My lab seeks to understand the cell biophysical mechanisms regulating transmembrane signal transduction, i.e. signal transfer from ligands to receptors to downstream effectors. There is mounting evidence that cell surface receptors and associated proteins exhibit a high degree of dynamic organization at the plasma membrane (PM), which is critical for their ligand binding and signaling. However, many fundamental questions remain unanswered. Toward filling this gap in our knowledge, my lab will pursue two research directions over the next five years. The first direction will investigate how focal adhesions, the actin cortex, and interactions with integrins regulate the spatiotemporal organization and signaling of the endothelial cell receptor VEGFR2. VEGFR2 is the main promoter of angiogenesis (the formation of new blood vessels from existing vessels) in both normal physiology and disease. Thus there is great interest and need to understand the mechanisms that regulate its signaling. These studies will reveal mechanisms that underlie the activation of multiple pathways downstream of VEFR2, and that underlie the integration of multiple external signals at the level of the PM. As VEGFR2 belongs to the large family of receptor tyrosine kinases, our studies are expected to reveal general principles of the regulation and signaling of this important family of receptors. The second direction will focus on the novel organizational principle of liquid-liquid phase separation (LLPS) for proteins at the PM, using the transmembrane protein LAT as a model system. LAT is critical for the activation and immune function of T cells upon encountering an antigen presenting cell. Recent in vitro reconstitution work suggests that signaling clusters composed of LAT and its downstream effectors are formed through LLPS. The cellular environment is however much more complex than an in vitro reconstituted system. Thus we will investigate to what extent LAT microclusters at the PM of T cells are formed through LLPS, and the mechanisms that regulate LAT cluster composition. These studies will shed light on the role of LLPS for protein organization within the cellular environment in general. Both research directions – by their very nature – require probing molecular activities with high specificity and resolution in their native cellular environment. To achieve this, we will develop integrative approaches based on live-cell single-molecule, super-resolution and activity biosensor imaging, combined with cutting-edge mathematical and statistical analysis tools to extract quantitative information from the experiments and to multiplex the complementary information that the different imaging modalities provide. These analytical tools will be critical for our studies because single-cell and single-molecule microscopy often reveal molecular and cellular heterogeneity that is difficult to digest without such tools. We expect our novel analytical tools to be broadly applicable to other...