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

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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.