IVEM TOMOGRAPHY OF ISOLATED EXTRACTED & RE CONSTRUCTED SPISULA CENTROSOMES: CLAM

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(Supported by NIH GMS 43264 to R. Palazzo and GMS 40198 to C. Rieder) This is an ongoing project in which IVEM and HVEM tomography are being used to analyze the structure of centrosomes isolated from Spisula (surf clam) oocytes prior to and after various experimental manipulations. This year we characterized the high-resolution 3-D structure of control and KI extracted centrosomes, and also extracted centrosomes that had regained their function by incubation in high speed extracts from oocytes. We found that heavily KI extracted centrosomes are non-functional and consist of an insoluble 15-nm filamentous "centromatrix" material. When this centromatrix is incubated in high speed extracts of activated egg oocytes, the extracted centrosome regains its functional abilities. The loss of centrosome function is correlated, at the IVEM tomographic level, with the loss of gamma tubulin containing 20-nm diameter ring-shaped structures, while regain of function is correla ted with the reappearance of these structures. The data reveal that the centromatrix serves as a scaffold for binding those proteins involved in microtubule nucleation. Because centrosomes are extremely small (0.25 by 0.5 um) and complex, the only way their morphology and replication can be studied is with 3-D EM. Over the past 12 years we have shown that IVEM/HVEM tomography of serial thick sections greatly facilitates such studies, and have published numerous papers on this topic-many recently with the Palazzo laboratory. We are now exploring the utility of combining high resolution antibody labeling with IVEM tomography of high pressure frozen and freeze substituted samples. Schnackenberg, B.J., A. Khodjakov, C. L. Rieder and R. E. Palazzo (1998). The disassembly and reassembly of functional centrosomes in vitro. Proc. Natl. Acad. Sci. USA 95:9295-9300.