Design of a light-switched, genetically-encoded regulator of actin assembly

描述（由申请人提供）：免疫细胞是一个复杂的动态系统。一个关键的例子是，t细胞激活需要在t细胞和它的靶细胞之间形成一个免疫突触。突触的形成和信号传递涉及跨膜受体、脂筏、可溶性蛋白和细胞骨架的局部重组在空间和时间上的协调。即使单个分子中细微的缺陷也会破坏这一过程，给免疫系统功能带来可怕的后果。现代光学显微镜，特别是与遗传编码的荧光报告相结合，已经成为跟踪免疫突触分子过程的关键实验工具。然而，到目前为止，我们一直局限于观察这一过程。我们的目标是开发分子工具，使我们能够以相同的空间和时间分辨率操纵免疫突触，我们现在可以观察它。具体来说，我们建议设计和测试遗传编码的、光开关版本的Wiskott-Aldrich综合征蛋白（WASP），它是免疫突触中肌动蛋白骨架重组的关键调节因子。我们的设计基于WASP和小细菌光感受器PYP（光活性黄蛋白）的构象平衡耦合，因此光激活减轻WASP的自抑制，从而通过激活Arp2/3复合物刺激肌动蛋白聚合。Wiskott-Alrdrich综合征蛋白是免疫细胞识别靶标、伤口愈合过程中的细胞迁移以及神经元发育和重塑过程中的树突生长的关键分子。因此，这种蛋白质的功能障碍导致一系列使人衰弱的疾病，包括但不限于同名的Wiskott-Aldrich综合征。我们建议开发新的研究工具，以更好地了解这种蛋白质的功能，并帮助开发治疗由其功能失调引起的疾病的方法。

DESCRIPTION (provided by applicant): Immune cells are complex and dynamic systems. A key example, T-cell activation requires the formation of an immunological synapse between the T-cell and its target. Formation and signaling across this synapse involves the spatially and temporally coordinated assembly of transmembrane receptors, lipid rafts, soluble proteins and the local reorganization of the cytoskeleton. Even subtle defects in individual molecules can disrupt this process with dire consequences for immune system function. Modern optical microscopy, in particular in its combination with genetically- encoded, fluorescence-based reporters, has been a key experimental tool to follow the molecular processes at the immunological synapse. However, until now we have been limited to observing this process. It is our goal to develop molecular tools that allow us to manipulate the immunological synapse with the same spatial and temporal resolution, with which we can now observe it. Specifically we propose to design and test genetically-encoded, light-switched versions of the Wiskott-Aldrich Syndrome Protein (WASP), a key regulator of actin skeleton reorganization at the immunological synapse. Our design is based on coupling the conformational equilibria of WASP and the small bacterial photoreceptor PYP (Photoactive Yellow Protein) so that light activation alleviates WASP's auto-inhibition and thus stimulates actin polymerization via activation of the Arp2/3 complex. The Wiskott-Alrdrich syndrome protein is a key molecular player in target recognition by immune cells, cell migration during wound healing and dendrite growth during neuronal development and remodeling. Consequently malfunction of this protein leads to a series of debilitating diseases including, but not limited to the eponymous Wiskott-Aldrich syndrome. We are proposing to develop new research tools to better understand the function of this protein and to help develop cures for the diseases caused by its malfunction.