CORRELATIVE MICROSCOPY OF GOLGI APPARATUS ASSEMBLY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Several lines of evidence from our research converge to provide evidence that Golgi membrane proteins cycle continuously between the Golgi apparatus and the ER in mammalian cells. This is a COPI coat protein independent pathway and is dependent of rab6 isoforms and rab33b. The Golgi apparatus may be a derivative of the ER. Specific mutants in rab proteins or the small GTPase, Sar1p that recruits COPII to ER membranes, result in Golgi apparatus disassembly. We are initiating reversibility studies, i.e., can the organelle be reassembled? Reassembly in vivo can be done by ¿reverting¿ mutations. For example, mutant Sar1p can be expressed followed by reversion by overexpression of wild type Sar1p in HeLa cells. This is a matter of careful titration of protein or plasmid amounts. A Tet regulated Sar1p cell line is being developed. A Tet rab6 or rab33b line is also possible. Present experiments are being done as microinjection. The correlative microscopy questions are, what is the initial material after Golgi dispersal? And, in a reassembly experiment, what is formed and what is the effect of various other mutations on formation? Correlative microscpy is essential here to identify starting material and what is formed