Understanding what modulates the function of ?-catenin, the key linker to the actin cytoskeleton within the cadherin-catenin complex (CCC) has widespread implications for understanding and treating defects during embryonic development, for modulating cell behavior in tissue engineering applications, and for diagnosing and treating metastatic tumors. Using functional genomics and proteomics, we identified the srGAP, SRGP-1, as an important regulator of the CCC during C. elegans morphogenesis. Mutations in srGAP orthologs lead to mental retardation in humans, but nothing is known about their roles in epithelial cell-cell adhesion. We will use genetics, innovative in vivo imaging, and biochemistry to test a model in which ?-catenin and SRGP-1 act together to promote maturation and maintenance of adherens junctions in the following specific aims:
Aim 1 : Role of the SRGP-1 plasma membrane interaction in AJ maturation and maintenance. We will (a) characterize the phospholipid binding and bending properties of SRGP-1 using biochemistry, (b) assess recruitment of SRGP-1 to areas enriched in key phospholipids in vivo using dynamic imaging;and (c) test the importance of SRGP-1's membrane bending activity vs. general membrane recruitment in vivo using point mutations in key residues predicted to mediate convex vs. concave membrane bending and heterologous membrane targeting motifs.
Aim 2 : Role of the SRGP-1 interaction with actin regulators in AJ maturation and maintenance. We will (a) use a novel tissue-specific inducible expression system to test whether SRGP-1 regulates Rac signaling at nascent and maturing junctions in the epidermis;(b) use epitasis tests and WVE-1/deletion constructs predicted to abrogate SRGP-1 recruitment to the WAVE complex to place SRGP-1 within a genetic hierarchy during epidermal enclosure and embryonic elongation, and (c) we will test whether WVE-1/Wave, components of the C. elegans Sra-1/Nap1/Abi complex, and MIG-10/lamellipodin interact with the homologous region of SRGP-1.
Aim 3 : Role of SRGP-1 binding to ?-catenin in AJ maturation and maintenance. We will (a) assess the temporal order of recruitment of SRGP-1 and the CCC using high-speed in vivo imaging, and test for their mutual dependence;(b) map the interacting domains within each protein, and assess the effects of the HMP-1/SRGP-1 interaction on their dynamics and function at AJs in vivo and on their known binding partners in vitro;and (c) we will validate candidate novel binding partners of the SRGP-1 C terminus, and perform large-scale screens for additional SRGP-1 physical interactors. As a result of these studies, we will gain new insight into how adherens junctions mature during epithelial morphogenesis in a living organism, a process crucial for diverse cellular events during human development and suppression of oncogenesis. We will also gain fundamental new insights in vivo into a class of proteins, the srGAPs, required for normal human development.
Understanding how cells attach to one another is important for understanding many common birth defects, and how cancer cells lose their connections to one another and invade the body. This proposal examines a key protein, ?-catenin, which regulates cell adhesiveness, and how this protein works together with a protein known as srGAP to ensure that cells make proper connections in the body. By studying how this protein works in living embryos, we will gain important information that can be used to understand and treat human disease.
|Walck-Shannon, Elise; Hardin, Jeff (2014) Cell intercalation from top to bottom. Nat Rev Mol Cell Biol 15:34-48|
|Maiden, Stephanie L; Harrison, Neale; Keegan, Jack et al. (2013) Specific conserved C-terminal amino acids of Caenorhabditis elegans HMP-1/ýý-catenin modulate F-actin binding independently of vinculin. J Biol Chem 288:5694-706|
|Hardin, Jeff; Lynch, Allison; Loveless, Timothy et al. (2013) Cadherins and their partners in the nematode worm Caenorhabditis elegans. Prog Mol Biol Transl Sci 116:239-62|
|Ikegami, Richard; Simokat, Kristin; Zheng, Hong et al. (2012) Semaphorin and Eph receptor signaling guide a series of cell movements for ventral enclosure in C. elegans. Curr Biol 22:1-11|
|Maiden, Stephanie L; Hardin, Jeff (2011) The secret life of ýý-catenin: moonlighting in morphogenesis. J Cell Biol 195:543-52|
|Hardin, Jeff (2011) Imaging embryonic morphogenesis in C. elegans. Methods Cell Biol 106:377-412|
|Simske, Jeffrey S; Hardin, Jeff (2011) Claudin family proteins in Caenorhabditis elegans. Methods Mol Biol 762:147-69|
|Neukomm, Lukas J; Frei, Andreas P; Cabello, Juan et al. (2011) Loss of the RhoGAP SRGP-1 promotes the clearance of dead and injured cells in Caenorhabditis elegans. Nat Cell Biol 13:79-86|
|Zaidel-Bar, Ronen; Joyce, Michael J; Lynch, Allison M et al. (2010) The F-BAR domain of SRGP-1 facilitates cell-cell adhesion during C. elegans morphogenesis. J Cell Biol 191:761-9|
|Grana, Theresa M; Cox, Elisabeth A; Lynch, Allison M et al. (2010) SAX-7/L1CAM and HMR-1/cadherin function redundantly in blastomere compaction and non-muscle myosin accumulation during Caenorhabditis elegans gastrulation. Dev Biol 344:731-44|
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