CAREER: Reorganization of the Vertebrate Golgi Apparatus

高尔基体是由一叠一叠的膜封闭池组成的，但人们对产生这种非凡结构的过程仍然知之甚少。传统模型将高尔基体表示为一系列稳定的、定义良好的子隔室。然而，新出现的数据支持“池成熟”模型，该模型认为高尔基体不是一个独立的细胞器，而是内质网的动态产物。高尔基池被认为是由内质网衍生的囊泡融合而重新形成的，这些囊泡是在称为过渡内质网（tER）位点的专门亚域产生的。这项研究的目的是了解产生tER位点和高尔基体的过程。作为这项努力的一部分，目前的建议侧重于描述调解高尔基体重组的机制。脊椎动物细胞的高尔基体在一定条件下经历了戏剧性的重排，包括微管的解聚和细胞通过有丝分裂的通道。基于池成熟模型，一些重组事件涉及高尔基体组分通过内质网的转运，随后在tER位点附近出现新的高尔基体结构，这似乎是合理的。这一假设将通过生化分析和荧光显微镜研究的结合来检验。本提案的研究部分有两个具体目的：1。开发一种生化试验，揭示高尔基蛋白在高尔基重组过程中是否通过内质网转运。通过内质网转运将通过内质网定位的葡萄糖苷酶的作用来检测。这些葡萄糖苷酶最初会被特定的抑制剂阻断，从而使高尔基体装载含有糖基化低聚糖的蛋白质。去除抑制剂后，将诱导高尔基重组，然后分析高尔基蛋白是否存在葡萄糖残基。利用荧光显微镜分析tER功能与高尔基重组的关系。池成熟模型预测tER位点的分布和活性将影响高尔基重组。这一预测将用荧光显微镜进行检验。例如，如果药物治疗最初导致高尔基蛋白返回内质网，重组的高尔基结构应该在tER位点附近找到。为了检查tER位点的动态，这些位点将被标记为绿色荧光蛋白（GFP）融合结构。这些实验将与格利克实验室正在进行的芽殖酵母分析协同进行。通过阐明ter -高尔基体系的保守性，这些研究旨在为高尔基体系的形成提供统一的分子描述。该研究计划与Glick博士的职业教育目标紧密结合。在他的本科和研究生细胞生物学课程中，高尔基体作为一个有用的例子，说明了三个关键的教训：1，细胞生物学过程最好通过综合来自不同模式生物的数据来理解；细胞不是由逻辑工程师设计出来的，而是机会主义进化的结果；第三，人们必须总是愿意重新考虑基本假设。更一般地说，在格利克博士对高尔基体的探索中，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Glick博士的教学中非常强调，他的教学注重培养学生的批判性思维能力。反过来，形成和呈现这些见解的练习激发了教师自己对研究相关问题的思考。

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.