CORRELATIVE MICROSCOPY OF GOLGI APPARATUS ASSEMBLY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们研究的几条证据汇集在一起，提供了高尔基体膜蛋白在哺乳动物细胞中的高尔基体和内质网之间不断循环的证据。这是一个不依赖于COPI外壳蛋白的途径，依赖于rab6亚型和rab33b。高尔基体可能是内窥镜的衍生物。rab蛋白或募集COPII到内质网膜的小GTPase Sar1p的特异性突变导致高尔基体解体。我们正在启动可逆性研究，也就是说，细胞器可以重新组装吗？体内重组可以通过“恢复”突变来完成。例如，突变型Sar1p可以在HeLa细胞中表达，然后通过过表达野生型Sar1p进行逆转。这是一个仔细滴定蛋白质或质粒量的问题。一种Tet调控的Sar1p细胞系正在开发中。Tet rab6或rab33b线也是可能的。目前的实验是以显微注射的方式进行的。相关的显微镜问题是，高尔基分散后的初始物质是什么？在重组实验中，形成了什么以及各种其他突变对形成的影响是什么？相关显微镜在这里是必要的，以确定起始物质和什么形成

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