Project title: Analysis of Mammalian Cilial Trafficking with Super-Resolution Imaging.
Cilia are small microtubule-based “organelles” with critical signalling and sensory roles in develop-ment and disease. Their small size (diameter of 0.2μm, length of ~1μm in the case of primary cilia) and dynamic nature makes them challenging to study by conventional light microscopy where the limits of resolution 250nm on X-Y and 500nm in Z-direction. While ultrastructure analysis by transmission electron microscopy (TEM) is excellent at resolving cellular structure, molecular charac-terization is difficult and offers only static snapshots of dynamic processes. The advent of super-resolution techniques which break Abbe’s barrier such as PALM, STORM and STED, offer the possibility of resolving molecular structures below 50nm. Trafficking events in and out of the cilium are focused at the ciliary base and peri-ciliary membrane. Given spatially the structures and events we need to resolve are focused within a ~0.5μm window at the ciliary base, we find ourselves at the limits of resolution by conventional microscopy. Moreover as cilia are dynamic, going through cy-cles of cilia formation, maintenance and resorbtion, live cell imaging of many cilia to get quantita-tive data to define population behaviour is also very important. In this project, I am developing and optimising tools and techniques for super resolution imaging of cilia trafficking in live and fixed cells.