Mapping protein interaction networks of the cell is the ultimate goal of systems-level proteomics. Recently, proximity-based approaches using the promiscuous biotin ligase BirA* have been developed to determine protein interactions directly in live cells, overcoming some of the conceptual and technical limitations of other protein-protein interaction assays. This application aims to develop a new general technique that combines the advantages of proximity-based biotinylation and split protein complementation to detect novel proteins that interact with known signaling complexes. To accomplish this, pairs of split BirA* fragments that restore enzymatic activity only when brought into close proximity will be defined and validated in cell-based biotin ligase assays. Candidate positive split BirA* fragments will then be used in vivo to identify novel proteins interacting with signaling receptors and planar cell polarity (PCP) complexes that are spatially segregated in the Xenopus embryonic neural plate, a new model of PCP. Xenopus embryos are uniquely suited for these studies, allowing rapid analysis of protein localization and function through a combination of biochemical, embryological and cell biological approaches. These studies will provide a versatile general platform to map protein interactions in different experimental settings, will advance our understanding of PCP signaling mechanisms and help establish a new vertebrate PCP model. Since PCP proteins have been implicated in neural tube defects, ciliopathies, polycystic kidney disease and metastatic cancers, the proposed research is relevant to human health and will advance the knowledge necessary for preventing PCP- associated diseases and congenital abnormalities. 1
This application will develop a new general approach to identify proteins interacting with known signaling complexes in live cells. This technique will be applied to study polarized protein complexes in the vertebrate neuroepithelium to establish a new animal model of tissue polarization. The proposed studies will contribute to the knowledge necessary for prevention of birth abnormalities, such as neural tube defects, and are relevant to metastatic cancers and other diseases linked to changes in cell polarity and behavior.
