CAREER: Reorganization of the Vertebrate Golgi Apparatus

高尔基体被组织成成堆的薄膜封闭的池子，但产生这种非凡结构的过程仍然鲜为人知。传统模型将高尔基体描述为一系列稳定的、定义明确的小舱。然而，新出现的数据支持“脑池成熟”模型，该模型认为高尔基体不是一个独立的细胞器，而是内质网的动态副产物。高尔基体池被认为是通过融合内质网衍生的小泡而形成的，这些小泡是在称为过渡性ER(TER)位点的专门亚域中产生的。本研究的目的是了解创建TER位点和高尔基体堆叠的过程。作为这项努力的一部分，目前的提案侧重于确定调解高尔基机构重组的机制的特点。脊椎动物细胞的高尔基体在某些条件下发生戏剧性的重排，包括微管的解聚和细胞通过有丝分裂。根据脑池成熟模型，其中一些重组事件似乎涉及高尔基体成分通过内质网的运输，然后在TER位点附近出现新的高尔基体结构。这一假说将通过生化分析和荧光显微镜研究相结合的方式进行验证。这项建议的研究部分有两个具体目标：1.建立一种生化分析方法，揭示高尔基体重组过程中高尔基蛋白是否通过内质网。通过内质网定位的葡萄糖苷酶的作用将检测到通过内质网的转运。这些葡萄糖苷酶最初会被特定的抑制剂阻断，从而使高尔基体含有含有糖基化低聚糖的蛋白质。去除抑制剂后，高尔基体重组将被诱导，然后将分析高尔基体蛋白是否存在葡萄糖残留物。用荧光显微镜分析TER功能与高尔基体重组之间的联系。脑池成熟模型预测，TER位点的分布和活性将影响高尔基体重组。这一预测将用荧光显微镜进行验证。例如，如果药物治疗最初导致高尔基体蛋白返回内质网，重组的高尔基体结构应该在TER位点附近找到。为了研究TER位点的动态，这些位点将被绿色荧光蛋白(GFP)融合结构标记。这些实验将与Glick实验室正在进行的对萌芽酵母的分析相配合。通过阐明TER-Golgi系统的保守方面，这些研究旨在为高尔基体的形成提供统一的分子描述。这项研究计划与Glick博士的职业教育目标紧密结合。在他的本科生和研究生细胞生物学课上，高尔基是一个有用的例子，说明了三个关键的教训：1，细胞生物学过程最好的理解是通过合成来自不同模型生物的数据；2，细胞不是由逻辑工程师设计的，而是由机会主义进化设计的；以及3，人们必须始终愿意重新考虑基本假设。更广泛地说，格利克博士探索高尔基的一个反复出现的主题是，从多个角度看待每个问题的重要性。这一点在格利克博士的教学中得到了强烈的强调，他的教学重点是培养学生的批判性思维能力。反过来，阐述和展示这些见解的练习刺激了教师自己对与研究相关的问题的思考。

The Golgi apparatus is organized into stacks of membrane-enclosed cisternae, but the processes that generate this remarkable structure are still poorly understood. Conventional models represent the Golgi as a series of stable, well-defined subcompartments. However, emerging data favor a 'cisternal maturation' model that views the Golgi not as an independent organelle, but as a dynamic outgrowth of the endoplasmic reticulum. Golgi cisternae are postulated to form de novo by the fusion of ER-derived vesicles, which are produced at specialized subdomains called transitional ER (tER) sites.The goal of this research is to understand the processes that create tER sites and Golgi stacks. As part of this effort, the current proposal focuses on characterizing the mechanisms that mediate reorganization of the Golgi apparatus. The Golgi of vertebrate cells undergoes dramatic rearrangements under certain conditions, including depolymerization of microtubules and passage of the cells through mitosis. Based on the cisternal maturation model, it seems plausible that some of these reorganization events involve the transit of Golgi components through the ER, followed by the emergence of new Golgi structures next to tER sites. This hypothesis will be tested by a combination of biochemical assays and fluorescence microscopy studies.The research portion of this proposal has two Specific Aims:1. TO DEVELOP A BIOCHEMICAL ASSAY THAT WILL REVEAL WHETHER GOLGI PROTEINS TRANSIT THROUGH THE ER DURING GOLGI REORGANIZATION. Transit through the ER will be detected by the action of ER-localized glucosidases. These glucosidases will initially be blocked with specific inhibitors, thereby loading the Golgi with proteins that contain glucosylated oligosaccharides. After removal of the inhibitor, Golgi reorganization will be induced, and then Golgi proteins will be analyzed for the presence or absence of glucose residues.2. TO ANALYZE THE LINK BETWEEN tER FUNCTION AND GOLGI REORGANIZATION USING FLUORESCENCE MICROSCOPY. The cisternal maturation model predicts that the distribution and activity of tER sites will influence Golgi reorganization. This prediction will be tested using fluorescence microscopy. For example, if a drug treatment initially causes Golgi proteins to return to the ER, the reorganized Golgi structures should be found near tER sites. To examine the dynamics of tER sites, these sites will be labeled with a Green Fluorescent Protein (GFP) fusion construct.These experiments will synergize with ongoing analysis of budding yeasts in the Glick laboratory. By elucidating conserved aspects of the tER-Golgi system, these studies aim to provide a unified molecular description of Golgi stack formation.This research plan is closely integrated with Dr. Glick's CAREER education objectives. In his undergraduate and graduate cell biology classes, the Golgi serves as a useful example for illustrating three key lessons: 1, cell biological processes are best understood by synthesizing data from diverse model organisms; 2, cells were designed not by a logical engineer, but by an opportunistic evolution; and 3, one must always be willing to reconsider basic assumptions. More generally, a recurring theme from Dr. Glick's explorations of the Golgi is the importance of viewing each question from multiple perspectives. This point is emphasized strongly in Dr. Glick's teaching, which focuses on developing students' capacity for critical thinking. In turn, the exercise of formulating and presenting these insights stimulates the teacher's own thinking about research-related questions.