Protein-membrane interactions in regulated exocytosis

项目总结 受调控的胞吐作用--依赖于刺激的胞外囊泡融合--介导了广泛的基础 生物过程包括营养动态平衡、激素信号、突触传递和消除 转化的或被病毒感染的细胞。这些胞外途径的不平衡导致人类主要形式的 糖尿病、神经疾病、免疫缺陷和癌症等疾病。这个项目的总体目标是 研究是利用葡萄糖的转运来建立调节胞吐作用的分子原理 Transporter GLUT4作为模型系统。在我们之前的研究中，我们描绘了BMP的分子机制。 已知的GLUT4途径中的胞外调节因子。在这项研究中，我们将重点关注一组新的监管 在我们最近的全基因组CRISPR筛查中确定了调查GLUT4胞吐的因素。我们会带着 开展深入的生化、生物物理、细胞生物学和遗传学研究，以解决两个关键问题：1)如何 胞外介质是否协同作用来驱动胞外囊泡融合？2)胞外囊泡融合是如何耦合的？ 达到综合反应的其他细胞过程？为了回答第一个问题，我们将定义 圈套结合调节剂单独和组合控制圈套的分子机制 拉链、膜系带、双层曲率生成、脂类混合和内容物混合。要回答这个问题 第二个问题，我们将确定GLUT4胞外途径是否以及如何受到刺激的影响- 依赖于SNARs的磷酸化，保守的SM蛋白，特殊的胞外调节因子，以及 网状蛋白介导的内吞作用的介体。我们还将研究它的功能作用和分子基础。 货物检索在建立完整的胞外反应中的作用。除了这些机械分析外，我们还将 继续根据我们的候选基因识别、验证和描述新的贩运监管机构 不偏不倚和有针对性的CRISPR屏幕。机理研究和CRISPR屏幕完全是 补充并将提供对胞吐调节的全面了解，这两种方法都不是 单单一个人就能产生。这项拟议研究的成功完成将填补关于 并将作为理解膜的一般原理的跳板 贩卖人口。最终，从这项工作中收集到的见解将有助于开发新的治疗方法 异常胞吐引起的疾病的治疗策略。

PROJECT SUMMARY Regulated exocytosis – stimulus-dependent exocytic vesicle fusion – mediates a broad range of fundamental biological processes including nutrient homeostasis, hormonal signaling, synaptic transmission, and elimination of transformed or virally infected cells. Imbalances in these exocytic pathways lead to major forms of human disease such as diabetes, neurological disorders, immunodeficiency, and cancer. The overall goal of this research is to establish the molecular principles of regulate exocytosis, using the trafficking of the glucose transporter GLUT4 as a model system. In our previous research, we delineated the molecular mechanisms of known exocytic regulators in the GLUT4 pathway. In this research, we will focus on a group of new regulatory factors identified in our recent genome-wide CRISPR screens investigating GLUT4 exocytosis. We will carry out in-depth biochemical, biophysical, cell biological, and genetic studies to address two key questions: 1) How do exocytic mediators act in concert to drive exocytic vesicle fusion? 2) How is exocytic vesicle fusion coupled to other cellular processes to achieve an integrated response? To answer the first question, we will define the molecular mechanisms by which SNARE-binding regulators, alone and in combination, control SNARE zippering, membrane tethering, bilayer curvature generation, lipid mixing, and content mixing. To answer the second question, we will determine whether and how the GLUT4 exocytic pathway is influenced by stimulus- dependent phosphorylations on SNAREs, conserved SM proteins, specialized exocytic regulators, and mediators of clathrin-mediated endocytosis. We will also investigate the functional roles and molecular basis of cargo retrieval in establishing an integrated exocytic response. Besides these mechanistic analyses, we will continue to identify, validate and characterize new trafficking regulators based on the candidate genes from our unbiased and targeted CRISPR screens. The mechanistic studies and CRISPR screens are fully complementary and will provide a comprehensive understanding of exocytosis regulation that neither approach alone could generate. Successful completion of this proposed research will fill major gaps in the knowledge of regulated exocytosis and will serve as a springboard for understanding the general principles of membrane trafficking. Ultimately, insights gleaned from this work will facilitate the development of new therapeutic strategies for diseases caused by dysregulated exocytosis.