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OnlineSeminar - Gaia Pigino, PhD - Towards a mechanistic understanding of motile and primary cilia with CLEM and cryo-electron tomography”

Research Group Leader, Max Plank Instituteof Molecular Cell Biology and Genetics, Dresden, Germany
When Jan 26, 2021
from 12:00 PM to 01:00 PM
Where Tigem On line
Contact Name
Contact Phone 08119230659
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Short CV

Research in my lab aims at understanding the molecular principles and processes that drive the self-organization of complex cellular machines, which are relevant for human health and disease. In this talk I will show how we combine EM and molecular cell biology methods to obtain a mechanistic understanding of the molecular machineries required for the assembly and the function of cilia/flagella: conserved organelles that are fundamental for most 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 of IFT trains is driven by the anterograde motor kinesin-2 and the retrograde motor dynein-1b, which are both bound to a large complex of about 25 IFT adaptor proteins. We developed a millisecond resolution 3D correlative light and electron microscopy (CLEM) approach to show that the spatial segregation of oppositely directed IFT trains on the two microtubules of each axonemal doublet ensures a collision free transport in the cilium. Then, it remained to be explained how competition between kinesin and dynein motors, both found on anterograde IFT trains, is avoided. In other bidirectional transport systems in the cell 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 this question, we used cryo-electron tomography and sub-tomogram averaging to resolve the 3D structure of IFT train complexes in the cilia/flagella of 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. We also show that dyneins are released from the train at the ciliary tip, but how this happens and how these motors are activated to power retrograde IFT is not yet understood. Protein complexes that form specialized structures at the ciliary tip are thought to be involved in this process. By mechanically manipulating IFT trains in the cilia/flagella of Chlamydomonas cells, we showed that the structures of the ciliary tip are not necessary for the conversion from anterograde to retrograde IFT. Thus, the conversion process is an intrinsic, calcium-optional ability of IFT machinery. 

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