Prostaglandin signaling temporally regulates actin cytoskeletal remodeling during Drosophila oogenesis

• Main Author: Spracklen, Andrew James
• Other Authors: Tootle, Tina L.
• Format: Others
• Language: English
• Published: University of Iowa 2014
• Subjects: actin; cytoskeleton; Drosophila; Enabled; oogenesis; prostaglandins; Cell Biology
• Online Access: https://ir.uiowa.edu/etd/1399; https://ir.uiowa.edu/cgi/viewcontent.cgi?article=5438&context=etd

Prostaglandins (PGs) are small, lipid signaling molecules produced downstream of cyclooxygenase (COX) enzymes. PG signaling regulates many processes including pain, inflammation, fertility, cardiovascular function and disease, and cancer. One mechanism by which PG signaling exerts its function is by regulating the dynamics of the actin cytoskeleton; however, the exact mechanisms remain largely undefined. Drosophila oogenesis provides an ideal system to determine how PG signaling regulates the actin cytoskeleton. Drosophila follicles, or eggs, pass through 14 well- characterized, morphologically defined stages of development. Each developing follicle is comprised of 16 interconnected germline-derived cells (15 nurse cells and 1 oocyte) that are surrounded by a layer of somatically derived epithelial cells. During Stage 10B (S10B), the nurse cells form a cage-like network of parallel actin filament bundles that extend from the nurse cell membranes inward, toward the nurse cell nuclei. During Stage 11 (S11), the nurse cells rapidly transfer their cytoplasmic contents into the oocyte in an actomysoin-dependent contraction termed nurse cell dumping. Previous work uncovered that the Drosophila COX-like enzyme, Peroxinectin-like (Pxt), and thus PG signaling, is required to promote both actin filament bundle formation during S10B and subsequent nurse cell dumping. This finding established Drosophila oogenesis as a genetically tractable model in which to elucidate the conserved mechanisms underlying PG- dependent actin remodeling. The research presented in this dissertation focused on identifying actin-binding proteins that are regulated by PG signaling during Drosophila oogenesis. To identify these downstream effectors, we performed a dominant modifier screen to uncover factors that could suppress or enhance the ability of COX inhibitors to block nurse cell dumping in vitro. This screen revealed a number of actin-binding proteins that enhance the dumping defects caused by COX-inhibition, including the actin bundling protein, Fascin (Drosophila Singed, Sn); the actin filament elongation factor, Enabled (Ena); and the actin filament capper, Capping protein (Drosophila Capping protein alpha, Cpa, and beta, Cpb). Through a collaborative effort between Christopher Groen and myself, Fascin was shown to mediate PG-dependent cortical actin integrity and actin bundle formation during Drosophila ooogenesis. Ena and Capping protein regulate actin filament elongation through opposing actions: Ena promotes their elongation, while Capping protein binds to, or caps, the growing end of actin filaments to prevent their further elongation. However, genetic reduction of either Ena or Capping protein enhance the nurse cell dumping defects caused by COX inhibition. These findings suggest that Ena activity must be balanced to promote proper actin remodeling during S10B. Ena localization to the growing ends of actin filament bundles is reduced in pxt mutants during S10B, suggesting that PG signaling is required to promote Ena localization at this stage. Together, these data support a model in which PG signaling promotes actin remodeling during S10B, at least in part, by modulating Ena-dependent actin remodeling. While PG signaling promotes parallel actin filament bundle formation during S10B, PGs also restrict actin remodeling during Stage 9 (S9). Loss of Pxt results in early actin remodeling, including the formation of extensive actin filaments and actin aggregate structures within the posterior nurse cells of S9 follicles. Wild-type follicles exhibit similar structures at a low frequency. Ena preferentially localizes to the early actin structures observed in pxt mutants and reduced Ena levels strongly suppress early actin remodeling in pxt mutants. These data indicate that PG signaling temporally restricts actin remodeling during Drosophila oogenesis, at least in part, through negative regulation of Ena localization or activity during S9. The data presented here support a model in which PG signaling coordinates the concerted activity of a number of actin-binding proteins to regulate actin remodeling during Drosophila oogenesis. Specifically, PG signaling temporally restricts actin remodeling during S9 of Drosophila oogenesis, but promotes parallel actin filament bundle formation during S10B. PG signaling achieves this temporal regulation, at least in part, through differential regulation of Ena-dependent actin remodeling. Based on prior pharmacologic studies, we hypothesize that PGE2 is required to restrict Ena-dependent actin remodeling during S9, while PGF2Α; is required to promote Ena-dependent actin remodeling during S10B. Determining how these signaling cascades achieve differential regulation of Ena throughout Drosophila oogenesis is an important area for future investigation. As both the actin cytoskeletal machinery and PG signaling are conserved across species, the data presented here provide new and significant insights into the likely conserved mechanisms by which PG signaling regulates actin remodeling across species.