Investigation of mechanotransduction by means of optical tweezers

In living cells and organisms there is a myriad of biomolecules that are able to activate specific cellular pathways, but in mechanotransduction the same physical stimulus - that is the force - modulates a broad range of cellular function. This is due to the fact that cells in a physiological environment are exposed to a series of mechanical stresses caused mainly by tension, compression and shear stress. Cells, independently from the phylogenetic branch, from arkea to eukarya, react to the mechanical stimulations by triggering responses ranging from cytoskeletal rearrangement to epigenetic remodeling and lineage specification. In mammals, the mechanical forces are mainly transduced by specialized sensory neurons, providing the basis of the touch, hearing and proprioception. Recent studies suggest that also other kind of non-specialized neurons, such as the olfactory neurons, and possibly all the cells of the body are able to transduce the mechanical stress. The main aim of this thesis is to study the neuronal mechanotransduction pathway at pN range, a physical level that cells experience in vivo. In order to exert controlled mechanical stimulations in the pN range, we established a new method using an optical tweezers with a polystyrene microbead in an oscillatory optical trap. In this way it is possible to touch the cell in the vertical direction and to analyze cellular responses to forces in the range of 5–20 pN. In the first paper we have demonstrated that weak mechanical forces in the range of 5-20 pN, can produce membrane nano-indentation and intracellular Ca2+ transients in mouse neuroblastoma NG 108-15 cells dependent on the strength stimuli and frequency. One of the quickest way a cell responds to the force is the opening of transmembrane ion channels, referred as mechanosensitive channels (MSCs) that allow an ionic flux, with highly selected permeability within few microseconds. By confirming the possible existence of MCS channels in the NG108-15 we have found a relatively high background expression of the mechanosensitive channel Piezo1. Through the utilization of the specific MCSs inhibitor, GSMTX-4, we have demonstrated that Ca2+ response is strictly dependent on the opening of MCSs. In the second work, we have investigated in a more detailed way the neuronal mechanotransduction pathway using rat hippocampal neurons. We have noticed that the forces applied at the level of hippocampal growth-cone behave as a repulsive stimulus, inducing both the retraction and turning. The same forces repeatedly applied to the soma evoke its shrinkage. In order to see how actin filaments reorganize themselves inside the neuron during mechanical indentation we transfected hippocampal neurons with LifeAct-mCherry. We found that the mechanical indentation lead to a rapid re-organization of the actin cytoskeleton. In particular, in response to mechanical stress the actin network gradually disappears in the indentation area, followed by a retraction of few microns of the actin network. We have also observed that hippocampal neurons display intracellular Ca2+ elevation in response to mechanical indentation. Interestingly, we found that OT indentation activates the small G protein RhoA potentially leading to a reorganization of the cytoskeleton that we previously observed. Finally, immunochemistry shows that Piezo1 channels are expressed over the entire membrane of hippocampal neurons. In the third work we have demonstrated the integration of the patch-clamp electrophysiology with a pulsed optical tweezers, showing preliminarly that piconewton forces applied vertically on the cell membrane induce detectable inward ion currents. All the results obtained in this thesis altogether, show that mechanical signaling operates for very weak forces and outline the molecular events underlying this exquisite mechanosensitivity.