Smart Tools for Mechanical Interrogation of the T cell
Biological ligand-receptor bonds are mechanosensitive - their kinetics and strength are force dependent. Living cells too are mechanosensitive. However, a big current challenge is to bridge our understanding of the molecular and cellular scales. This has particular importance in the context of T cell immune response since the T cell receptor, which is at the heart of the first steps of immune recognition, appears to be force sensitive. A missing piece in this puzzle is reliable measurement of the force generated by T cells. Very few reports exist – probably because the forces are feeble, and these cells are very sensitive to perturbations. New tools are desperately needed to enable measurement of feeble traction forces, under physiologically relevant conditions, at the same time permitting high resolution optical microscopy to reveal the architecture and dynamics of the different molecular players in force generation. At the heart of this project will be development of new tools to measure sub-nano-Newton forces applied by T cells during early contact with antigen presenting surfaces. Two approaches will be taken: “see-through printed force gauge” - based on extension of classical traction force microscopy using tools of nanolithograghy and “LCD for Watching Force” – a totally novel way to measure tiny forces using an oriented liquid-crystalline matrix.
smart substrates, innovative biophysics tools, T cell recognition and activation traction force.
Development of new tools to measure sub- nano-Newton cell forces.
Tool 1. A see-through printed force gauge, to improve resolution of traditional traction force microscopy (TFM), and to allow the simultaneous use of high-resolution surface imaging techniques (RICM, TIRF) - not possible currently.
Tool 2. LCD for Watching Force, to bypass microscopic marker based detection, and thus access much weaker forces while still allowing microscopy of all types (see above).
Proposed approach (experimental / theoretical / computational)
The two new experimental tools will be developped:
1. A see-through printed force gauge: See-trough soft protein printed substrates will be developed and the tools of TFM already developed will be adapted to measure the forces exerted on the substrate. Since the patterns will be only at the surface, the reduction in the quality of the imaging due to blurring seen in TFM, will be absent. Implement simultaneous high resolution quantitative microscopy to image the cell membrane, cell surface molecules and internal structures including the cytoskeleton.
2. LCD for Watching Force: A second aim will be to develop a totally new way to measure feeble forces by-passing microscopic marker based detection. This will remove the need of using a fluorescent channel to measure the deformation of the soft substrate, which sometimes preclude interesting use of markers for the biological object (ex. Dual membrane / cytoskeleton labelling, or TCR /actin labelling etc) Instead, detection will be based on interaction of polarized light with a deformable anisotropic medium whose distortion in response to cell-applied forces will be optically detected. For that, we plan to incorporate in our soft PDMS substrates liquid crystals that will be preordered magnetically, and will, like a LED watch, reveal deformation of the substrate upon changes in orientation, without the need of any marker, freeing one channel for bio-labelling the cells to reveal simultaneously, membrane and cytoskeletal structures in real-time.
In this collaboration, CINaM (Physics) will bring expertise on material science and the techniques of micro and nano-engineering; LAI (Biology) will bring expertise on immunology. Neither partner can hope to achieve the goals alone. LAI lacks relevant expertise in materials and patterning and CINaM lacks competence to access important biological tools. In addition, we shall benefit from the technology platform of Centuri for help with fabrication as well as computational support that will be needed for quantifying the distortion and converting detected distortions to a measured force. We hope to use this PhD as a stepping-stone to being the newly developed tools to the Centuri community.
The candidate should have an academic background in physics/engineering or biophysics. Candidates with previous experience in optical microscopy will be given preference. Reasonable competence in computer programming is expected. We look for highly motivated candidates willing to do experimental and computational work at the interface of physics and biology, in two interdisciplinary laboratories gathering physicists, biologists and medical doctors.
Is this project the continuation of an existing project or an entirely new one? In the case of an existing project, please explain the links between the two projects
2 to 5 references related to the project
 Ligand-Mediated Friction Determines Morphodynamics of Spreading T Cells, Pierre Dillard, Rajat Varma, Kheya Sengupta, Laurent Limozin. Biophys. J. 107 (11) 2629–2638 (2014).
 Biphasic mechanosensitivity of TCR mediated adhesion of T lymphocytes. A. Wahl, C. Dinet, P. Dillard, P-H. Puech, L. Limozin, and K. Sengupta. PNAS (2019) 116 (13) 5908-5913.
 Membrane tube extrusions as mechanical probes of the cytoskeleton-receptor interaction, Fabio Mancaa, Gautier Eicha, Omar N’Dao, Lucie Normand, Kheya Sengupta, Laurent Limozin and Pierre-Henry Puech. In preparation.
 Size-Tunable Organic Nanodot Arrays: A Versatile Platform for Manipulating and Imaging Cells, Fuwei Pi, Pierre Dillard, Ranime Alameddine, Emmanuelle Benard, Astrid Wahl, Igor Ozerov, Anne Charrier, Laurent Limozin, and Kheya Sengupta, Nano Letters, 15 (8) 5178-5184 (2015).
 Protocol for measuring weak cellular traction forces using well-controlled ultra-soft polyacrylamide gels. Farah Mustapha, Kheya Sengupta, and Pierre-Henri Puech. Cell Press STAR Protocols (accepted, Dec. 2021).
3 main publications from each PI over the last 5 years
1. First-Principle Coarse-Graining Framework for Scale-Free Bell-Like Association and Dissociation Rates in Thermal and Active Systems, J. A. Janes, C. Monzel, D. Schmidt, R. Merkel, Rudolf, U. Seifert, K. Sengupta, A-S. Smith. Phys. Rev. X (2022) 12 (3) 031030.
2. Physics of Organelle Membrane Bridging via Cytosolic Tethers is Distinct From Cell Adhesion Frontiers in Physics (2022).
3. Ligand Nanocluster Array Enables Artificial-Intelligence-Based Detection of Hidden Features in T-Cell Architecture, Nano Letters (2021). DOI: 10.1021/acs.nanolett.1c01073.
1. Mechanotransduction as a major driver of cell behaviour: mechanisms, and relevance to cell organization and future research Puech Pierre-Henri* and Bongrand Pierre*. Open Biol. 2021 Nov;11(11):210256. doi: 10.1098/rsob.210256
2. Controlling T cells shape, mechanics and activation by micropatterning. A. Sadoun, M. Biarnes-Pelicot, L. Ghesquiere-Dierickx, A. Wu, O. Théodoly, L. Limozin, Y. Hamon*, P.-H. Puech*, Sci Rep. 2021 Mar 24;11(1):6783. doi: 10.1038/s41598-021-86133-1
3. Single-cell immuno-mechanics: rapid viscoelastic changes are a hall-mark of early leukocyte activation Alexandra Zak, Sara Violeta Merino Cortés, Anaïs Sadoun, Farah Mustapha, Avin Babataheri, Stéphanie Dogniaux, Sophie Dupré-Crochet, Elodie Hudik, Hai-Tao He, Abdul I Barakat, Yolanda R Carrasco, Yannick Hamon, Pierre-Henri Puech, Claire Hivroz, Oliver Nüsse, Julien Husson, Biophys J. 2021 May 4;120(9):1692-1704. doi: 10.1016/j.bpj.2021.02.042