Protein-membrane interactions in regulated exocytosis

项目摘要 受调节的胞吐作用-刺激依赖的胞吐囊泡融合-介导广泛的基础免疫应答。 生物过程，包括营养平衡、激素信号、突触传递和消除 转化或病毒感染的细胞。这些胞吐途径的不平衡导致了人类免疫缺陷的主要形式。 疾病，如糖尿病、神经系统疾病、免疫缺陷和癌症。这个项目的总体目标是 研究是利用葡萄糖的运输来建立调节胞吐作用的分子原理 转运蛋白GLUT4作为模型系统。在我们以前的研究中，我们描绘了 GLUT4通路中已知的胞吐调节因子。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筛选中确定的因素，研究GLUT4胞吐作用。创新实施 深入的生物化学，生物物理学，细胞生物学和遗传学研究，以解决两个关键问题：1）如何 胞吐介质是否协同作用以驱动胞吐囊泡融合？2)胞吐囊泡融合是如何偶联的 到其他细胞过程来实现综合反应？为了回答第一个问题，我们将定义 SNARE结合调节剂单独和联合控制SNARE的分子机制 拉链化、膜栓系、双层曲率产生、脂质混合和内容物混合。回答 第二个问题，我们将确定GLUT 4胞外途径是否以及如何受到刺激的影响- 依赖于SNARE的磷酸化，保守的SM蛋白，专门的胞外调节因子， 网格蛋白介导的内吞作用的介质。我们还将研究的功能作用和分子基础， 在建立整合的胞吐反应中的货物回收。除了这些机制分析，我们将 继续根据我们的候选基因识别、验证和描述新的贩运调节剂， 无偏见和有针对性的CRISPR筛选。机制研究和CRISPR筛选完全 互补，并将提供一个全面的了解胞吐调节，既不接近 独自一人可以产生。成功完成这项拟议的研究将填补知识的主要空白， 调节胞吐作用，并将作为理解膜的一般原则的跳板 贩卖人口最终，从这项工作中收集的见解将促进新的治疗方法的开发。 由失调的胞吐作用引起的疾病的策略。

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.