The growth of the germline ring canals during Drosophila melanogaster oogenesis

Butler University, Indianapolis IN

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Abstract

PROJECT SUMMARY/ABSTRACT Infertility and impaired fecundity affect millions of Americans; studying normal gamete formation is essential to developing new treatments. Intercellular bridges are highly conserved structures that are essential for fertility in organisms from insects to mammals, and they allow for the coordinated behavior and sharing of cytoplasmic contents between neighboring cells. Although the structure of intercellular bridges has been studied for over 50 years, there is still much to be learned about their formation and regulation. The long-term goal of this project is to determine the molecular mechanisms that drive intercellular bridge formation, stabilization, and growth. One of the best-studied examples of intercellular bridges in gametogenesis is in the developing fruit fly egg chamber, which gives rise to the mature egg. These intercellular bridges, or ring canals, connect the 15 supporting nurse cells to the developing oocyte. They are rich in f-actin and actin binding proteins which allow them to expand in size to a final diameter of ~10 µm. The actin-nucleating Arp2/3 complex has been implicated in ring canals growth; however, the molecular mechanism by which the complex is localized to and activated at the ring canals is not known. Preliminary studies have identified two novel proteins at the germline ring canals of the developing egg chamber ? the Ste20 family kinase, Misshapen (Msn), and the SH2/SH3 adaptor protein, Dreadlocks (Dock). Altering the levels of Msn or Dock leads to defects in ring canal structure and failure of bulk cytoplasmic transfer from the nurse cells to the oocyte. The objective of this proposal is to determine the role of Msn, Dock, and the Arp2/3 complex in ring canal growth. Because Msn and Dock function within the same pathway in other developmental contexts, and both have been linked genetically or biochemically to the Arp2/3 complex or its activators, this has led to the central hypothesis that Msn and Dock act cooperatively to promote ring canal growth through direct or indirect regulation of the Arp2/3 complex. Aim 1 will use clonal mutant analysis, RNAi, over-expression, molecular epistasis, and immunofluorescence to determine the role for Msn in growth of the germline ring canals. Aim 2 will use clonal mutant analysis, RNAi, genetic epistasis, immunofluorescence, and biochemistry to test whether Msn regulates the Arp2/3 complex during ring canal growth. In Aim 3, clonal mutant analysis, RNAi, epistasis, and immunofluorescence will be used to determine the role for Dock in ring canal growth. This work will provide important insight into the role for these highly conserved proteins in regulation of intercellular bridge growth. Identifying additional bridge components in Drosophila could impact our understanding of normal human gametogenesis as well as infertility. Both Msn and Arp2/3 homologs have been implicated in cancer metastasis, so characterizing their basic cellular functions would provide insight into their role in other diseases. This work will expose undergraduate researchers to cutting edge techniques and a powerful genetic model organism while answering important questions in developmental biology.