The Tootle Lab’s long-term objective is to understand how particular prostaglandin signaling events result in specific biological outcomes. Prostaglandins are locally acting, transient hormones that mediate a wide variety of biological activities, from female reproduction to cancer development and progression. Prostaglandins are synthesized at their sites of action by cyclooxygenase (COX) enzymes, the targets of Aspirin and Advil. As prostaglandin signaling is transient and locally acting, to determine the molecular mechanism of prostaglandin action a cell-cell signaling model is needed. Drosophila is an excellent system to use, as Drosophila genetics has been routinely employed to identify and characterize signaling cascades at single cell resolutions.
Previously, we developed Drosophila oogenesis as a new and powerful model for studying prostaglandin signaling. Using both pharmacology and genetics, we discovered that prostaglandins mediate Drosophila follicle development, identified the Drosophila COX1 enzyme, Pxt, and revealed that genetic perturbation of prostaglandin signaling can be used to exam the function of prostaglandins. This research reveals that prostaglandin signaling modulates actin/membrane dynamics, cell migration, stem cell activity, and the timing of gene expression during Drosophila follicle development.
Prostaglandin signaling regulates the actin cytoskeleton
Prostaglandin signaling regulates actin dynamics during follicle development. Both pharmacologic and genetic studies reveal that prostaglandin signaling is required for nurse cell dumping, a process in which the germline derived nurse cells push all of their cytoplasmic contents into the oocyte. In mutants with no prostaglandin signaling the actin structures required for nurse cell dumping are substantially reduced and often completely eliminated. By using a multifaceted experimental approach that combines Drosophila genetics, cell biology, live imaging, and biochemistry we are determining where the prostaglandin signal is coming from, which prostaglandins are involved, whether this is by the canonical signaling pathway, how prostaglandin signaling interacts with known actin regulators, and the downstream changes in gene expression during nurse cell dumping. The results from these studies are likely to provide general insight into how prostaglandins regulate the cytoskeleton at a cellular level. Such mechanisms of prostaglandin action are likely to be reutilized throughout development, including mediating the cytoskeletal changes that occur during cancer progression and metastasis.
Through these studies the lab has identified a number of actin binding proteins as new downstream effectors of prostaglandin signaling. We identified Enabled as an effector of prostaglandins. Our data suggests that prostaglandins inhibit Enabled early in oogenesis to prevent early and aberrant actin filament and aggregate formation. Later, prostaglandins promote Enabled activity to drive bundle formation necessary for nurse cell dumping. We also found that Fascin, an actin bundling protein, is required downstream of prostaglandins to regulate bundle formation necessary for nurse cell dumping and to maintain cortical actin integrity. Importantly, this work provided the first link between prostaglandins and Fascin. Importantly, prostaglandins and the mammalian homologs of Fascin and Enabled (i.e. Mena) are known to play critical roles in cancer development and metastasis. Thus, the same mechanisms by which prostaglandins regulate Drosophila Fascin and Enabled are likely conserved in the instance of cancer. Current efforts are focused on uncover the mechanisms by which prostaglandins regulate these and other actin binding proteins.
In our efforts to determine how prostaglandins regulate Fascin we made the surprising discovery that Fascin is not solely cytoplasmic, but localized to the outside of the nuclear envelope and inside the nucleus. The localization of Fascin to these new cellular sites is conserved from flies to humans. As Fascin is a critical contributor to cancer, understanding these novel Fascin activities is critically important. At the nuclear periphery, Fascin interacts with the Linker of the Nucleoskeleton and Cytoskeleton (LINC) Complex to mediate force transmission to the nucleus. This activity is critical for invasive migration. Inside the nucleus, Fascin regulates nucleolar structure and as discussed below, nuclear actin. Fascin’s localization to both the nuclear periphery and the nucleus, and its nucleolar activity are regulated by prostaglandin signaling.
Recent collaborative work with the Welte Lab at the University of Rochester, has been exploring the relationship between prostaglandin signaling and lipid droplets. We found that the lipid droplet-associated Adipose Triglyceride Lipase (ATGL) cleaves triglycerides to release the substrate for prostaglandin production, initiating the prostaglandin signaling that drives the actin remodeling necessary for follicle morphogenesis. Similar mechanisms have been observed in a few human cell types, suggesting this is a more common mechanism of activating prostaglandin signaling than previously appreciated, and may contribute to the increased inflammation observed in metabolic syndromes. Current efforts are focused on defining the roles of other lipid droplet-associated proteins in prostaglandin-dependent actin remodeling.
Invasive, collective cell migration
Border cell migration is an excellent model of collective, invasive cell migration. This type of migration is widely observed during development and also mediates cancer metastasis. During Stage 9 of Drosophila oogenesis, 8-10 border cells delaminate from the follicular epithelium and migrate, invasively between the much larger nurse cells to the nurse cell-oocyte boundary. Thus the nurse cells are the substrate for and microenvironment of border cell migration.
Our recent work uncovered that two prostaglandins are required for on-time border cell migration. PGF2a is produced by the synthase Akr1B in the border cells, whereas PGE2 is produced by the nurse cells, the substrate on which the border cells migrate, by the synthase cPGES to promote on-time migration. These prostaglandins act by similar and distinct mechanisms – PGF2a promotes integrin-based adhesions and myosin activity within the border cells, and PGE2 drives myosin activation in both the substrate (nurse cells) and the border cells. Current efforts are focused on defining the signaling pathways by which these prostaglandins act.
Other current cell migration projects include: 1) Defining the nucleoskeletal changes needed for border cell migration, and the roles of prostaglanding signaling in controlling these changes; 2) Uncovering the roles of lipid droplets and their associated proteins in border cell migration; and 3) Dissecting the roles of extracellular matrix components in border cell migration.
Nuclear actin and actin binding proteins
Actin is not solely cytoplasmic but localizes and functions in the nucleus. The nuclear localization of actin is highly regulated. In the cytoplasm actin bind to Cofilin, and this complex is recognized by Importin 9 and translocated to the nucleus. Additional nuclear import factors also promote the nuclear localization of actin. Inside the nucleus, actin-Profilin complexes are recognized by Exportin 6 and taken out of the nucleus. In the nucleus, actin has been implicated in regulating transcription, DNA damage repair, and nuclear structure. However, much remains to be learned including what are the structures of actin in the nucleus, how does the structure impact its function, how is nuclear actin regulated, and what is it doing during development.
We have identified multiple reagents that allow us to examine nuclear actin during Drosophila oogenesis. Through characterization of the subnuclear localization, cell-type specificity, and developmental timing of these different actin pools we are developing testable hypotheses for the functions of nuclear actin during follicle development. Our recent work uncovered that prostaglandins limit nuclear actin levels and control is form to restrict nucleolar activity. Current efforts are focused on the role of Fascin in modulating nuclear actin and nucleolar function.
