CORRELATIVE MICROSCOPY OF GOLGI APPARATUS ASSEMBLY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们研究的几条证据汇聚在一起，提供了哺乳动物细胞高尔基体和内质网之间高尔基体膜蛋白持续循环的证据。这是一个不依赖于COPI外壳蛋白的途径，依赖于rab6亚型和rab33b。高尔基体可能是内质网的衍生品。Rab蛋白或小GTP酶Sar1p中的特定突变会将COPII招募到内质网膜上，导致高尔基体的拆解。我们正在启动可逆性研究，即细胞器能否重新组装？体内的重组可以通过逆转突变来完成。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而逆转。这是一个仔细滴定蛋白质或质粒数量的问题。Tet调控的Sar1p细胞系正在建立中。Tet rab6或rab33b系也是可能的。目前的实验是以微量注射的形式进行的。相关的显微镜问题是，高尔基体扩散后的初始物质是什么？在重组实验中，形成了什么，各种其他突变对形成的影响是什么？在这里，相关显微图像对于识别起始材料和形成的物质是必不可少的

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