The Drosophila genes cappuccino and spireinteract with Rho family GTPases to regulate the cytoskeleton during oogenesis
AuthorJames, Brian Patrick
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PublisherThe University of Arizona.
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AbstractThe genes cappuccino (capu) and spire (spir) are required for establishment of the anterior/posterior (A/P) and dorsal/ventral (D/V) axes during oogenesis in Drosophila melanogaster. In the oocytes of capu and spir mutant females, axis-defining determinants are either mislocalized or not localized at all. Mounting evidence suggests that this localization defect is due to misregulation of the microtubule and actin cytoskeletons, and by extension, suggests that the wild type function of capu and spir is to regulate the cytoskeleton during oogenesis. In support of this hypothesis, previous data from two-hybrid experiments have suggested that SPIR binds to actin through its two WH2 domains. Here I show the interaction between SPIR and ACTIN is direct using in vitro binding assays. Both genetic and yeast two-hybrid evidence suggested that CAPU and SPIR also interact with Rho family GTPases, which include RHOA, RHOL, CDC42 and RAC1. GST pull-down experiments, performed to test the directness of these interactions, revealed that CAPU and SPIR both bind strongly to RHOA and weakly to CDC42. SPIR also binds strongly to RHOL and weakly to RAC1. I also present here the first evidence for capu function outside of oogenesis and for three splice variants of capu. Searches of the Drosophila genome database reveal that two splice variants of capu are expressed in the adult head, and in situ hybridization results reveal that capu message is expressed in the developing larval brain. Additionally, phenotypes in the adult wing are described. One representative of each of two classes of EST from the database that appeared to define two novel classes of capu splice forms are sequenced and compared to the existing capu splice form. Taken together, the data described here help demonstrate that capu and spir play a role in early axis determination in Drosophila, and in regulation of the cytoskeleton. It remains to be determined if capu and spir act to regulate the actin cytoskeleton which in turn regulates the microtubule cytoskeleton, or if these genes directly regulate the microtubule cytoskeleton.
Degree ProgramGraduate College
Molecular and Cellular Biology