Non-Canonical Pathways of Golgi Apparatus Protein Recycling

高尔基体蛋白质回收的非规范途径

• 批准号: 0549001
• 负责人: Brian Storrie
• 金额: --
• 依托单位: University of Arkansas Medical Sciences Campus
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-15 至 2010-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0549001&HistoricalAwards=false
• 关键词: Non; Canonical; Pathways; Golgi; Apparatus

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.

智力优点 该项目建立在累积观察的基础上，即在哺乳动物细胞的细胞分裂之间，高尔基体不断组装和解构。 高尔基体蛋白通过内质网 (ER) 循环返回。 这种循环至内质网的高尔基体受到生理调节，并且可以响应某些细胞培养条件而被诱导。 工作假设是，大部分高尔基体蛋白质再循环到内质网是通过它们从反式高尔基体/反式高尔基体网络移动到邻近的内质网而发生的。 与此一致的是，反式高尔基体蛋白再循环至内质网的速率比内侧或顺式蛋白更快。 这种假设的高尔基蛋白从高尔基体的远端（反式）端到内质网的运动，而不是从最近的（顺式）端运动，以前从未观察到过；因此，它被称为“非规范”回收。 该项目的具体目标是： 1． 确定系链/SNARE 复合物在高尔基体蛋白再循环至内质网中的作用。 根据初步研究和文献，ZW10/RINT1/Syntaxin 18 复合物被选为最有可能在高尔基体蛋白再循环至内质网中发挥决定性作用的复合物。 初步数据表明，选择性 siRNA 敲低 ZW10 会破坏 HeLa 细胞中的高尔基体结构，导致免疫荧光可视化的高尔基体膜网络扩展，并抑制组成型糖基转移酶蛋白再循环至内质网 3 倍。 该复合体的各个成员的功能将接受测试，以确定他们是否参与了 ​​Storrie 实验室通过实验定义的非规范回收。2。当非规范回收受阻时，高尔基体的结构如何变化？ 迄今为止，非经典高尔基体蛋白质回收的证据来自通过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断该途径的特定分子变化的反应。 该目标将探索一种不同的方法。 正常条件下高尔基体的三维组织已通过电子显微镜断层扫描确定。 在这些实验中，非规范途径将被阻断，然后所得的高尔基体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扫描仪布拉德·马什(Brad Marsh)合作进行的。所提出的概念，即驻留高尔基体蛋白从细胞器的反侧直接转运到内质网，挑战了现有的范式，因为传统观点要求蛋白质从反式高尔基体向后移动到中间高尔基体，再到顺式高尔基体，然后到达内质网。 该项目中进行的实验将为非规范途径假说提供进一步的测试，并且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 资助的研究主题。 这将涉及研究生教育、研究生培训以及本科生和高中生的暑期实习参与。 虽然 UAMS 是一所医科大学，而 Storrie 博士之前就读的大学（弗吉尼亚理工大学）是一所拥有大量本科生的院校，但该项目实际暑期本科生或高中生参与的机会更大。 生物科学研究基础设施网络 (BRIN) 项目由 UAMS 生理学和生物物理学系领导。 该项目征集并赞助小石城 UAMS 的正式夏季本科课程。 在高中阶段，小石城中央高中等机构中有一批有抱负的非洲裔美国人。 Storrie 博士过去在指导本科生的研究项目方面取得了成功，如出版物列表中所引用的那样，他在 UAMS 的新职位应该为继续指导提供更多的机会。