Directional cell motility is required for the development of an organism with proper polarity such as dorso-ventral, anterior-posterior, and left-right symmetry. We have found in Xenopus laevis that depletion of TRPM7, the first ion channel discovered to have its own kinase domain, results in embryos with severe gastrulation and neural fold closure defects, making TRPM7 the first ion channel shown to have a dramatic effect on early vertebrate development. A possible explanation for this effect is our recently reported discovery that TRPM7 controls the activity of the calcium-dependent protease m-calpain to regulate cell adhesion. Although a compelling picture is emerging of TRPM7's role in cell motility, important details are still missing, namely, the mechanism by which TRPM7's channel is activated, regulation of the kinase, and a full understanding of how and under what conditions TRPM7 controls cell motility. Finally, the specific aspect(s) of gastrulation affected by TRPM7 and the roles played by its kinase and channel in these events have not been defined. We propose two specific aims to clarify TRPM7's function and regulation on the cellular level and in vivo during Xenopus development. In the first specific aim, we will take an electrophysiological approach to investigate the hypothesis that PDGF-receptor activation of TRPM7's channel is dependent upon PIP2 synthesis. Cell surface biotinylation experiments will be used to test whether PDGF-mediated activation of TRPM7 relies upon the recruitment of the channel to the plasma membrane from intracellular sites. In addition, we've created TRPM7-knockdown fibroblast cell lines to investigate the regulation of TRPM7's kinase and its phosphorylation and regulation of myosin II by the PDGF receptor. Finally, we will test whether the PDGF receptor utilizes both TRPM7 and the ERK signaling pathway to regulate m-calpain and focal adhesion turnover. In the second specific aim we will employ channel- and kinase-dead mutants we've created in a combined loss-of-function/gain-of-function approach to define the roles of TRPM7's channel and kinase in early pattern formation in Xenopus laevis. These investigations will include an examination of TRPM7's influence on convergent extension movements and blastopore and neural fold closure. Collectively, the proposed experiments should greatly advance our understanding of TRPM7's function in vivo. Study of this bifunctional channel could deepen our understanding of many physiological processes including neural crest cell migration and could potentially lead to new strategies for treating pathological conditions dependent on cell motility such as inflammation during heart disease, cancer cell metastasis, and spinal cord injuries.
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