Dr. Gaia Pigino received her PhD in Evolutionary Biology from the University of Siena, Italy in 2007 for her studies on bio-indicators for contaminated soil. Electron microscopy (EM), one of the tools she used for this work, quickly became the central method for her research ever since. After her first postdoc in the EM Lab of the Department of Evolutionary Biology in Siena, she moved to ETH Zurich and the Paul Scherer Institute in Switzerland and was awarded an EMBO fellowship to continue her work on the cryo-EM structural analysis of ciliary components. In 2012, Dr. Pigino started her own lab at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden, Germany. The work of her lab is centered around the question how complex cellular machines self-organize. Cilia remain the main focus, where the Pigino Lab investigates fundamental functional aspects of the dynamic process required for the assembly of the cilium. Learning about self-organization and functional implications of structural aspects naturally requires to combine and further the latest imaging technologies (3D cryo-EM, tomography, correlative light and electron microscopy (CLEM), and various light microscopy techniques) with tools from biochemistry, in vitro reconstitution, and genetic engineering.
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, 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. These findings show how tightly coordinated structural changes mediate the behavior of such a complex cellular machine.