Research

Oogenesis Background

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The production of Drosophila eggs is accomplished in “assembly lines” called ovarioles that contain developing egg chambers. Oogenesis initiates in the germarium located at the anterior tip of each ovariole when a germline stem cell daughter undergoes four rounds of mitosis to produce a cluster of 16 cells. The mitotic divisions are characterized by incomplete cytokinesis and stabilization of arrested cleavage furrows into intercellular bridges called ring canals. Somatic follicle cells form an epithelium around the germline cells to complete egg chamber formation. One of the 16-germline cells becomes the oocyte, while the other 15 cells, called nurse cells, provide the oocyte with maternal protein, mRNA and organelles by intercellular transport through ring canals.

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In the Drosophila egg chamber, intercellular bridges called ring canals allow for the movement of cytoplasmic material into the developing oocyte from supporting nurse cells. The ring canals expand in diameter from ~1µ to ~10µ as the egg chamber develops. This growth is driven by a robust and dynamic actin cytoskeleton that polymerizes at the plasma membrane in order to push the ring canal rim outward. The Kelch protein is required to maintain the organization of the ring canal actin cytoskeleton by reversibly cross-linking actin filaments (F-actin). 

RC Growth 

In kelch mutants, the ring canal lumen is partially occluded with disorganized F-actin, reducing the flow of cytoplasm into the oocyte, causing the production of small eggs and sterile females. Kelch also binds Cullin-3 (Cul-3) and forms a Cullin-RING E3 ubiquitin ligase. Cullin-based E3 ubiquitin ligases typically add Lys-48-linked poly-ubiquitin chains to target proteins, signaling their destruction by the proteasome. Interestingly, the level of Kelch protein itself is regulated in a Cullin-3-dependent manner. In Cul-3 mutant cells, Kelch accumulates in the lumen of the ring canal, and ring canals contain disorganized F-actin. This suggests the interesting possibility that Cul-3-mediated, autocatalytic Kelch turnover is a novel mechanism for regulating the actin cross-linking activity of Kelch. We are using biochemical and genetic approaches to investigate the mechanism of Cul-3-dependent turnover of Kelch and how it contributes to F-actin organization during ring canal growth.

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Ring canals form through the stabilization of arrested mitotic cleavage furrows, and provide direct cytoplasmic connections among sibling cells. Ring canals are well documented for their participation the development of germline cells in Drosophila and many other animals, but little is known about their role in the several Drosophila somatic tissues in which they are also found. 


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In the Cooley lab, we use a variety of genetic and imaging tools to investigate the impact of ring canals in live and fixed Drosophila somatic tissues. To date, we have successfully demonstrated robust intercellular exchange of proteins through ring canals, quantified the rate of movement, and presented evidence that somatic ring canals aid epithelial biogenesis by equilibrating protein levels among groups of transcriptionally mosaic cells. Our continuing work is focused on defining the essential functions and identifying the structural components of somatic ring canals. In our current work we are disrupting the movement of cytoplasm between these ring canals so that we can characterize their function. Additionally, few structural components of the somatic ring canal have been identified to date. We are working on purifying the ring canal complex in order to gain a better understanding of ring canal formation, as well as Drosophila cytokinesis in general.

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High fecundity is one of the many advantages of using Drosophila melanogaster as a model system. Females devote significant resources to reproduction, producing up to 60 eggs per female per day on a nutrient-rich diet. Mechanisms used by fruit flies to maximize reproductive capacity also provide important insights into how animals protect their germline under varying environmental conditions. We are investigating the signaling mechanisms used in egg chambers to control their cellular response to starvation, and the consequences of interfering with the ability of egg chambers to respond to starvation.

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Many maternal mRNAs become oocyte-enriched during oogenesis. They reside within ribonucleoprotein complexes (mRNPs) containing proteins that repress mRNA translation until they reach their destination in the oocyte. Enrichment of mRNPs in the oocyte depends on the microtubule (MT) network that extends from the oocyte through the cyst either by dynein-mediated transport or by diffusion and localized retention. 

The distribution of germline mRNPs and MTs, as well as the general development of egg chambers, is highly sensitive to nutrition. In protein-poor (starved) conditions, egg chambers do not complete development and instead undergo apoptosis during stage 8 just before the onset of yolk uptake, or vitellogenesis. This prevents the female from making a large metabolic investment (yolk and eggshell production) when nutrients are limited and progeny survival is uncertain. Starvation conditions also result in a fourfold decrease in follicle stem cell and germline stem cell proliferation, thus slowing the overall rate of egg chamber production (Daniela Drummond-Barbosa). We found that germline cells in pre-vitellogenic egg chambers (stages 2-7) from starved females have aggregates of mRNP components that resemble Processing bodies (P bodies), as well as cortically condensed MTs. Importantly, providing starved females with rich food (yeast) reverses the effects of starvation within two hours, suggesting the starvation response is tightly regulated. We also observed that culturing egg chambers with insulin for one hour reversed the germline starvation response, suggesting the involvement of insulin signaling in controlling the egg chamber’s response to nutrition. We are investigating the role of the Insulin/TOR signaling pathway in controlling egg chamber physiology in response to food availability.

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The circular muscle sheath surrounding ovarioles contracts peristaltically to move developing egg chambers toward the oviduct. The muscle cells are unusual for striated muscles because they have a single nucleus rather than being formed by fusion of myoblasts. We are taking advantage of this to use using mosaic analysis to study the effects of mutations on muscle function.