Decoding the mechanotransduction: spatial and temporal coordination of the mechanical and biochemical signals – DeMeTr

To attain this goal, it is essential to have the dynamical control of both mechanical and biochemical cues on the same cell. To the extent of my knowledge, the measurement and the spatiotemporal control of the mechanical strains and of the biochemical activity have never been achieved together on the same experimental system. It is therefore necessary to develop and combine different state-of-the-art experimental approaches to achieve the ultimate goal of the project which is to test the signal transduction in both ways on the same cell. Practically, active traction force microscopy will be developed to exert and measure forces through the cell’s substrate. Combined with the visualization and quantification of the biochemical activity with a fluorescent sensor, this system will allow the testing of the mechanotransduction from the mechanical cues towards the biochemical response. By varying the input parameters and measuring the response, we will establish the stimulus/response dictionary in the first direction. To test the transduction in the other direction, we will develop optogenetics on this system to control spatially and temporally the activity of a signaling protein. Hence, the stimulus/response dictionary will also be established from biochemical to mechanical cues. With these results, we will be able to identify the key components defining the codes of the mechanotransduction and in particular to define the role of the mechanical characteristics of the intracellular milieu. The role of the cytoskeleton will be specifically addressed with an original nanoimaging technique allowing the visualization of nanometer deformations of the actin stress fibers which are tension cables between the adhesion sites. We will go further and with the help of adhesive micro-patterns imposing the cell morphology, we will test the impact of the cell geometry and history on the biochemical response to a mechanical stress.

After 18 months, we have achieved the first proof of principle of the active traction force microscopy: we are able to deform locally a continuous soft material and hence exert a force an adherent cell. We can arrange micromagnetic elements inside the substrate and actuate them with an electromagnet with reasonable currents. We have seen that an adherent cell is deformed through the deformation of its adherent substrate. This is a confirmation of the major hypothesis of the project: we can apply mechanical stresses to the cells by deforming their adherent substrate.

Now that our approach has been validated, the next step is to enhance the method and make it reproducible. We want to align the patterns of micromagnets with the adhering patterns for cells so as to have one actuator per cell, located at a defined distance and position with respect to the cell. We are still working on the fluorescence microscope developing ALEX-FRET on live cells and combining it with a confocal type excitation for optogenetics.

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Mechanotransduction is a very large and complex process which allows cells and tissues to sense and respond to their physical environment. Through a number of dynamical mechanisms, physical properties such as substrate rigidity, deformations or forces are translated into biochemical signals. The different biochemical signals are in turn integrated by the cells which consequently modulate functions which are crucial for their viability (motility, adhesion, proliferation …). Defects in the mechanotransduction machinery are implied in numerous diseases like cancer growth, cardiac and bone pathologies. The signal transduction has been shown to occur in both directions from the mechanical cues to the biochemical signals and vice versa, the overall process implying feedback loops. However, the coordination in space and time of the mechanical and biochemical signals is not yet clear and rarely addressed. In this fundamental work, we will address the mechanotransduction from an original point of view: we will consider the cell as an active and dynamic system which filters and transforms signals in both directions. The main goal of this project is to decode the mechanotransduction, in other words to establish the dynamical dictionary between mechanical and biochemical cues.
To attain this goal, it is essential to have the dynamical control of both mechanical and biochemical cues on the same cell. To the extent of my knowledge, the measurement and the spatiotemporal control of the mechanical strains and of the biochemical activity have never been achieved together on the same experimental system. It is therefore necessary to develop and combine different state-of-the-art experimental approaches to achieve the ultimate goal of the project which is to test the signal transduction in both ways on the same cell. Practically, active traction force microscopy will be developed to exert and measure forces through the cell’s substrate. Combined with the visualization and quantification of the biochemical activity with a fluorescent sensor, this system will allow the testing of the mechanotransduction from the mechanical cues towards the biochemical response. By varying the input parameters (frequency, amplitude …) and measuring the response, we will establish the stimulus/response dictionary in the first direction. To test the transduction in the other direction, we will develop optogenetics on this system to control spatially and temporally the activity of a signaling protein. Hence, the stimulus/response dictionary will also be established from biochemical to mechanical cues. With these results, we will be able to identify the key components defining the codes of the mechanotransduction and in particular to define the role of the mechanical characteristics of the intracellular milieu. The role of the cytoskeleton will be specifically addressed with an original nanoimaging technique allowing the visualization of nanometer deformations of the actin stress fibers. We will go further and with the help of adhesive micro-patterns imposing the cell morphology, we will test the impact of the cell geometry and history on the biochemical response to a mechanical stress. Hence, we can tackle the question of the mechanotransduction universality by looking whether the response is particular or not to cell geometry, history or cell types.
In a word, this work will allow the understanding of the dynamical parameters which regulate the mechanotransduction and will clarify the relationship between mechanical properties and specific cellular responses. We hope that the decoding of mechanotransduction will pave the way for a new way of treating unhealthy cells beyond the conventional biochemical drugs.