Regulation of Actin Assembly and Cell Motility by IQGAPs

描述(申请人提供)：本申请的总体目标是确定IQGAP1和其他哺乳动物IQGAP如何通过Arp2/3复合体调节肌动蛋白细丝成核，进而调节细胞运动性。在像人类这样的多细胞动物中，细胞的运动和形状的变化是生物过程的基础，包括器官发育、伤口愈合、肿瘤转移和突触可塑性。因此，解开允许细胞移动和改变形状的分子机制一直是一个主要目标。现在看来，许多形式的动物细胞的运动和形态发生是由Arp2/3复合体成核的分支肌动蛋白细丝的极化组装引起的。Arp2/3复合体的这种活性不是连续的。相反，它依赖于激活因子，如N-WASP和WASP/WAVE家族的其他成员。同样，N-WASP对Arp2/3复合体的最大刺激需要额外的因子将N-WASP从自抑制状态释放出来。在过去的几年里已经发现了一些这样的因子，最引人注目的是激活了CDC42和PIP2。最近，申请人的实验室发现，IQGAP1可以通过需要N-WASP和Arp2/3复合体的机制在体外有效地刺激肌动蛋白组装。这些开创性的观察表明，IQGAP1是体内Arp2/3复合体的一个以前未知的主要调节因子，并作为本应用的基础。提出了三个具体目标。1)IQGAP1通过Arp2/3复合体调节肌动蛋白组装的分子机制将通过体外重组来确定。2)哺乳动物IQGAP1以外的IQGAPs也调节Arp2/3复合体的假设将得到验证。其中包括已知存在于蛋白质水平的IQGAP2和根据基因组DNA和表达序列标签(EST)推断的IQGAP3。将使用免疫学方法、质谱学、原位杂交和逆转录聚合酶链式反应来寻找IQGAP3在蛋白质水平表达的证据。3)将对IQGAP1刺激细胞内肌动蛋白组装的假设进行检验，该假设是通过将IQGAP1上的靶向结构域与配体激活的细胞表面受体的细胞质尾部结合而触发的。IQGAP1对肌动蛋白组装的调节及其对细胞运动的影响将通过光镜方法和细胞蛋白复合体的免疫沉淀来研究。对其他哺乳动物IQGAP的类似研究将通过追求特定的目标2来通过Arp2/3复合体调节肌动蛋白组装而启动。

DESCRIPTION (provided by applicant): The overall goal of this application is to determine how IQGAP1 and other mammalian IQGAPs regulate actin filament nucleation by the Arp2/3 complex, and by extension, cellular motility. In multicellular animals like humans, cell movements and shape changes underly biological process as diverse as organismal development, wound healing, tumor metastasis, and synaptic plasticity. Unraveling the molecular mechanisms that allow cells to move and change shape has therefore been a major goal. It now seems clear that many forms of animal cell motility and morphogenesis are caused by the polarized assembly of branched actin filaments nucleated by the Arp2/3 complex. This activity of Arp2/3 complex is not consitutive. Instead, it relies on activating factors, like N-WASP and other members of the WASP/WAVE family. Likewise, maximal stimulation of Arp2/3 complex by N-WASP requires additional factors that release N-WASP from an autoinhibited state. A few such factors have been discovered within the past few years, most notably activated Cdc42 and PIP2. Recently, the applicant's laboratory discovered that IQGAP1 can potently stimulate actin assembly in vitro by mechanisms that require both N-WASP and Arp2/3 complex. These seminal observations suggest that IQGAP1 is a major, previously unrecognized regulator of the Arp2/3 complex in vivo, and serves as the foundation of the present application. Three Specific Aims are proposed. 1) The molecular mechanisms by which IQGAP1 regulates actin assembly through Arp2/3 complex will be determined by in vitro reconstitution. 2) The hypothesis that mammalian IQGAPs other than IQGAP1 also regulate Arp2/3 complex will be tested. These include IQGAP2, which is known to exist at the protein level, and IQGAP3, which has been inferred from genomic DNA and expressed sequence tags (ESTs). Evidence for expression of IQGAP3 at the protein level will be sought using immunological methods, mass spectrometry, in situ hybridization, and RTPCR. 3) A test will be made of the hypothesis that stimulation of actin assembly by IQGAP1 in cells is triggered by binding of a targetting domain on IQGAP1 to the cytoplasmic tails of ligand-activated cell surface receptors. The regulation of actin assembly by IQGAP1, and the consequences for cell motility will be studied in cultured mammalian cells by light microscopic methods and immunoprecipitation of cellular protein complexes. Similar studies will be initiated for other mammalian IQGAPs shown by pursuit of Specific Aim 2 to regulate actin assembly through the Arp2/3 complex.