Cell cortex regulation by Ca(2+) binding proteins of the S100 family

在膜下和富含肌动蛋白的细胞皮层中的动态重组控制着许多细胞过程，包括细胞形状和细胞迁移的调节。许多这些重组是由Ca2+结合蛋白家族介导的细胞内Ca2+水平的短暂升高引发的。S100蛋白是这些Ca2+效应蛋白家族中最大的一个，其成员存在于皮质细胞骨架中。我们开发了一种特殊的亲和力方法来寻找S100蛋白的生理相关靶点，并可以识别出两个细胞皮质调节因子ezrin和IQGAP1，它们由Ca2+结合的S100P相互作用控制。Ezrin作为质膜脂、蛋白和肌动蛋白丝之间的连接物，而IQGAP1代表皮层支架蛋白，在细胞刺激下组装不同的信号中间体。在这两种情况下，至少在体外，S100P结合以Ca2+依赖的方式调节皮质靶蛋白的活性。在ezrin的情况下，与Ca2+/S100P的相互作用释放分子内自抑制，从而引发暴露膜蛋白和f -肌动蛋白结合位点的构象激活。在IQGAP1的情况下，Ca2+/S100P的结合可以调节IQGAP1介导的信号传导，以响应生长因子刺激。因此，通过S100P结合Ca2+信号可以直接影响皮质细胞骨架中的重要调控因子，从而控制细胞极化和迁移等过程中的细胞形状变化。该项目旨在了解调节皮质细胞骨架动力学响应Ca2+信号的分子机制。基于我们之前的发现，特别强调的是S100家族的Ca2+结合蛋白的作用。具体而言，将解决以下问题：1。在活细胞中是否观察到Ca2+结合的S100P与ezrin和/或IQGAP1之间的复合物形成，这是否是靶蛋白皮质易位所必需的？Ca2+依赖性S100P-ezrin和S100P-IQGAP1相互作用在二维表面和组织中细胞迁移中的作用是什么？相关的S100蛋白是否与其他皮质靶蛋白形成复合物？这些复合物的功能是什么？为了回答这些问题，我们将分析Ca2+调控的S100/靶蛋白复合物在培养的上皮细胞和发育中的斑马鱼胚胎的原始生殖细胞迁移过程中的动力学和功能。

Dynamic reorganizations in the membrane-underlying and actin-rich cell cortex control many cellular processes that include the regulation of cell shape and cell migration. Many of these reorganizations are triggered by transient elevations of intracellular Ca2+ levels that are mediated by families of Ca2+ binding proteins. S100 proteins represent the largest of these families of Ca2+ effector proteins with members being present in the cortical cytoskeleton. We developed a special affinity approach to search for physiologically relevant targets of S100 proteins and could identify two cell cortex regulators, ezrin and IQGAP1, which are controlled by interaction with Ca2+ bound S100P. Ezrin serves as a linker between plasma membrane lipids and proteins and underlying actin filaments, whereas IQGAP1 represents a cortical scaffold protein assembling different signalling intermediates in response to cell stimulation. In both cases S100P binding regulates activities of the cortical target proteins in a Ca2+ dependent manner, at least in vitro. In the case of ezrin, interaction with Ca2+/S100P releases an intramolecular autoinhibition and thereby triggers a conformational activation that exposes the membrane protein and F-actin binding sites. In the case of IQGAP1, binding of Ca2+/S100P can modulate IQGAP1-mediated signalling in response to growth factor stimulation. Thus, via S100P binding Ca2+ signalling can directly affect important regulators in the cortical cytoskeleton and thereby control cell shape changes in processes such as cell polarization and migration.The project aims at understanding the molecular mechanisms that regulate cortical cytoskeleton dynamics in response to Ca2+ signalling. Based on our previous findings a particular emphasis is on the action of Ca2+ binding proteins of the S100 family. Specifically, the following questions will be addressed:1. Is complex formation between Ca2+ bound S100P and ezrin and/or IQGAP1 observed in living cells and is it required for cortical translocation of the target protein?2. What is the role of Ca2+ dependent S100P-ezrin and S100P-IQGAP1 interactions in cell migration both, on two-dimensional surfaces and in tissues?3. Do related S100 proteins form complexes with other cortical target proteins and what is the function of these complexes?To answer these questions we will analyse the dynamics and function of Ca2+-regulated S100/target protein complexes during the migration of cultured epithelial cells and of primordial germ cells in developing zebrafish embryos.