T lymphocyte recognition
P. Bongrand, L. Limozin, PH. Puech, P. Robert
Collab. C. Boyer (CIML), A Van der Merwe (Oxford)
The detection of foreign material by T lymphocytes is both a key step of immune responses and a model of cell decision triggering. We addressed this model by several parallel approaches based on quantitative advanced biophysical methods. We measured the dynamics of T cell membrane interaction with planar surface with nearly nanometric and subsecond resolution, using reflection interference contrast microscopy (RICM) and evanescent wave illumination (TIRF). We found that T cells could detect the presence of foreign material within less than 10 seconds with highly motile microvilli and pulling motion (J. Immunol. 2013), starting ultimately active spreading, the earliest reporter of bona fide cell activation (J. Immunol. Methods 2011). T cell could quantitatively discriminate between specific antigens of varying potency within 45 seconds after initial contact (Eur. J. Immunol 2015). Quantitative analysis of TIRF images revealed that T cell plasma membranes displayed transverse undulations of several tens of nanometer amplitude at one hertz frequency (Cell Mol Bioengineering, 2015), an efficient way of mechanosensing the nearby surface through the TCR. The importance and significance of addressed problems was discussed in two review papers (Frontiers Immunol. 2012; Annual Rev. Immunol. 2015).
Coupling AFM force mode and fluorescence microscopy for mechanotransduction studies
PH Puech, A. Sadoun
Collab. Y. Hamon, CIML.
We designed a simple method to mechanically stimulate immune cells while recording calcium pulses, and observe T cell mechanical response upon photoactivation of a small G protein (Ultramicroscopy 2016).
Biosensors in T cell activation/migration
We determined that ZAP-70 is activated upon LFA-1 engagement during T cell migration. Furthermore, we demonstrated that the kinase is involved in regulating the recruitment of talin to high affinity LFA-1 and that this process requires binding of the ZAP-70 SH2 domains to a yet to be identified partner (Biophys J 2014, Biotech. J 2014, BBRC 2015). Aside of those reporters, we developped photoactivable proteins, such as PA-Rac, in order to use the power of optogenetics to perturb cell cytoskeleton and function (Ultramiscroscopy 2016).