Tina L. Tootle

Drosophila Eggshell Production: Identification of New Genes and Coordination by Pxt

Drosophila ovarian follicles complete development using a spatially and temporally controlled maturation process in which they resume meiosis and secrete a multi-layered, protective eggshell before undergoing arrest and/or ovulation. Microarray analysis revealed more than 150 genes that are expressed in a stage-specific manner during the last 24 hours of follicle development. These include all 30 previously known eggshell genes, as well as 19 new candidate chorion genes and 100 other genes likely to participate in maturation. Mutations in pxt, encoding a putative Drosophila cyclooxygenase, cause many transcripts to begin expression prematurely, and are associated with eggshell defects. Somatic activity of Pxt is required, as RNAi knockdown of pxt in the follicle cells recapitulates both the temporal expression and eggshell defects. One of the temporally regulated genes, cyp18a1, which encodes a cytochromome P450 protein mediating ecdysone turnover, is downregulated in pxt mutant follicles, and cyp18a1 mutation itself alters eggshell gene expression. These studies further define the molecular program of Drosophila follicle maturation and support the idea that it is coordinated by lipid and steroid hormonal signals.

Fascin Regulates Nuclear Movement and Deformation in Migrating Cells

Summary Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. Here, we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion.

The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.

Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools--Utrophin, Lifeact, and F-tractin--for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool within the tissue and cell type of interest in order to identify the tool that represents the best compromise between acceptable labeling and minimal disruption of the phenomenon being observed. In this case, we find that F-tractin, and perhaps Utrophin, when Utrophin expression levels are optimized to label efficiently without causing actin defects, can be used to study F-actin dynamics within the Drosophila nurse cells.

Drosophila Fascin is a novel downstream target of prostaglandin signaling during actin remodeling

Prostaglandins (PGs) regulate the actin cytoskeleton. However, their mechanisms of action are unknown. Use of Drosophila oogenesis—specifically nurse cell dumping—as a model shows that PGs regulate the actin bundler Fascin to control parallel actin filament bundle formation and cortical actin integrity.

Genetic insights into the in vivo functions of prostaglandin signaling

Prostaglandins (PGs) are lipid signals that are produced at their sites of action by cyclooxygenase (COX) enzymes, the targets of non-steroidal anti-inflammatory drugs (NSAIDs), and PG-type specific synthases. Active PGs serve as ligands for G protein-coupled receptors (GPCRs). The functions of PGs have largely been elucidated using pharmacologic, expression-based (synthesis and signaling components), and genetic studies. In this review, we discuss the in vivo roles of PGs in cancer, development, and reproduction that have been characterized using genetic knockout/knockdown and overexpression approaches in mice, zebrafish, and invertebrate model systems, and how pharmacologic inhibition of PG synthesis affects cardiovascular health/disease and cancer incidence and progression.

Prostaglandins regulate nuclear localization of Fascin and its function in nucleolar architecture

Fascin, a conserved actin-bundling protein, is not only cytoplasmic but also localizes to the nucleus and nuclear periphery in both Drosophila and mammalian cell contexts. In Drosophila, prostaglandin signaling regulates this localization. In addition, Fascin plays a critical role in nucleolar architecture in both Drosophila and mammalian cells.

Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis

Tight regulation of actin remodeling is essential for development, and misregulation results in disease. Cytoskeletal dynamics are regulated by prostaglandins (PGs)—lipid signals. PGs temporally regulate actin remodeling during Drosophila oogenesis, at least in part, by modulating the activity of the actin elongation factor Enabled.

Fascin regulates nuclear actin during Drosophila oogenesis

Study of Drosophila oogenesis reveals that the nuclear localization of actin is controlled by both development and Fascin. Fascin regulates both endogenous nuclear actin and ectopic nuclear actin rod formation by controlling Cofilin.

Drosophila: A Model for Studying Prostaglandin Signaling

Prostaglandin (PG) synthesis and signaling are conserved in Drosophila melanogaster. PGs are produced downstream of cyclooxygenase or COX enzymes, the targets of nonsteroidal anti-inflammatory drugs. Almost 20 years ago, biochemical studies suggested that Drosophila possess COX activity. Recent efforts utilizing a combination of pharmacological and genetic approaches revealed that PGs have critical functions in Drosophila oogenesis or follicle development. Pxt was identified as the COX-like enzyme and is required for multiple aspects of female fertility, including temporal regulation of both gene expression and actin cytoskeletal remodeling. Here we review the PG synthesis and signaling machinery, the evidence for PG activity in Drosophila, the roles of PGs in flies, primarily focused on oogenic activities, and the conservation of PG function in higher animals. We also point out how studies on PGs in a genetic model system, such as flies, can significantly advance our understanding of the molecular actions of PGs.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.