Biology puzzles are diverse, and a combination of biochemical and biophysical approaches are often required to solve them. The elucidation of protein-protein interactions using nanobiology and super resolution microscopy will be discussed.
Nuclear pore complexes (NPCs) act as effective and robust gateways to the nucleus and allow only the passage of select macromolecules across the nuclear envelope. This scaffold anchors proteins termed FG-nups, whose natively disordered domains line the passageway and form an effective barrier to the diffusion of most macromolecules. However, cargo-carrying transport factors overcome this barrier by transient binding to the FG-nups. We constructed an artificial NPC mimic by lining a polycarbonate passageway with only FG-nups. Our device faithfully reproduces key features of selective trafficking through the NPC, including transport factor-mediated cargo import.
The super-resolution method of photoactivated localization microscopy (PALM) can be used to analyze the distribution and dynamics of single molecules within bigger structures, which makes it an ideal tool for mechanistic investigation of biological processes. We developed a new method to analyze the distribution of single molecules obtained with PALM by separating contributions from stochastic clustering (corresponding to multiple appearances of a single protein) and protein clustering (corresponding to homo- and hetero-oligomers) using autocorrelation analysis. We probed six different plasma membrane proteins that have distinct anchoring mechanisms and lipid affinities. We showed that our approach provides insight into the organization of proteins with ~25 nm resolution and identifies conditions that significantly change their distribution.