15 Novembre – Thesis defense - Ani Augustine Jose

09 h30 Amphi - Institut d'Optique d'Aquitaine (Talence)

RESOLFT Nanoscopy to study cellular adhesions.

Cells can adjust their adhesive and cytoskeletal organizations depending on the changes in the biochemical and physical nature of their surroundings. In return, cells can also control their microenvironment by adhering and generating forces on neighboring cells and extracellular matrices. Integrin-dependent adhesion sites (AS) are the converging zones of these interactions, integrating biochemical and biomechanical signals between intracellular and extracellular components. Various cellular functions linked to AS are associated with the formation and cohesion of tissues. Cancer cells that spread through the circulatory system use mechanisms associated with cell adhesion to establish new tumors in the body.
Nanoscopic techniques have revolutionized the study of biological structures by bringing sub-diffraction imaging into the realm of light microscopy. RESOLFT (Reversible Saturable Optical Fluorescent Transitions) break the diffraction barrier by suppressing the volume of emission through a reversible on-off process. We studied the photophysics of a reversible switchable protein called rsEGFP2 expressed in living cells. The parameters for photoswitching obtained from these studies were used to implement a RESOLFT nanoscope. Using this system, we have demonstrated an imaging resolution close to 50 nm, a 4-fold improvement over the diffraction limit. RESOLFT nanoscopy requires less intensity to obtain a super-resolved image, thereby reducing the effect of photobleaching and enabling long term live cell imaging.
We then used the RESOLFT nanoscope to study the initiation, stabilization, and disassembly of AS. Using this technique, we were able to study the dynamic reorganization of various proteins involved in the formation of AS on mouse embryonic fibroblasts and ovarian tissues of drosophila melanogaster. We observed clusters of β3-integrin-rsEGFP2 within the AS that are smaller than the diffraction limit, demonstrating RESOLFT's ability to study the nanoscale organization of proteins in the AS. We also observed a rapid reorganization of the β3-integrin-rsEGFP2 clusters within the AS, with the cluster lifetime only being a few tens of seconds. These data suggest a model of constant remodeling of AS protein clusters. In the case of Talin rsEGFP2, we observed a rearward flow of clusters towards the interior of the cell. The speed of this flow was measured and was found to be close to the speed measured by other techniques like single-molecule tracking. We also studied the dynamics of zyxin nanoclusters in AS. We recorded nanoscale fluctuations of zyxin levels in AS, and our experiments indicate a link between this fluctuation and mechanical forces existing inside AS.
To enable faster imaging, we parallelized our RESOLFT setup using optical lattices. Optical lattices are periodic patterns of light formed through interference. Compared to our first implementation of RESOLFT, which probes only a single point at a time, optical lattices can be used to probe multiple points simultaneously. We also implemented a second lattice for fluorophore on switching, which helped us to reduce the effects of photobleaching during imaging. The dual lattice setup allowed us long term imaging (>40 frames) of nanostructures with resolution close to 55 nm inside living cells, at a frame rate of 0.3kHz over a 15x15μm field of view.

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