Magnetic Manipulation of Intracellular Signaling

Abstract

In vivo imaging has shown how the establishment and maintenance of cell polarity relies on complex mechanisms by which signaling cascades become regulated at sub-cellular levels. How signaling networks are spatio-temporally coordinated into a polarized cell is not elucidated. In this context, we developed a new tool to locally probe and perturb signaling pathways inside living cells.In our approach, magnetic nanoparticles (MNPs) functionalized with active proteins are inserted in the cytosol of mammalian cells where they behave as solid signaling platforms. The precise control of MNPs surface chemistry allows to probe the self assembly of macromolecular complexes directly inside the cell. By exerting magnetic forces, MNPs are then manipulated in the cytosol to position their signaling activity at different subcellular locations. The cellular response to this spatially resolved biochemical perturbation is finally quantified in term of effector recruitment and cytoskeleton/membrane dynamics.We show that MNPs of different sizes, from 50nm to 500nm in diameter, can be used to generate different spatial perturbation patterns. While the biggest MNPs are trapped in internal structures of the cell and require large forces (>10 pN) to be displaced, they allow us to create precise point-like perturbations at the cell periphery. At the opposite, smallest MNPs diffuse fast in the cytosol (∼1μm²/s) and are used to create gradient of signaling activity spanning over the whole cell.We apply our technique to the Rho-GTPase signaling network which orchestrate cell polarity and migration. MNPs were functionnalized with GEF or active GTPase. The effect of intracellular GTPase gradient (rac, cdc42) on cell polarity was analyzed. We demonstrated that the pathway linking Rac1 to actin polymerization is spatially restricted to the protrusive areas of the cell by transporting TIAM1 particles at different subcellular locations while monitoring GTPase activation and actin polymerization.