Regulation of cell migration during dorsal appendage morphogenesis. Sandra G. Zimmerman, Celeste A. Berg. Department of Gemone Sciences, University of Washington, Seattle, WA.

   Cell motility is critical for normal development and homeostasis. Abnormalities in these processes can produce birth defects or drive cancer cell metastasis. An excellent model for studying the regulation of cell migration is dorsal appendage (DA) morphogenesis in the ovary of Drosophila melanogaster. The DAs form from two patches of follicle cells that lie dorsal to the oocyte; these cells reorganize into tubes and elongate by crawling over the squamous stretch follicle cells, which lie over the nurse cells. Mutations in the transcription factor Bullwinkle (BWK) lead to cell adhesion defects and aberrant cell migration, resulting in broad, moose-antler-like DAs. BWK, which functions in the nurse cells, acts upstream of tyrosine kinases SHARK and SRC42A in the overlying somatic stretch cells to regulate DA cell migration. Interestingly, shark RNA localizes in the stretch cells in patches over the nurse cell nuclei, perhaps to localize SHARK translation. This shark mRNA distribution, which is absent in bwk egg chambers, may localize SHARK protein, possibly to facilitate phosphorylation activity. A major unanswered question is: what are the other components of this pathway? To identify new factors that regulate cell migration through their function in the BWK-SHARK-SRC42A pathway, we used liquid chromatography coupled with tandem mass spectrometry and label-free quantitation to compare protein expression and phosphorylation in stretch cells from wild-type vs. bwk egg chambers. To purify stretch cells, we adapted a published protocol for magnetic bead cell separation for a new use with mass spectrometry. We identified >100 proteins with at least a 2-fold difference in relative abundance between wild-type and bwk egg chambers. We selected a small subset of the most interesting of these candidate proteins for in vivo functional analysis using RNAi, protein and RNA expression analysis, and clonal analysis. Characterization of these newly identified factors will delineate their function in the BWK-SHARK-SRC42A pathway and advance our understanding of the regulation of cell migration.