent feature of mammalian neuroendocrine and exocrine cells and are also well developed in the ciliates. In Tetrahymena thermophila, the ability to generate mutants in dense-core granule biosynthesis and fusion presents a versatile system for dissecting steps in regulated exocytosis. We have previously shown that defective granules in such mutants could be characterized by several biochemical criteria, including buoyant density, which increases during maturation, and the degree of proteolytic processing of the content precursors. We have now used indirect immunofluorescence, taking advantage of a monoclonal antibody directed against a granule protein, to visualize the morphology and distribution of both granules and putative granule intermediates in mutant and wild-type cells. The results are consistent with the biochemical analysis and extend our characterization of the mutants, allowing us to distinguish four classes. In addition, the assay represents a powerful technique for diagnosis of new mutants.
Turkewitz
A P
AP
Department of Biochemistry and Biophysics and Hormone Research Institute, University of California, San Francisco.
Kelly
R B
RB
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D003594:Cytoplasmic Granules / Q000276:immunology / Q000378:metabolism*; D005089:Exocytosis; D005455:Fluorescent Antibody Technique; D009077:Mucins / Q000276:immunology / Q000378:metabolism; D009154:Mutation; D015800:Protozoan Proteins / Q000276:immunology / Q000378:metabolism; D016808:Tetrahymena thermophila / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D009077:Mucins; D015800:Protozoan Proteins