Role of SNARE-mediated Trafficking in Cell-ECM Interaction

细胞-细胞外基质(ECM)的动态相互作用，包括细胞黏附和迁移，需要整合素结合ECM蛋白和随后的细胞内生化信号。整合素的功能依赖于整合素及其相关蛋白在细胞内的转运，这种转运是由称为SNARES(可溶性NSF附着蛋白受体)的膜锚定蛋白介导的。我们最近发现，正常的整合素介导的细胞-ECM相互作用需要一种名为Synaxin4(Stx4)的SNARE功能，并且相关蛋白Munc18c在这种情况下调节Stx4。Stx4和Munc18c的相互作用代表了细胞-ECM相互作用的一个新的调节点，对这一调控的分析将揭示组织结构和功能发展和维持的分子机制。在当前的提案中，我们旨在定义Stx4和Munc18c控制细胞黏附和迁移的机制，并确定在这种情况下Stx4和Munc18c的功能是如何调节的。该提案包含三个主要目标。*(1)阐明Synaxin4和Munc18c在细胞黏附和迁移中的作用。*将分析B1整合素的运输和回收，以及FA成分(如Src、FAK、paxlin)对细胞外基质接触部位的靶向。我们已经开发了稳定的细胞系，表达抑制Stx4或Munc18c的结构，以便于生化和显微分析，以及基于细胞的分析来检查细胞与细胞外基质的相互作用。*(2)研究Synaxin4和Munc18c的磷酸化调控。*将确定Stx4和/或Munc18c的磷酸化与细胞-ECM相互作用之间的关系。Stx4或Munc18c将从处于“静止”状态(包被多聚L-赖氨酸)或“粘着”状态(包被细胞外基质)的细胞中进行免疫沉淀，并将通过蛋白质组分析确定Synaxin4和Munc18c中的磷酸化位点(S)。在细胞-ECM相互作用过程中，这些位点的磷酸化将用生化和分子方法进行表征。*(3)确定控制细胞-ECM相互作用的SNARE功能的新调节因子。*细胞将被电镀在ECM底物上，然后SNARE复合体将被免疫沉淀。与SNARE复合体一起捕获的蛋白质，在ECM和对照细胞上电镀的细胞中表现不同的蛋白质将通过蛋白质组分析进行鉴定，并随后进行表征。*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。这项研究将通过培训高素质的人员，并通过促进我们对组织和器官中细胞功能的分子基础的理解，使细胞生物学家和加拿大国内外的其他研究人员受益。********

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. ********