Implementation of High-Throughput Approaches
To understand signaling network dynamics at a systems level in mammalian cells and how this might control cell processes and contribute to disease, we have developed and are implementing a diverse variety of high throughput screening approaches at the Network Biology Collaborative Center at the Lunenfeld-Tanenbaum Research Institute of Mount Sinai Hospital.
Protein-protein interaction screens
In the early 2000’s, we developed a method known as LUMIER to systematically map protein-protein interactions in mammalian cells. In LUMIER, a luciferase-tagged bait protein of interest is screened for interactions with Flag-tagged proteins expressed from a cDNA library comprised of genes encoding diverse signaling proteins. This was first applied to the analysis of the TGFβ pathway and led to our discovery of a novel TGFβ polarity signalling network that controls apical-basal polarity and epithelial-to-mesenchymal transition. This approach has been extended, by our groups and in collaborative efforts with other investigators, to the study of WNT, BMP, Hippo and other pathways as well as for understanding how alternative-splicing events alter protein networks and contribute to disease. For some examples, see:
We also conduct a variety of functional assays to uncover new biological insights. We have used custom and genome-wide siRNA libraries to assay for ligand-induced signalling, cell and tissue characteristics, somatic cell reprogramming and other processes. For example, our studies have revealed an intimate connection between WNT and the cell growth control Hippo pathway and in separate work, a key role for BMP-driven mesenchymal-to-epithelial transition for the initiation of somatic cell reprogramming. We have also implemented small molecule screens to identify positive and negative chemical inhibitors of TGFβ, WNT and other pathways. Ongoing high throughput efforts include genome-wide and directed CRISPR/Cas9-based screens in cells and organoids, live-cell imaging screens and genome-wide transcriptomic analysis (RNAseq). See research projects for more details.