Functional Analyses of Endophilin B Isoforms in Drosophila Brain and Ovary

The Endophilin proteins, a highly conserved family, are composed of an N-terminal N-BAR domain and a C-terminal SH3 domain. Two subgroups in the family: Endophilin A cooperates with dynamin through its SH3 domain and participates in clathrin-coated endocytosis. Endophilin B is reported to involve in the process of membrane curvature. In the FlyBase data base two Endophilin B protein isoforms (PA and PB isoforms) are encoded by one Drosophila Endophilin B gene through alternative splicing. We found that the isoform PA is enriched in the fly brain, whereas the isoform PB is mainly expressed in the ovary. Our previous study (manuscript in preparation, and please see the preliminary results) has demonstrated that isoform PB plays a crucial role in the vitellogenesis during Drosophila oogenesis, and in endophilin B null mutant the yolk content and egg production are reduced. Endophilin B-PB is localized at the oocyte posterior pole, which depends on Oskar L form. The precise localization of Endophilin B-PB at ultra-structural level was examined under Electronic Microscope, D-Endophilin B-PB was mainly localized at the vesicular and tubular structures and was partially co-localized with the yolk receptor, Yolkless. Furthermore, in immuno-gold labeling data Endophilin B-PB was partially co-localized with Oskar, but not Vasa. Consistently, D-endophilin B does not affect the pole plasm assembling but is required for maintaining the endocytic activity at the oocyte posterior which was demonstrated by FM4-64 incorporation assay. Reduction of yolk content and egg production defects were fully rescued by Endophilin B isoform PB expressing in the germ cells. Taken together, these data suggest that D-Endophilin B-PB plays a role in regulating fertility through yolk uptake by Oskar mediated endocytosis. In addition, we observed defects of germ line stem cell (GSC) maintenance in the ovary of endophilin B null mutant female. Therefore, we intend to find out how endophilin B plays a role in GSC function. We would like to determine which isoform is responsible for the GSC defect. If PA in the brain can affect GSC in the ovary, one possible mechanism is through regulation of Drosophila insulin like peptide (DILP) activity which is required for GSC maintenance and differentiation. The release of neuropeptides, such as DILP, is thought to be regulated by exocytosis/endocytosis events in the brain, which is similar to the mechanism controlling the release of neurotransmitters. However, it is possible that other neuropeptides rather than insulin in the brain is controlled by Endophilin B-PA and affect GSC in the ovary. If this is the case, we will try to identify them and find their responding signal pathways in the ovary. If Endophilin B-PB is responsible for the GSC defect, the mechanism by which Endophilin B-PB regulates GSC maintenance will be investigated in details.

Project ID:PA10101-1608
External Project ID:NSC100-2311-B182-001-MY3