Reported here is the isolation and molecular characterization of two novel alleles of the DIP 1 gene; GE89 and GE77. As well, a third deletion of the DIP 1 gene, EY*4, isolated by our collaborators in France was characterized. PCR and sequencing analysis confirms all three alleles to be molecular deletions of the DIP 1 gene. However, in none of these cases is the entire gene excised. Also, immunohistochemistry of ovaries from each of these strains does not demonstrate a complete lack of DIPl protein expression in any of the deletion strains. Thus, it appears that some protein product is being formed in each case. However, it is not clear whether this protein is functional. An assay was also conducted to investigate a function for DIPl in mechanisms of epigenetic gene silencing. Although the findings of these experiments are incomplete, it appears that DIPl may play a functional role in heterochromatin formation and/or post-transcriptional gene silencing. Interestingly, appendage formation phenotypes were observed in the original P-element insertion line as well as a female sterility phenotype in the GE77 allele. Overall, DIP 1 is an interesting double stranded RNA binding protein. Newly isolated alleles of the DIP 1 gene will be useful tools for further investigation of the functional role of this gene. / Thesis / Master of Science (MSc)

Assembly of branched actin filaments produces dynamic structures required during membrane associated processes including cell motility and endocytosis. The Actin Related Protein 2/3 (Arp2/3) complex is the only known regulator capable of nucleating actin branches. To specify the sub cellular localization and timing of actin assembly the complex is tightly regulated. Canonical activation of the Arp2/3 complex by Wiskott-Aldrich Syndrome proteins (WASP), requires preformed actin filaments, ensuring the complex nucleates new actin filaments off the sides of preformed filaments. WASP proteins can therefore propagate branch formation but cannot initiate a Y-branch without performed filaments. A key question, then, is what is the source of preformed filaments that seed branched actin network formation in cells? It is unclear how activation of Arp2/3 by multiple regulators is balanced to specify actin filament architectures that are productive in vivo. In this dissertation, we identified WISH/DIP1/SPIN90 (WDS) family proteins as activators of the Arp2/3 complex that do not require preformed filaments, and evaluated whether WDS proteins seed branching nucleation. In chapter II, we dissected the biochemical properties of WDS proteins and found they activate the Arp2/3 complex using a non-WASP like mechanism. Importantly, we discovered WDS-mediated Arp2/3 activation produces linear, unbranched filaments, and this activity is conversed from yeast to mammals. These observations highlight that WDS proteins have the biochemical capacity to seed actin branches. In chapter III, we observed WDS-generated linear filaments can seed WASP-mediated branching directly using single molecule microscopy with fluorescently labeled Dip1. We find that WDS-mediated nucleation co-opts features of branching nucleation. In chapter IV, we investigated how WDS activity is balanced with WASP. We discovered WDS proteins use a single turnover mechanism to activate Arp2/3 and this is conserved during endocytosis. In contrast, WASP-mediated activation is multi-turnover, highlighting a crucial difference between WDS proteins and WASP. Our observations explain how Arp2/3 may limit linear filament production to initiate networks and favor branches during network propagation. Finally, we use fission yeast to show that increasing Dip1 is sufficient to cause defects in actin assembly and the timing of actin patches at sites of endocytosis.