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.