Tomas Kirchhausen, M.S., Ph.D. - "Cellular Dynamics imaged in real time and in 3D: the mechanism of intraluminal vesicle formation mediated by ESCRT-III and Vps4"
Mar 02, 2017
from 12:20 PM to 01:30 PM
|Where||Tigem Auditorium "Vesuvius"|
|Contact Name||Andrea Ballabio|
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Four years ago it would have been unheard off. Three years ago, it was a dream. Now, it’s a reality. By using ultra-thin sheets of light to rapidly illuminate biological samples with extremely low photon doses, 3D experiments previously limited to seconds or minutes by photo-bleaching or by photo-toxicity, can now be extended to hours or days. The lattice light sheet microscope invented by Eric Betzig is a new fluorescence visualization tool that can image living cells in action, from single molecules to organelle biogenesis to cell migration, with unprecedented duration at diffraction limited resolution and high-temporal resolution. The talk will illustrate our most recent efforts mainly using the lattice light sheet microscope to ‘see’ in three dimensions processes that mediate and regulate the movement of vesicular carriers throughout cells and the biogenesis of organelles in both, isolated cells maintained in tissue culture conditions and cells within tissues of a living zebrafish embryo. It will also illustrate our analysis of the recruitment dynamics of ESCRT-III and Vps4 revealed the mechanism of reverse membrane budding and fission on endosomes. The data is consistent with a model whereby that Vps4 acts as a ATP-dependent molecular ratchet to stochastically mediate the clustering of relatively short curved ESCRT-III filaments; at early stages, the uncoordinated ATP-dependent ratchet activity of several Vps4 hexamers promotes membrane bulging, i.e. ILV budding; at later stages, the rachet activity facilitates the apposition of the opposing membrane leaflets of the bulged cone leading to membrane fusion, release of the ILV and creation of MVBs.