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CHARACTERIZATION OF THE ROLES OF RAPOSTLIN AND REVERSION INDUCED LIM DOMAIN PROTEIN (RIL) AS EFFECTORS FOR DAAM1 AND NON-CANONICAL WNT SIGNALING

by
Courtney Mezzacappa
B.S., The College of New Jersey - 2006

Thesis Advisor: Raymond Habas, Ph.D
Graduate Program Biochemistry & Molecular Biology


BioLife Building, Room 234
Temple University
1900 North 12th Street
Philadelphia, PA 19122

Monday, November 21, 2011
4:00 p.m.


Abstract

The Wnt signaling pathway plays crucial roles in a plethora of early developmental processes including cell polarization, motility, and cell fate determination. Dramatic changes to the actin cytoskeleton via non-canonical Wnt signaling mediate cell movement and polarity and are required for proper vertebrate gastrulation. The exact mechanism by which Wnt signaling modulates the actin cytoskeleton remains poorly understood. Determination of molecular components required in this essential signaling pathway will help clarify the role Wnts play during vertebrate gastrulation.
Daam1, a Formin homology protein identified by our laboratory, is a central component of the non-canonical Wnt signaling pathway and is required for Wnt-induced cytoskeletal changes during development, though its exact mechanism remains unknown. A yeast two-hybrid screen with the C-terminus of Daam1 as bait yielded two potential binding partners, among others: Rapostlin and Reversion Induced Lim domain protein (RIL). Both are highly conserved vertebrate proteins with protein domains implicated in actin interaction.
To characterize the role of these proteins in non-canonical Wnt signaling, binding of Rapostlin and RIL to Daam1 was confirmed via co-immunoprecipitation and GST pull-down. Immunofluorescence experiments in mammalian cells show specific subcellular localization of these proteins in response to Wnt stimulation, and colocalization with actin and focal adhesions. In the Xenopus embryo, both RIL and Rapostlin are expressed throughout development, and become spatially refined to areas of highly dynamic actin reorganization during gastrulation, such as the neural tube. Overexpression and knockdown of RIL and Rapostlin in Xenopus embryos cause severe gastrulation defects hallmarked by open neural tubes. These defects are specific to each protein, and can be rescued. Importantly, suboptimal doses of Daam1 and either RIL or Rapostlin synergize to induce severe gastrulation defects in the embryo. Interestingly, RIL and Rapostlin interact and synergize to produce severe gastrulation defects as well.
Taken together, these studies suggest that Rapostlin and RIL are novel downstream effectors in non-canonical Wnt signaling, directly involved in regulation of the actin cytoskeleton to control gastrulation in vertebrate embryos. Elucidation of their mechanisms will provide insight into human birth defects including spina bifida.


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