Role of SNARE-mediated Trafficking in Cell-ECM Interaction

SNARE 介导的运输在细胞-ECM 相互作用中的作用

• 批准号: RGPIN-2017-05199
• 负责人: Coppolino, Marc
• 金额: $ 1.89万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=681658
• 关键词: Role; SNARE; mediated; Trafficking; Cell

Dynamic cell-extracellular matrix (ECM) interactions, including cell adhesion and migration, require binding of ECM proteins by integrins and subsequent intracellular biochemical signalling. Integrin function is dependent on the intracellular trafficking of integrins and associated proteins, and this traffic is mediated by membrane-anchored proteins called SNAREs (Soluble NSF-Attachment protein Receptors). We recently discovered that the function of a SNARE called Syntaxin4 (Stx4) is required for normal integrin-mediated cell-ECM interactions, and that an associated protein, Munc18c, regulates Stx4 in this context. The interaction of Stx4 and Munc18c represents a novel regulatory point in cell-ECM interactions, and analysis of this regulation will reveal molecular mechanisms that underlie the development and maintenance of tissue architecture and function.******In the current proposal, we aim to define the mechanisms by which Stx4 and Munc18c control cell adhesion and migration, and to determine how the functions of Stx4 and Munc18c are regulated in this context. The proposal contains three major aims.******(1) To elucidate the functions of Syntaxin4 and Munc18c in cell adhesion and migration.***Trafficking and recycling of B1 integrins, and the targeting of FA components (e.g. Src, FAK, paxillin) to sites of ECM contact, will be analyzed. We have developed stable cell lines, which express constructs to inhibit Stx4 or Munc18c, to facilitate both biochemical and microscopic analyses, as well as cell-based assays to examine cell-ECM interactions. ******(2) To examine the regulation of Syntaxin4 and Munc18c by phosphorylation.***The relationship between phosphorylation of Stx4 and/or Munc18c and cell-ECM interactions will be defined. Stx4 or Munc18c will be immunoprecipitated from cells in a resting' state (plated on poly-L-lysine) or an adhesive' state (plated on ECM substrate), and the phosphorylated site(s) in Syntaxin4 and Munc18c will be determined through phosphoproteomic analysis. Phosphorylation at these sites during cell-ECM interactions will then be characterized using biochemical and molecular approaches.******(3) To identify novel regulators of SNARE function that control cell-ECM interactions.***Cells will be plated on ECM substrates, and SNARE complexes will then be immunoprecipitated. Proteins, captured along with SNARE complexes, that are differentially represented in cells plated on ECM vs. control cells will be identified by proteomic analyses and subsequently characterized.******The above aims include several projects that offer collaborative training opportunities for graduate and undergraduate students. The research will benefit cell biologists, and other researchers across Canada and abroad, both through the training of highly qualified personnel and by advancing our understanding of the molecular underpinnings of cellular function within tissues and organs. ********

动态的细胞-细胞外基质（ECM）相互作用，包括细胞粘附和迁移，需要ECM蛋白通过整合素和随后的细胞内生化信号传导的结合。整联蛋白的功能依赖于整联蛋白和相关蛋白的细胞内运输，这种运输由称为SNARE（可溶性NSF附着蛋白受体）的膜锚定蛋白介导。 我们最近发现，一种称为Syntaxin 4（Stx 4）的SNARE的功能是正常的整合素介导的细胞-ECM相互作用所必需的，并且相关蛋白Munc 18 c在这种情况下调节Stx 4。 Stx 4和Munc 18 c的相互作用代表了细胞-ECM相互作用中的一个新的调节点，对这种调节的分析将揭示组织结构和功能的发展和维持的分子机制。在目前的提案中，我们的目标是确定Stx 4和Munc 18 c控制细胞粘附和迁移的机制，并确定Stx 4和Munc 18 c的功能在这种情况下是如何调节的。 该提案包含三个主要目标。* (1)阐明Syntaxin 4和Munc 18 c在细胞粘附和迁移中的功能。将分析B1整联蛋白的运输和再循环，以及FA组分（例如Src、FAK、桩蛋白）靶向ECM接触部位。我们已经开发了稳定的细胞系，其表达抑制Stx 4或Munc 18 c的构建体，以促进生物化学和显微镜分析，以及基于细胞的测定来检查细胞-ECM相互作用。 **（2）研究磷酸化对Syntaxin 4和Munc 18 c的调节。*将定义Stx 4和/或Munc 18 c的磷酸化与细胞-ECM相互作用之间的关系。Stx 4或Munc 18 c将从处于“静止”状态（接种在聚-L-赖氨酸上）或“粘附”状态（接种在ECM底物上）的细胞中免疫沉淀，并且将通过磷酸蛋白质组学分析来确定突触融合蛋白4和Munc 18 c中的磷酸化位点。 然后将使用生物化学和分子方法表征细胞-ECM相互作用期间这些位点的磷酸化。** (3)鉴定控制细胞-ECM相互作用的SNARE功能的新型调节剂。*将细胞接种在ECM基质上，然后将SNARE复合物免疫沉淀。 将通过蛋白质组学分析鉴定与SNARE复合物一起沿着捕获的蛋白质，其在ECM上铺板的细胞与对照细胞中差异表达，并随后表征。*上述目标包括为研究生和本科生提供合作培训机会的几个项目。这项研究将有利于细胞生物学家，以及加拿大和国外的其他研究人员，通过培训高素质的人员，并通过推进我们对组织和器官内细胞功能的分子基础的理解。********