Regulation of Membrane-Cortical Cytoskeleton Crosstalk by WASH

描述(申请人提供)：质膜和下面的皮质细胞骨架经历连续的动态相互作用，这是许多基本细胞过程所必需的，包括信号转导、迁移、细胞内外和细胞内附着/黏附。因此，调节这些相互作用及其功能的蛋白质在生物学中具有普遍的重要性。Wiskott Aldrich综合征蛋白家族作为Rho家族GTP酶的效应者，通过Arp2/3复合体聚合肌动蛋白。WASs家族的两个成员WASP和SCAR/WAVE参与了膜-细胞骨架的相互作用，在膜/囊泡运输、形态发生、血管生成、炎性免疫反应等过程中起重要作用，当调控不当时，参与病原体感染、出血/瘀伤、免疫缺陷和癌症转移。我们最近已经确定了第三个AS家族蛋白亚家族，命名为WASH，它从内阿米巴到人类都是保守的。这项提议的长期目标是描绘WASH的细胞质和核功能(S)以及这些过程出错的发育后果。这项建议的具体目的是：1)利用遗传学、发育、分子/生化和细胞生物学方法，确定WASH在肌动蛋白和微管细胞骨架协调和细胞-膜串扰中的作用(S)；2)描述WASH功能在血细胞发育和趋化迁移中的机制；以及3)结合比较生物信息学、细胞生物学和高分辨率显微镜方法，研究WASH对核结构和组织的影响。果蝇丰富的技术、复杂的活体成像和可用的试剂使其成为研究WASH在体内功能的极好的、可遗传操作的生物。从这些项目中获得的结果预计将产生广泛的影响，因为WASH在整个生物体中都是保守的。WASH还将作为揭示其他肌动蛋白结合蛋白和细胞质(细胞骨架)蛋白/家族作用的一般组织原则和/或机制的参考蛋白，并将为控制核内外普遍的分子和细胞生物学事件的机制提供新的见解。在这些项目中收集的信息对于了解WASH如何在正常发育或疾病状态下调控不当时控制关键细胞过程，以及从长远来看，对于潜在开发新的抗病药物和/或疗法，或提高现有治疗的有效性，将是重要的。 公共卫生相关性：Wiskott-Aldrich综合征是一种X连锁的遗传传播疾病，临床症状从轻度湿疹到严重瘀伤/出血、免疫缺陷和癌症。这项拟议的研究将研究新发现的WAS族蛋白亚家族的功能，称为WASH，它的功能是在细胞中重组细胞骨架和膜结构，以响应各种信号，从而导致细胞改变形状和/或移动。由于WASH是高度保守的，从果蝇那里学到的信息可以推断到更高级的生物，包括人类。这些研究有望提供有关其作用的新信息(S)，不仅在正常过程中，而且在疾病状况和癌症发生/进展中也是如此，并可能为治疗发展的新途径提供基础。

DESCRIPTION (provided by applicant): The plasma membrane and the underlying cortical cytoskeleton undergo continuous dynamic interplay that is necessary for many essential cellular processes, including signal transduction, migration, endo-/exo-cytosis, and inter- and intra-cellular attachments/adhesion. Thus, the proteins that regulate these interactions and their function are of general importance in biology. The Wiskott Aldrich Syndrome (WAS) protein family, which act as effectors of Rho family GTPases, polymerize actin through the Arp2/3 complex. The two founding WAS family members, WASP and SCAR/WAVE, are involved in membrane-cytoskeletal interactions important for processes including membrane/vesicle trafficking, morphogenesis, angiogenesis, inflammatory immune response, and when mis-regulated, in pathogen infection, bleeding/bruising, immune deficiency and cancer metastasis. We have recently identified a third subfamily of WAS family proteins, designated WASH, that is conserved from Entamoeba to humans. The long-term goal of this proposal is to delineate both the cytoplasmic and nuclear function(s) of WASH and the developmental consequences of these processes going awry. The specific aims of this proposal are 1) to determine the role(s) of WASH in the regulation of actin and microtubule cytoskeletal coordination and cytoskeletal-membrane crosstalk using genetic, developmental, molecular/biochemical, and cell biological approaches; 2) to delineate the mechanisms of WASH function in hemocyte developmental and chemotactic migrations; and 3) to investigate the effect of WASH on nuclear architecture and organization using a combination of comparative bioinformatic, cell biological, and high-resolution microscopic approaches. The wealth of techniques, sophisticated live imaging, and reagents available in Drosophila make it an excellent, genetically manipulable, organism for studying the functions of WASH in vivo. Results obtained from these projects are expected to have wide implications as WASH is conserved throughout organisms. WASH will also serve as a reference protein for revealing general organizational principles and/or mechanisms for the roles of other actin-binding and cytoplasmic (cytoskeletal) proteins/families and will provide new insight into mechanisms controlling universal molecular and cell biological events both in and out of the nucleus. The information gathered in these projects will be important for understanding how WASH functions to control key cellular processes in normal development or when mis-regulated in disease states, and in the longer term, for the potential development of novel anti- disease drugs and/or therapies, or to enhance the effectiveness of existing treatments. PUBLIC HEALTH RELEVANCE: Wiskott-Aldrich Syndrome (WAS) is an X-linked genetically transmitted disease that presents clinically with symptoms ranging from mild eczema to severe bruising/bleeding, immune deficiency, and cancer. The proposed studies will examine the functions of a newly identified subfamily of WAS family proteins, named WASH, which functions to reorganize cytoskeleton and membrane structures in cells in response to various signals, thereby causing cells to change shape and/or move. As WASH is highly conserved, information learned from Drosophila can be extrapolated to higher organisms, including humans. These studies are expected provide new information on its role(s) not only in normal processes, but also in disease conditions and cancer onset/progression, and may provide a basis for new avenues of therapeutic development.