The migration of cranial neural crest (CNC) cells is a critical process for proper formation of the face and jaw during embryo development. Failure of these cells to migrate to the correct locations can result in severe craniofacial abnormalities at birth, yet there is a significant gap in our knowledge of how to prevent or treat these defects. This proposal aims to increase our current understanding of CNC cell migration and the mechanisms by which it is regulated. In Xenopus laevis, CNC migration requires the cell surface metalloprotease ADAM13, which has a dual function in the CNC. The extracellular protease cleaves Cadherin-11 to shed its adhesive domain from the surface to promote migration, while the cytoplasmic domain of ADAM13 is cleaved off and regulates gene expression in the nucleus. The long-term goal of this research is to elucidate the mechanisms by which ADAM13 controls CNC cell migration. The objective of this proposal is to determine how the activity of the cytoplasmic domain is regulated. Based on preliminary data, the hypothesis is that the kinase GSK3 primes the cytoplasmic domain of ADAM13 for subsequent phosphorylation by Polo-like kinase (Plk), and that these events are essential for the downstream nuclear activity of ADAM13. This hypothesis will be tested by determining the contribution of Plk (Aim 1) and GSK3 (Aim 2) to the function of ADAM13 in the CNC. In vivo migration assays will be employed to determine if Plk and GSK3 are required for migration and if constitutively phosphorylated forms of ADAM13 can promote CNC migration in the absence of Plk and GSK3 kinase activity. Immunoprecipitation and western blot analysis will be used to examine the effect of blocking phosphorylation by GSK3 or Plk on both the cleavage and nuclear translocation of the cytoplasmic domain, as well as the requirement of GSK3 phosphorylation on Plk binding. Additionally, real time PCR will used to analyze the ability of non-phosphorylatable forms of ADAM13 to regulate gene expression in the CNC. The results of this study are expected to determine if GSK3 and Plk regulate the activity of the ADAM13 and how this contributes to CNC migration. This will better define how ADAM proteins function in specific tissues during development and ultimately provide a clearer understanding of the mechanisms of CNC cell migration in vivo.
The proposed study is relevant to public health because it addresses a critical need to elucidate the molecular processes that contribute to craniofacial development. Improving our understanding of how cranial neural crest migration is controlled will provide valuable insight into how craniofacial abnormalities can arise and potentially be prevented or treated. In addition, increasing our knowledge of ADAM function in cell migration is also relevant as they are often upregulated in invasive cancers.
|Langhe, Rahul P; Gudzenko, Tetyana; Bachmann, Michael et al. (2016) Cadherin-11 localizes to focal adhesions and promotes cell-substrate adhesion. Nat Commun 7:10909|
|Abbruzzese, Genevieve; Gorny, Anne-Kathrin; Kaufmann, Lilian T et al. (2015) The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity. J Cell Sci 128:1139-49|
|Abbruzzese, Genevieve; Cousin, HÃ©lÃ¨ne; Salicioni, Ana Maria et al. (2014) GSK3 and Polo-like kinase regulate ADAM13 function during cranial neural crest cell migration. Mol Biol Cell 25:4072-82|