Thesis presented October 17, 2001
Abstract: The deletion of the rotund (rn) locus of
Drosophila melanogaster induces a short adult appendage phenotype and male sterility. The molecular characterization of the
rn region indicates that it encodes two major transcription units: the
rn unit that has been implicated in appendage morphogenesis (S. Saint Pierre, personal communication), and the
rnRacGAP transcription unit that is essential during spermatogenesis (Bergeret
et al., 2001). We showed using molecular biological analyses and fertility tests, that
rn mutants having an intact and functional
rnRacGAP transcription unit, presented partial fertility. Moreover, microscopic analysis of the reproductive apparatus of these mutants indicated that they are capable of producing motile spermatozoa, stocked in seminal vesicles. By transgenic experiments and inducing multiple recombination events, we showed that the fertility of these mutants is sensitive to rnRacGAP dosage and the genetic background. The protein product RotundRacGAP (RnRacGAP) shows high sequence similarity with the human MgcRacGAP (Male Germ Cell RacGAP) protein, which is a GAP (GTPase Activating Protein) known to have a specific activity
in vitro, toward the Rho-GTPases Rac and Cdc42. Therefore, in a second step, we undertook biochemical analyses to test whether RnRacGAP showed GAP activity toward Rho-GTPases. We showed that the Rho-GTPases Rac and Cdc42 are the specific substrates of RnRacGAP,
in vitro. Moreover, the loss of a dose of
rnRacGAP enhances the phenotype induced by over-expression of Rac and Cdc42 in the eye, indicating that RnRacGAP negatively regulates these two targets,
in vivo. We have also used, as a model system, dorsal closure during embryogenesis, which is known to be controlled by the Rho-GTPases, to dissect the effects of the over-expression of RnRacGAP on Rho-GTPase-dependent cell signaling. We showed that the expression of RnRacGAP in the embryo affects actin cytoskeleton organisation and, under certain conditions regulates gene expression in the Rac/Jun
N-terminal kinase signalization pathway. Finally, to define connections between RnRacGAP and other cell signaling pathways and the molecular mechanisms of their interactions, we have performed a misexpression screen and identified new genetic partners of RnRacGAP.
Keywords:Drosophila melanogaster, genetic, signalization, GTPase activating protein, Rac, Rho, Cdc42, cytoskeleton
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