Joint GRE/CDB Mechanistic understanding of cilia assembly and transport by cryo-EM Seminar details July 5, 2019, 2:00 pm @ Small Lecture Theatre, School of Life SciencesDr. Gaia Pigino, MPI-CBG, Dresden Host: Jason Swedlow and Jens Januschke Abstract New cryo-EM technologies enable to investigate protein structures in the native physiological context of the cell. We use these technologies to study the self-organized assembly of cilia, ubiquitous organelles of eukaryotic cells. Assembly of the cilium requires the rapid bidirectional intraflagellar transport (IFT) of building blocks to and from the site of assembly at its tip. This bidirectional transport is driven by the anterograde motor kinesin-2 and the retrograde motor dynein-1b, which are both bound to a large complex of 25 IFT adaptor proteins. We have recently developed a millisecond resolution 3D correlative light and electron microscopy (CLEM) approach to show that anterograde and retrograde IFT trains use separated microtubule tracks along the microtubule doublets of the cilium (Stepanek & Pigino, Science 2016). With this method at hand we showed that the spatial segregation of oppositely directed trains ensures a collision free transport in the cilium. However, it remained to be explained how competition between kinesin and dynein motors, which are both found on the same anterograde trains, is avoided. In bidirectional transport systems in the cell, other than IFT, the presence of opposing motors leads to periodic stalling and slowing of cargos moving along the microtubule. No such effect occurs in IFT. To address these questions, we take advantage of the most advanced technologies in cryo-electron tomography and sub-tomogram averaging. After obtaining the 3D structure of IFT train complexes in the cilia of intact Chlamydomonas cells, we showed that a tug-of-war between kinesin-2 and dynein-1b is prevented by loading dynein-1b onto anterograde IFT trains in an inhibited conformation and by positioning it away from the microtubule track to prevent binding (Jordan et al., NCB 2018). These findings show how tightly coordinated structural changes mediate the behavior of such a complex cellular machine.