A Rab8-Like GTPase Functionally Interacts with SCAR, a WASp Family Protein

多细胞生物的进化已经允许特化细胞和组织的形成。多细胞性进化的关键步骤是获得细胞与其他细胞相互作用的能力，并进行发育调节的细胞外基质（ECM）蛋白的分泌。各种细胞膜相关蛋白，统称为细胞粘附分子（CAM），使许多植物和动物细胞相互紧密粘附。CAM在细胞外与相邻细胞上的其他CAM和ECM中的分子相互作用，并且在细胞内与肌动蛋白细胞骨架相互作用。在发育过程中分泌的蛋白质在组织形态发生的信号转导中也起重要作用。关于成为粘附的一部分或ECM的一部分的蛋白质的类型已知很多;然而，关于这些分子如何被递送到细胞表面的知之甚少。据推测，这些蛋白质的运动依赖于囊泡运输。Ras相关的Rab GTP酶是低分子量GTP酶的超家族，被认为是囊泡运输的主控制器。一种深入了解CAM和分泌分子的机制的方法。在发育过程中被贩卖的动物的研究是探索这些过程的进化起源，并研究非后生动物系统和动物共有的特征。盘基网柄藻是一种很有前途的生物体，因为它可以很容易地进行遗传操作，因为它具有一个简单的发育周期。Temesvari博士的实验室已经获得的证据表明，两个Rab 8样GTPases的Dictyosteopathy，被称为Sas 1和Sas 2，可能是细胞粘附，肌动蛋白细胞骨架和发育调节分泌的关键调节因子。这些网骨藻Rab 8样GTP酶与人Rab 8具有75%的序列同一性。表达组成型活性版本的Sas 1（Sas 1CA）的突变细胞系表现出许多肌动蛋白丰富的膜突起，无法聚集（在网骨藻发展的第一步）。在其他细胞中，WASP相关蛋白如SCAR调节膜延伸的形成。由于突变细胞系表现出许多膜突起，因此假设活性Sas 1与包括WASP相关蛋白（如SCAR）在内的信号通路进行通信。为了测试这一点，活化的Sas 1在其中SCAR基因被破坏（SCAR-null）的网骨藻细胞系中表达。有趣的是，发育缺陷在双突变体中被逆转，表明这是通过SCAR的异常信号传导的结果。Western blot和real-time RT-PCR结果显示，在表达激活的Sas 1的细胞中，SCAR蛋白和信使的表达水平均升高。这提供了Rab 8样GTsp和WASp家族蛋白之间功能性相互作用的任何系统中的第一个遗传证据，并证明活化Rab 8样蛋白的表达足以诱导SCAR蛋白水平的增加。这也为Rab 8样GTP酶如何影响肌动蛋白细胞骨架提供了一个可能的解释。在这个项目中，我们将使用这个模型细胞系来进一步了解这些独特的Rab 8相关的GTP酶。为了进一步阐明Sas 1与SCAR的通讯机制，本项目将有两个目标：（i）通过检测SCAR及其下游信号分子Rac和Arp 2/3的激活状态，进一步阐明Sas 1与SCAR的通讯机制;（ii）鉴定和表征Sas 1（Rab 8a）相互作用蛋白以及与Sas 1阳性囊泡相关的蛋白。这些研究的智力价值在于，它们将为Rab 8样GTP酶的功能提供重要的见解，从而增加关于肌动蛋白细胞骨架调节的知识。这些研究还将提供丰富的突变体来源，作为本科教学的工具，以证明囊泡运输，肌动蛋白细胞骨架和发育之间的关系，从而将有更广泛的影响。主要研究者（PI）预计将允许本科生在细胞生物学实验室表征这些突变体作为课堂项目的一部分。PI致力于研究生和本科生水平的科学教育，并强烈认为为本科生提供研究经验是促进科学研究和教育专业的绝佳方式。

The evolution of multicellular organisms has permitted the formation of specialized cells and tissues. Key steps in the evolution of multicellularity were the acquisition of the ability of cells to interact with other cells and to carry out developmentally regulated secretion of extracellular matrix (ECM) proteins. Various cell membrane-associated proteins, collectively termed cell adhesion molecules (CAMs), enable many plant and animal cells to adhere tightly to one another. CAMs interact extracellularly with other CAMs on neighboring cells and with molecules in the ECM, and intracellularly with the actin cytoskeleton. Proteins secreted during development also play an important role in signal transduction during tissue morphogenesis. Much is known about the types of proteins that become part of adhesions or part of the ECM; however, less is known about how these molecules are delivered to the cell surface. Presumably the movement of these proteins relies on vesicle trafficking. The Ras-related Rab GTPases are a superfamily of low molecular weight GTPases, proposed to be master controllers of vesicle trafficking. One approach to gaining insight into the mechanisms by which CAMs and secreted molecule. are trafficked during development is to explore the evolutionary origins of these processes and to examine the features that non-metazoan systems and animals share. Dictyostelium discoideum is a promising organism for such studies because of the ease with which it can be manipulated genetically and because it possesses a simple developmental cycle. Dr. Temesvari's laboratory has acquired evidence that suggests that two Rab8-like GTPases of Dictyostelium, known as Sas1 and Sas2, may be key regulators of cell adhesion, the actin cytoskeleton and developmentally regulated secretion. These Dictyostelium Rab8-like GTPases share 75% sequence identity with human Rab8. A mutant cell line expressing a constitutively active version of Sas1 (Sas1CA) exhibited numerous actin-rich membrane protrusions and was unable to aggregate (the first step in Dictyostelium development). In other cells, WASp-related proteins such as SCAR regulate the formation of membrane extensions. Because the mutant cell line exhibited numerous membrane protrusions, it was hypothesized that active Sas1 was communicating with signaling pathways that included WASp-related proteins such as SCAR. To test this, activated Sas1 was expressed in a Dictyostelium cell line in which the SCAR gene was disrupted (SCAR-null). Interestingly, the developmental defect was reversed in the double mutant suggesting that it was a result of aberrant signaling through SCAR. Western blot and real-time RT-PCR indicated that the levels of both SCAR protein and message were increased in cells expressing activated Sas1. This provides the first genetic evidence in any system of a functional interaction between a Rab8-like GTPase and WASp family proteins and demonstrates that expression of an activated Rab8-like protein is sufficient to induce increases in the level of SCAR protein. It also provides a possible explanation of how Rab8-like GTPases may influence the actin cytoskeleton. In this project, we will use this model cell line to gain further insight into these unique Rab8-related GTPases. In order to further discern the mechanism by which Sas1 communicates with SCAR this project will have two objectives: (i) to further discern the mechanism by which Sas1 communicates with SCAR by examining the activation status of SCAR and putative downstream signaling molecules including Rac and Arp2/3, and (ii) to identify and characterize Sas1(Rab8a)-interacting proteins as well as proteinsassociated with Sas1-positive vesicles. The INTELLECTUAL MERIT of the studies lies in the fact that they will provide significant insight into the function of Rab8- like GTPases, and thus increase knowledge regarding actin cytoskeletal regulation. These studies will also provide a rich source of mutants to use as tools in undergraduate teaching to demonstrate the relationship between vesicle trafficking, the actin cytoskeleton, and development and thus will have BROADER IMPACTS. The Principal Investigator (PI) anticipates allowing undergraduate students in a cell biology lab to characterize these mutants as part of a class project. The PI is committed to science education at the graduate and undergraduate levels and feels strongly that providing research experiences for undergraduates is an excellent way to promote professions in scientific research and education.