Non-Canonical Pathways of Golgi Apparatus Protein Recycling

智力价值该项目建立在累积观察的基础上，即高尔基体在哺乳动物细胞分裂之间的时间内不断地组装和解构。高尔基体蛋白质通过内质网(ER)循环。这种高尔基体循环到内质网是受生理调节的，并可以在某些细胞培养条件下被诱导。工作假说认为，高尔基体蛋白向内质网的大部分循环是通过它们从跨高尔基体/跨高尔基体网络到邻近的内质网的运动而发生的。与此一致的是，反式高尔基体蛋白质循环到内质网的速度比中间或顺式蛋白质快。高尔基体蛋白质从高尔基体的远(反)端到内质网，而不是从最近的(顺式)端移动的假设，以前从未观察到；因此，它被称为“非正则”循环。该项目的具体目标是：1.确定系绳/圈套复合体在定位高尔基体蛋白再循环到内质网中的作用。在初步研究和文献的基础上，ZW10/RINT1/Synaxin 18复合体被选为最有可能在高尔基体蛋白循环到内质网的靶向中发挥决定作用的复合体。初步数据显示，ZW10的选择性siRNA敲除破坏了HeLa细胞的高尔基体结构，导致免疫荧光显示的高尔基体膜的延伸网络，并将组成性糖基转移酶蛋白循环到内质网的能力抑制了3倍。根据斯托里实验室的实验定义，将测试建筑群中个别成员的功能，以确定他们是否参与了非规范回收。当非规范的循环被阻止时，高尔基体的结构是如何改变的？到目前为止，非规范高尔基蛋白质循环的证据来自荧光显微镜对单个蛋白质的跟踪，以及有限数量的标记蛋白质对阻止该途径的特定分子变化的反应。这个目标将探索一种不同的方法。用电子显微镜断层扫描确定了正常条件下高尔基体的三维结构。在这些实验中，非正则路径将被阻断，然后所产生的高尔基结构将在三维水平上被定义。这些实验是与澳大利亚昆士兰大学经验丰富的断层摄影家布拉德·马什合作进行的。居留的高尔基体蛋白质直接从细胞器的反面运输到内质网的概念挑战了现有的范式，因为传统的观点要求蛋白质从反式到内侧-顺式-高尔基体再到内质网。这个项目中进行的实验将提供对非正则路径假说的进一步测试，并应该对高尔基体的结构和组织产生新的见解。标准2该项目以研究教育为中心，这是NSF赞助的研究的一个主题。这将包括研究生教育、研究生培训和本科生和高中生参加暑期实习。尽管UAMS是一所医科大学，斯托里博士之前的大学(弗吉尼亚理工大学)是一所拥有大量本科生的机构，但对于这个项目来说，夏季本科生或高中生参与的实际机会更大。生物科学研究基础设施网络(BRIN)项目由UAMS的生理学和生物物理系领导。这个项目征集并赞助了位于小石城的UAMS的一个正式的夏季本科生项目。在高中阶段，有一群有抱负的非裔美国人在小石城的中央高中等机构就读。斯托里博士过去在指导本科生的研究项目方面取得了成功，他在UAMS的新职位应该会为他提供更多继续指导的机会。

Intellectual Merit The project builds on cumulative observations that the Golgi apparatus is assembled and deconstructed constantly in the time between cell divisions in mammalian cells. The Golgi proteins recycle back through the endoplasmic reticulum (ER). This Golgi apparatus recycling to the ER is subject to physiological regulation and can be induced in response to certain cell culture conditions. The working hypothesis is that much of Golgi protein recycling to the ER occurs by their movement from trans Golgi apparatus/ trans Golgi network to adjacent ER. Consistent with this, the rate of recycling of trans Golgi proteins to the ER is faster than that of medial or cis proteins. This hypothesized movement of Golgi proteins from the distant (trans-) end of the Golgi apparatus to the ER, rather than from the closest (cis-) end, has never been observed previously; for that reason it is termed "non-canonical" recycling. The Specific Aims of the project are:1. Determination of the role of tether/SNARE complexes in the targeting of Golgi protein recycling to the ER. On the basis of Preliminary Studies and literature, the ZW10/RINT1/Syntaxin 18 complex was chosen as most likely to play a determining role in targeting of Golgi apparatus protein recycling to the ER. Preliminary Data show that selective siRNA knockdown of ZW10 disrupts Golgi apparatus structure in HeLa cells, resulting in an extended network of Golgi membranes visualized by immunofluorescence, and inhibits constitutive glycosyltransferase protein recycling to the ER by 3-fold. The functions of individual members of the complex will be tested for their participation in non-canonical recycling as defined experimentally by the Storrie laboratory.2. How does the structure of the Golgi change when non-canonical recycling is blocked? To date, evidence for non-canonical Golgi protein recycling has come from the tracking of individual proteins by fluorescence microscopy, and the response of a limited number of marker proteins to specific molecular changes that block the pathway. This Aim will explore a different approach. The three dimensional organization of the Golgi apparatus under normal conditions has been defined by electron microscopic tomography. In these experiments, the non-canonical pathway will be blocked, and then the resulting Golgi structure will be defined at the three dimensional level. These experiments are in collaboration with Brad Marsh, an experienced tomographer at the University of Queensland in Australia.The proposed concept that resident Golgi proteins are transported directly to the ER from the trans side of the organelle challenges existing paradigms because the conventional views require that, instead, proteins move backwards from trans- to medial- to cis-Golgi and then to the ER. The experiments carried out in this project will provide further tests of the non-canonical pathway hypothesis, and should yield new insights into Golgi structure and organization. Criterion 2 The project is centered on education-through-research, a theme of NSF sponsored research. This will involve graduate education, graduate student training and summer internship participation for undergraduates and high school students. Although UAMS is a medical university and Dr. Storrie's previous university (Virginia Tech) was an institution with a large undergraduate population, the actual opportunities for summer undergraduate or high school student participation are greater for this project. A Biosciences Research Infrastructure Network (BRIN) project is led by the Department of Physiology and Biophysics at UAMS. This project solicits and sponsors a formal summer undergraduate program at UAMS in Little Rock. At the high school level, there is a population of aspiring African-Americans at institutions such as Central High School in Little Rock. Dr. Storrie has been successful in the past in the mentorship of undergraduates in research projects as cited in the publication list, and his new position in UAMS should provide expanded opportunities for continuing mentorship.