Super-resolution Microscopy Study of Molecular Transport Mechanisms

PROJECT SUMMARY Our laboratory has developed unique tools that have enabled further progress towards understanding molecular transport mechanisms involving three sub-cellular organelles in eukaryotic cells: the nucleus, cytoplasm, and primary cilium. Macromolecular trafficking among these compartments is suggested to be gated by two unique mechanisms. One is the nuclear pore complex (NPC) embedded in the nuclear envelope that mediates bidirectional trafficking of proteins and RNAs between the cytoplasm and the nucleus; the other is the transition zone (TZ) located at the base of primary cilium that regulates the entry of membrane and cytosolic proteins into the cilium. Progress is intimately linked to technical improvements in biophysical research putting us in the unique position to elucidate the fast kinetics and 3D transport routes of macromolecules as they transport through the sub-micrometer NPC or TZ in live cells. In the first four years of the current five-year MIRA grant, we have completed the development of single-point edge-excitation sub- diffraction (SPEED) microscopy and successfully applied the new method to solve several critical questions pertaining to nuclear transport and cytoplasmic-ciliary transport. We have included our major findings in thirty manuscripts and published them in prestigious scientific journals such as Nature Structural & Molecular Biology, Nature Communications, Protein Science, and Nature Protocols. Currently we are highly motivated to work on the following new research topics: 1) Development of a new optical imaging system termed enhanced photon microscopy aiming at doubling photon collection efficiency from fluorophores, which will significantly improve optical resolutions when integrated with epifluorescence microscopy, laser scanning confocal microscopy, SPEED microscopy and other super-resolution microscopy; 2) Mapping the spatial organization of dynamic disordered protein motifs in the NPC to distinguish between, and advance upon, FG- organizational models; and 3) Understanding the gating mechanism for the cytoplasmic-ciliary transport of membrane proteins. In summary, during the next funding period of the MIRA grant, we will continue to develop new microscopy imaging techniques and solve long-standing unanswered questions in both nucleocytoplasmic and cytoplasm-cilium transport mechanisms.