CHARACTERIZATION OF THE CASK PROTEIN IN DROSOPHILA OVARIES

Abstract

CASK (Ca2+/Calmodulin Serine Threonine Kinase) is a member of the MAGUK family of proteins. The protein contains the canonical MAGUK domains (PDZ domain, the GK domain and the SH3 domain) as well as an N‐terminal CamK‐like domain. CASK and other MAGUK family proteins, including Dlg, have been shown to function in the establishment and maintenance of cell polarity in a variety of cell types. Interestingly, a recent screen has identified CASK as regulator of border cell migration, a model of collective cell migration that occurs during Drosophila oogenesis. Border cell migration occurs during Stage 9 of Drosophila egg development and is a process by which two non‐motile polar cells located at the anterior of the egg chamber signal 4–8 adjacent follicular epithelial cells to transform into motile border cells. Once signaled, the border cells will transport the polar cells to the anterior end of the oocyte within the egg chamber where they will form the micropyle. The movement of this border cell cluster to the anterior end of the oocyte is completed by stage 10 of Drosophila ovary development. Despite the putative role for CASK in border cell migration, very little work has been done to characterize CASK's function in the Drosophila ovaries. The current study aims to begin to characterize CASK's basic gene expression in Drosophila ovaries and optimize a protocol for immunohistochemistry such that border cell migration assays can be conducted on dissected stage 10 egg chambers. RT‐PCR analysis was conducted with primers specific to each of the seven CASK isoforms known to be present in Drosophila (CASK‐A, ‐B,‐D, ‐E, ‐F, ‐G, ‐H). Thus far expression of the CASK‐A, CASK‐B, CASK‐H and CASK‐E isoforms have been confirmed in Drosophila ovaries. Studies were also conducted to determine the optimal time in which the flies should be fed yeast to yield the highest percentage of stage 10 ovaries for subsequent border cell migration assays. Feeding flies a wet yeast paste is known to provide a nutritive enhancement for female oogenesis. Yeast time trials were conducted in which 3 lines of wild‐type flies and the p18 CASK knockout fly line were allowed to feed on yeast for 12, 13 or 14 hours. Results indicated that the 13‐hour yeasting time yielded the highest number of stage 10 ovaries. This time was consistent across genotypes. Future work will continue to assess CASK gene expression via RT‐PCR with primers specific to the CASK‐D, CASK‐F and CASK‐G isoforms. Defects in border cell migration will also be assessed via immunohistochemical analysis with the singed antibody on Drosophila utilizing the yeast time trial results attained in this study. Characterization of CASK's gene expression and assays of border cell migration in CASK knockout flies will serve to elucidate the molecular mechanisms by which CASK contributes to group cell movement. Furthermore, this work will also contribute to a greater understanding of the types of proteins required for mediating cell migration during biological processes such as wound healing, inflammation and tumor cell metastasis.