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The parallel actin filament bundling protein Fascin is a critical protein in both disease and development. Overexpression of Fascin is linked to increased aggressiveness in a number of cancer types, including breast and colon carcinomas. Importantly, Fascin is not normally expressed in adult epithelial cells from which many of these cancers arise. Therefore, Fascin is increasingly cited as both a potential biomarker and therapeutic target in many types of cancer. Fascin is most commonly associated with the formation of filopodia and invadapodia (parallel actin filament bundle structures) to drive migration and invasion. However, Fascin activity and regulation remain poorly understood. In order for Fascin to be an effective target for cancer therapeutics, a better understanding of the mechanisms regulating Fascin activities in the cell is necessary. Prostaglandins (PGs) are short-lived lipid signaling molecules that mediate a wide range of biological activities. PGs act through G protein-coupled receptors to initiate signaling cascades that affect downstream targets, including actin cytoskeletal remodeling. Importantly, the key enzymes in the synthesis of PGs, cyclooxygenase (COX) 1 and 2, are the targets of non-steroidal anti-inflammatory drugs like aspirin. Interestingly, like Fascin, PGs have been independently implicated in cancer development and metastasis and aspirin may reduce the risk of aggressive cancer. However, the exact mechanisms by which PGs mediate cancer development are unknown. The work presented in this thesis focused on novel PG-dependent regulation and activity of Fascin. The research presented here utilized Drosophila oogenesis as a model system to analyze PG-dependent Fascin activity. Drosophila oogenesis is an ideal model in which to study the activity and regulation of actin binding proteins like Fascin. Oogenesis consists of 14 morphologically defined stages, which are observable many times over within a single isolated pair of ovaries. A developing follicle consists of 16 germline cells – 15 nurse, or support cells, and a single oocyte. The nurse cells are of particular interest because they are the sites of dynamic actin remodeling during mid-late oogenesis. During stage 10B, an array of radially-aligned actin filament bundles form at the nurse cell membranes and extend inwards towards the nucleus. A network of cortical actin is also strengthened during this stage. These actin structures are essential for the completion of oogenesis, and ultimately female fertility. Importantly, PGs and Fascin are required for this actin remodeling; genetic loss of Fascin or the Drosophila COX-like enzyme Pxt (Peroxinectin-like) leads to disruption of cytoplasmic actin remodeling, and ultimately, female sterility. Using this model system, work presented here describes the discovery of Fascin as a downstream target of PGs to promote actin bundle formation, described in Chapter 2. Additionally, Fascin is required for strengthening of the cortical actin network downstream of PGs. This observation is one of the first to describe a role for Fascin in a branched actin network. Additionally, Fascin is regulated by a specific PG – PGF2α – during S10B to promote follicle development. Finally, Chapter 2 shows that PGs target specific actin binding proteins to promote cytoskeletal remodeling; Villin, another actin bundling protein, does not interact with PGs. Chapter 3 describes the novel observation that Fascin localizes to the nucleus and the nuclear periphery in Drosophila nurse cells. This finding is significant, as it is the first to describe Fascin in a context other than cytoplasmic. Fascin localization in and around the nucleus is specific and dynamic, and changes throughout late stage oogenesis, suggesting regulated functions at these sites. Fascin localization is regulated by PGs, and loss of Pxt leads to reduced nuclear Fascin localization and failure to localize to the nuclear periphery. Additionally, Fascin has novel potential functions in the nucleus and at the nuclear periphery. Loss of Fascin leads to disruption of nucleolar morphology in the nurse cell nuclei. Additionally, loss of PGs, which cause reduced nuclear Fascin levels, also causes abnormal nucleolar morphology. These data suggest that PGs regulate Fascin to control nucleolar organization. At the nuclear periphery, Fascin localization requires components of the protein complex that links the nucleoplasm to the cytoplasm, termed the LINC complex. Loss of an essential LINC complex protein, Koi, leads to a loss of nuclear periphery Fascin localization. These data suggest that Fascin may be a novel component of the LINC complex. Finally, Chapter 4 describes regulation of Fascin by phosphorylation at conserved serine residues. PGs affect Fascin phosphorylation, and loss of PGs leads to more heavily phosphorylated Fascin. Additionally, phosphorylation of Fascin alters localization to the nucleus and to the nuclear periphery. These data suggest that one mechanism by which PGs regulate Fascin is to control its phosphorylation status to affect subcellular distribution. In summary, the work presented in this thesis has demonstrated novel regulation and function of the actin bundling protein Fascin using Drosophila oogenesis as a model. Importantly, these functions and regulation of Fascin are likely conserved in mammals, and may have implications in human health and disease. Continued study of the activity and regulation of actin binding proteins like Fascin in Drosophila will likely have great effect on our understanding of many human diseases.

While actin was discovered in the nucleus over 50 years ago, research lagged for decades due to strong skepticism. The revitalization of research into nuclear actin occurred after it was found that cellular stresses both induce the nuclear localization and alter the structure of nuclear actin. These studies provided the first hints that actin has a nuclear function. Subsequently, it was established that the nuclear import and export of actin is highly regulated. While the structures of nuclear actin remain unclear, it can function as monomers, polymers, and even rods. Furthermore, even within a given structure, distinct pools of nuclear actin that can be differentially labeled have been identified. Numerous mechanistic studies have uncovered an array of functions for nuclear actin. It regulates the activity of RNA polymerases, as well as specific transcription factors. Actin also modulates the activity of several chromatin remodeling complexes and histone deacetylases, to ultimately impinge on transcriptional programing and DNA damage repair. Further, nuclear actin mediates chromatin movement and organization. It has roles in meiosis and mitosis, and these functions may be functionally conserved from ancient bacterial actin homologs. The structure and integrity of the nuclear envelope and sub-nuclear compartments are also regulated by nuclear actin. Furthermore, nuclear actin contributes to human diseases like cancer, neurodegeneration, and myopathies. The work presented in this thesis aims to describe the nuclear localization and functions of actin during Drosophila oogenesis. Drosophila oogenesis, i.e. follicle development, provides a developmental system with which to study nuclear actin. Follicles are composed of roughly 1000 somatic follicle cells and 16 germline cells, including 15 nurse or support cells and a single oocyte. Follicles progress through a series of 14 morphological stages, from the germanium to Stage 14 (S14). Ovary staining using the anti-actin C4 antibody reveals one pool of nuclear actin during early oogenesis (germarium through S9), including in the germline and somatic stem cells, a subset of mitotic follicles cells, and a subset of nurse cells during S5-S9. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged actin results in nuclear actin rod formation. These findings indicate that nuclear actin is tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of C4 nuclear actin, but does not alter the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate C4 nuclear actin. Evidence indicates that Fascin positively regulates C4 nuclear actin through Cofilin. Indeed, loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high C4 nuclear actin levels. Thus, through Cofilin, Fascin positively regulates C4 nuclear actin. These studies identified Fascin as a novel means of nuclear actin regulation. Having established Drosophila oogenesis as an in vivo, developmental system to study nuclear actin, I sought to identify the functions of nuclear actin. To uncover the functions of nuclear actin, I manipulate nuclear actin levels by blocking its nuclear import (Importin 9) and export (Exportin 6). Knockdown of Importin 9, results in female sterility and defects within the germarium, supporting a role for nuclear actin in stemness. Additionally, reduced Importin 9 levels cause chromatin organization defects. Loss or knockdown of Exportin 6 causes reduced female fertility, abnormal nucleolar morphology, alterations in the nuclear envelope, and aberrant heterochromatin status. These data suggest several functions for nuclear actin in the ovary: nuclear actin is essential for stem cell differentiation, proper chromatin organization and dispersal, nucleolar structure and likely function, nuclear envelope morphology, heterochromatin status and likely gene expression. Ultimately, nuclear actin is absolutely required for the highly conserved process of follicle development. These studies provide insight into the regulation and function of nuclear actin in Drosophila oogenesis. The data presented here indicate that nuclear actin is critical for chromatin organization, nucleolar morphology, nuclear envelope shape, and heterochromatin status and suggest that nuclear actin ultimately impacts transcription, a process essential for all cells. Considering the high level of sequence and functional conservation of actin, studies in Drosophila oogenesis will provide insight into the conserved functions of nuclear actin in follicle development across higher organisms. The study of nuclear actin in the many cell types of the Drosophila ovary provide insight into the functions of nuclear actin for all cell types across evolution. Further, aberrant nuclear actin regulation has been implicated in several disease states. The studies in Drosophila provide insight into the regulation of nuclear actin and how misregulation contributes to disease states. Together, the data presented in this thesis advance our understanding of the nuclear localization and functions of actin.