Reconstitution and Characterization of Membrane Proximal Signaling Downstream of the T Cell Receptor
- DeGrandchamp, Joseph B
- Advisor(s): Groves, Jay T
Abstract
Early warning signals in the adaptive immune system originate from T Cell recognition of pathogenic antigens on the surface of Antigen Presenting Cells (APCs). T Cell activation and the production of warning molecules (cytokines) proceeds downstream of antigen binding to T Cell Receptor (TCR) at T Cell:APC membrane interfaces. Complex proofreading mechanisms are required to control T Cell activation, and this process is thought to take place primarily via TCR proximal signaling events on the inner T Cell membrane. A number of critical components for the origination of T Cell activation signals are well known, including the membrane-associated scaffold LAT that becomes phosphorylated following TCR-antigen binding events. LAT recruits a web of cytosolic adapters, like Grb2, and signaling enzymes, such as Sos1 and PLCγ1. LAT has been recognized as a signaling hub wherefrom both proliferation signals, through Sos1 activation of Ras, and cytokine production signals, through PLCγ1-mediated Calcium flux, diverge. Additionally, this hub has been established as a biomolecular membrane protein condensate, with multivalent crosslinking through Grb2 and Sos1 interactions as well as more recently discovered crosslinking through Grb2 dimerization and the SH array of PLCγ1. The consequences of condensation are not yet fully understood, but in new accounts from our lab, it has been suggested to encode information relevant for T Cell activation. Vital to the study of this system, we have established an in vitro platform for the study of fluorescently labeled proteins on supported bilayers with visualization through TIRF microscopy. LAT functionalized to these bilayers is capable of both recruitment of solution proteins and forming condensates, and signals from recruited enzymes can be detected using labeled biosensors. This allows for the piece-wise investigation of mechanisms in the formation of this central hub. Herein, I cover our recent efforts to increase reproducibility in the use of this platform and expound on the recent developments in the modes of LAT crosslinking leading to condensation. Then, I demonstrate the effect of LAT condensation on active PLCγ1 from mammalian cell lysate, most notably a marked decrease in activity in pLAT:Grb2 condensates. I conclude with single particle tracking experiments of two Ras isoforms that reveal functional differences in their interactions with the membrane as a function of lipid composition. These stand to be important contributions to the understanding of TCR signaling and will empower future discovery.