Understanding what modulates cadherin-mediated adhesion at adherens junctions (AJs) has widespread implications for understanding and treating defects during embryonic development, and for diagnosing and treating metastatic tumors. One key unresolved issue in the field is how catenin recruits and connects to the actin cytoskeleton via its C terminus. Another is how catenin- dependent and -independent actin recruitment pathways interact to strengthen AJs. C. elegans is currently the only genetic model system for rapid functional analysis of catenin in a living embryo, and provides key advantages for addressing these two unresolved issues. We will use in vitro assays, genetics, and innovative in vivo imaging to address the following specific aims:
Aim 1 : Role of the C terminus of HMP-1/catenin in recruiting actin to cadherin-based adhesions. Our data suggest that a mutation in an absolutely conserved region in the C terminus of catenin increases its affinity for actin, and that it is regulated by adjacent regions. We will test this hypothesis using biochemical assays and highly time-resolved imaging in vivo. We will also take a structure-function approach to identify other important domains in catenin, and to determine whether the C terminus of catenin, acting as a direct linker, can carry out its functions in vivo.
Aim 2 : Role of catenin in recruitment of MAGI to cadherin-based adhesions. Our genome- wide screen for enhancers of a weak catenin mutant, hmp-1(fe4), identified the MAGUK, MAGI-1. We will test whether HMP-2/catenin physically recruits MAGI-1 to AJs, using biochemistry, genetics, and in vivo imaging of MAGI-1 constructs. We will also test whether MAGI-1 recruits the Rap-GEF, PXF-1/PDZ-GEF, to cadherin-based junctions via direct physical interaction. These experiments represent the first analysis of a MAGI in cadherin-based adhesion in a living embryo.
Aim 3 : Role of Raps and AFD-1/AF-6 in MAGI-1-dependent maturation of cadherin-based adhesions. Loss of Rap and AFD-1/AF-6 function also enhances hmp-1(fe4). We will determine whether MAGI-1 leads to Rap activation at AJs. We will test this model using biochemistry, genetics, and innovative in vivo assessment of Rap activity. We will also test whether AFD-1/AF-6 acts as a Rap effector in concert with MAGI-1. Such analysis represents the first dynamic analysis of Raps and AF-6 in a living embryo. As a result of these studies, we will clarify how the cadherin/catenin complex recruits actin to nascent cell-cell contacts during epithelial morphogenesis in a living organism, and we will gain fundamental new insights into a process fundamental for events during human development and oncogenesis.

Public Health Relevance

Understanding how cells stick 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, called catenin that regulates cell adhesiveness, and how this protein works together with other proteins 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058038-11
Application #
7808905
Study Section
Special Emphasis Panel (ZRG1-CB-J (02))
Program Officer
Deatherage, James F
Project Start
1998-08-01
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
11
Fiscal Year
2010
Total Cost
$334,653
Indirect Cost
Name
University of Wisconsin Madison
Department
Zoology
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Loveless, Timothy; Qadota, Hiroshi; Benian, Guy M et al. (2017) Caenorhabditis elegans SORB-1 localizes to integrin adhesion sites and is required for organization of sarcomeres and mitochondria in myocytes. Mol Biol Cell 28:3621-3633
Shao, Xiangqiang; Kang, Hyunook; Loveless, Timothy et al. (2017) Cell-cell adhesion in metazoans relies on evolutionarily conserved features of the ?-cateninĀ·?-catenin-binding interface. J Biol Chem 292:16477-16490
Kang, Hyunook; Bang, Injin; Jin, Kyeong Sik et al. (2017) Structural and functional characterization of Caenorhabditis elegans ?-catenin reveals constitutive binding to ?-catenin and F-actin. J Biol Chem 292:7077-7086
Hardin, Jeff (2016) Regulating cell-cell junctions from A to Z. J Cell Biol 213:151-3
Walck-Shannon, Elise; Lucas, Bethany; Chin-Sang, Ian et al. (2016) CDC-42 Orients Cell Migration during Epithelial Intercalation in the Caenorhabditis elegans Epidermis. PLoS Genet 12:e1006415
Walck-Shannon, Elise; Reiner, David; Hardin, Jeff (2015) Polarized Rac-dependent protrusions drive epithelial intercalation in the embryonic epidermis of C. elegans. Development 142:3549-60
Choi, Hee-Jung; Loveless, Timothy; Lynch, Allison M et al. (2015) A conserved phosphorylation switch controls the interaction between cadherin and ?-catenin in vitro and in vivo. Dev Cell 33:82-93
Hardin, Jeff (2015) Getting to the core of cadherin complex function in Caenorhabditis elegans. F1000Res 4:
Callaci, Sandhya; Morrison, Kylee; Shao, Xiangqiang et al. (2015) Phosphoregulation of the C. elegans cadherin-catenin complex. Biochem J 472:339-52
Walck-Shannon, Elise; Hardin, Jeff (2014) Cell intercalation from top to bottom. Nat Rev Mol Cell Biol 15:34-48

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