Massive Parallelization of STED Nanoscopy to study the fast spatiotemporal orchestration of proteins in cell adhesion – FastNano
Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on neighboring cells and extracellular matrices (ECM) cells control their microenvironment, shape and movement. Integrin-dependent adhesion sites (AS) are the converging zones integrating biochemical and biomechanical signals arising from the extracellular environment and the intracellular actin cytoskeleton. Thus, assembly/disassembly of AS and actin networks control critical cellular functions including adhesion, mechano-transduction, migration, growth, differentiation and apoptosis. All those functions are implicated in tumor progression, which are often associated with integrin dysregulation. Integrin signaling is increased in tumors and reduced when ECM stiffening is prevented, indicating functional links between integrins and tumor mechanics. Acquisition of a metastatic phenotype is also accompanied by changes in the expression of glycans and glycoproteins. Such molecules dictate the physical properties of the glycocalyx, the primary space across which information flows from the microenvironment to membrane receptors. Since both integrins and their ligands are tethered their interactions are strongly influenced by mechanical constraints, and therefore the glycocalyx could influence integrin spatial organization and function. Yet, whether the molecular mechanisms of integrin activation are fundamentally different in tumoral cells is still unknown.
At the molecular, sub-cellular, and cellular levels, cell shaping and migration proceed through cycles lasting from seconds to minutes. During those cycles, critical proteins undergo motions and transient interactions that are essential to their functions. Therefore, the most challenging problem is to decipher how protein nanoscale dynamic organization and interactions could merge into those specific deterministic cellular responses.
The development of super-resolution microscopy techniques has revolutionized biomolecular imaging in cells. The observation of single biomolecule has enabled their localization and tracking at the nanometer scale. The close synergistic collaboration between our groups (Lounis, Giannone) over the last 4 years led to the development of new strategies for super-resolution imaging and single protein tracking (spt). Using these approaches, we unraveled the key spatiotemporal molecular events leading to integrins activation by their activators in mature AS. However super-resolution microscopies based on single molecule localization have very limited imaging speed, preventing to access the global nanoscale dynamic organization of macromolecular structures, occuring at second time scale.
Freed from the constraint of sequentiality of single molecules emission, STED (Stimulated Emission Depletion) and RESOLFT (Reversible Saturable OpticaL Fluorescence Transitions) nanoscopies could in principle offer better temporal resolution. However, they remain point-scanning techniques, which need parallelization for fast wide field imaging.
In this project, we will develop new fast wide-field nanoscopies. Massive parallelization of STED and RESOLFT will be achieved using well-designed optical lattices for depletion and off-switching respectively. We will study protein nanoscale reorganization and interactions in AS using 1- and 2-color fast nanoscopies. Fast nanoscopy will be combined with spt to understand how protein dynamics control AS life-cycle. Using parallelized STED we will develop a fast traction force nanoscopy technique to map forces within an AS at the nanoscale. By combining these techniques, we aim to: unravel the mechanisms controlling the initiation, stabilization and disassembly of early AS; reveal how the dynamic landscape of interaction in AS lead to mechano-transduction; and understand how the extracellular glycocalyx foster the formation of AS in cancer cells.
Project coordination
Brahim Lounis (Laboratoire Photonique Numérique et Nanosciences)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partner
IOGS (Institut d'Optique théorique et Appliquée) Laboratoire Photonique Numérique et Nanosciences
IINS Institut Interdisciplinaire de Neurosciences
Help of the ANR 466,988 euros
Beginning and duration of the scientific project:
September 2014
- 48 Months