Molekulare und funktionelle Analyse des Gens rings lost (CG4420) in der Entwicklung von Drosophila melanogaster

Abstract

Oogenesis of Drosophila is an attractive model system to study the arrangement of the actin-cytoskeleton. At the beginning of oogenesis a 16-cell cyst is formed and all cells of these cyst are interconnected via actin-rich cytoplasmic bridges, the so-called ring canals. By immunofluorescence microscopy proteins could be identified that localize with F-actin at the inner ring of the ring canals as well as proteins that are positioned closer to the plasma membrane, the outer ring. Together, these proteins regulate the stability and growth of the ring canals. Also subcortical actin filaments at the plasma membrane of the germ line cells and cytoplasmic actin filaments that are formed from stage 10 of oogenesis were described within oogenesis. In this work, a previously uncharacterized gene was studied and the protein coded by this gene has an influence on the regulation of the actin-cytoskeleton during oogenesis of Drosophila. Germ line clones of this gene show a loss of ring canals in the germ line and therefore we named it rings lost (rngo). The zygotic null mutant of rngo is lethal in pupal stages, which means that rngo is essential for the survival of the fly. This lethality can be rescued completely by transgenes encoding GFP-fusion proteins of Rngo. Besides the loss of ring canals rngo germ line clones show defects in the growth control of the ring canals and a mislocalization of ring canal-associated proteins. We could also observe a degeneration of the plasma membrane of germ line cells and as a consequence of this a migration of nurse cell nuclei into the former oocyte. We could also quite frequently observe an atypical morphology of nurse cell nuclei and the oocyte nucleus. In rngo-mutant egg chambers oogenesis arrests before stage 9-10, but without showing apoptosis. Rngo shows, through its ubiquitin-like (UBQ)- and ubiquitin-associated- (UBA) domain, the typical structure of an extraproteasomal polyubiquitinreceptor. In this study we showed that Rngo, like other extraproteasomal polyubiquitinreceptor, can bind to the proteasome and, mediated through its UBA-domain bind to ubiquitin. Moreover, it was shown by immunofluorescence microscopy that Rngo colocalizes with ubiquitin and the proteasome in ovaries. These results suggest that Rngo is involved in the proteasomal degradation of proteins that are necessary for the maintenance of the actin-cytoskeleton. Among the extraproteasomal polyubiquitinreceptors Rngo takes a special role because the UBQ-UBA-domains are flanking a retroviral aspartate protease domain (RVP). Due to the homodimerization of proteins that have such an RVP-domain, the catalytic center is formed by two aspartates. So far no proteolytic activity of these RVP-domains was described for the human homologues (HDdi1/HDdi2) and the yeast homologue (Ddi1/Vsm1) of Rngo. As already shown for Ddi1/Vsm1, Rngo also forms homodimers mediated by the RVP-domain. The potential catalytic Aspartat257 is not required. However, in the present study overexpression of GFP fusion proteins in which the catalytic Aspartat257 was replaced with an alanine show dominant negative effects, which point to a proteolytic function of the RVP-domain of Rngo. It is likely that dominant-negative effects of overexpression of the D257A-GFP fusion protein, similar to the rngo-germ line clones, lead to a degeneration of the plasma membrane in the germ line cells. In the present study a previously uncharacterized protein was described, which is responsible for the maintenance and arrangement of the actin-cytoskeleton. Furthermore, it was Rngo as the first protein of Ddi1 family for which a catalytic activity of RVP-domain was assigned.