Signalingcomplexes are out-of-equilibrium, heterogeneous multi-molecular structures andsites for intracellular signal transduction. Although they play a crucial rolein cellular activation, current research techniques have been unable to resolvetheir structure and formation mechanisms in
intact cells. I will briefly review recent advancements in far-field super-resolutionoptical microscopy and present a multi-color Single Molecule LocalizationMicroscopy (SMLM) approach for imaging multiple types of single molecules infixed and live cells, with resolution down to ~20nm. I will further describe astatistical framework to determine the nanoscale organization and cooperativityof molecular interactions in signaling complexes. Using these techniques weobserved that signaling complexes that determine immune (T) cell activationshowed surprising patterns of dynamic nanoscale organization and a hierarchicalnetwork of cooperative interactions between the constituent molecules.

Inthe second part of my talk, I will describe our study of protein mobility atthe plasma-membrane using single particle tracking (SPT). I will further presenta methodological approach to resolve mixed subdiffusion mechanisms acting simultaneouslyon polydispersed samples and complex media such as cell membranes. Using thesetools, we find that protein mobility of a short trans-membrane protein is highlyheterogeneous, subdiffusive and ergodic-like. Individual trajectories can bebroken into multiple mobility states having distinct underlyingsubdiffusion mechanisms. These mechanisms include viscoelasticity, fractality,and confinement.