Biology subjects

O'Dair, M. K.

Publications and source records attributed to O'Dair, M. K..

3 recordsLinked to original sources

bioRxiv2024

Discrete protein condensation events govern calcium signal dynamics in T cells

Calcium level variations, which occur downstream of T cell receptor (TCR) signaling, are an essential aspect of T cell antigen recognition. Although coordinated ion channel activities are known to drive calcium oscillations in other cell types, observations of nonperiodic and heterogeneous calcium patterns in T cells are inconsistent with this mechanism. Here, we track the complete ensemble of individual molecular peptide-major histocompatibility complex (pMHC) binding events to TCR, while simultaneously imaging LAT condensation events and calcium level. Individual LAT condensates induce a rapid and additive calcium response, which quickly attenuates upon condensate dissolution. No evidence of cooperativity between LAT condensates or oscillatory calcium response was detected. These results reveal stochastic LAT protein condensation events as a primary driver of calcium signal dynamics in T cells. One-Sentence SummaryCa2+ fluctuations in T cells reflect stochastic protein condensation events triggered by single molecular antigen-TCR binding.

biophysics↗

bioRxiv2021

Discrete LAT condensates encode antigen information from single pMHC:TCR binding events

LAT assembly into a two-dimensional protein condensate is a prominent feature of antigen discrimination by T cells. Here, we use single-molecule imaging techniques to resolve the spatial position and temporal duration of each pMHC:TCR molecular binding event while simultaneously monitoring LAT condensation at the membrane. An individual binding event is sufficient to trigger a LAT condensate, which is self-limiting, and neither its size nor lifetime is correlated with the duration of the originating pMHC:TCR binding event. Only the probability of the LAT condensate forming is related to the pMHC:TCR binding dwell time. LAT condenses abruptly, but after an extended delay from the originating binding event. A LAT mutation that facilitates phosphorylation at the PLC-{gamma}1 recruitment site shortens the delay time to LAT condensation and alters T cell antigen specificity. These results identify a role for the LAT protein condensation phase transition in setting antigen discrimination thresholds in T cells.

biophysics↗

bioRxiv2021

Height, but not binding epitope, affects the potency of synthetic TCR agonists

Under physiological conditions, peptide-MHC (pMHC) molecules can trigger T-cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCR can also be activated by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit non-physiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCR{beta} Fab antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab-DNA ligand efficiently activates TCR as a monomer when membrane-associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for effective TCR triggering and cellular activation by Fab-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex, but that length of the DNA tether is important. Increasing the intermembrane distance spanned by Fab-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for construction of synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand. STATEMENT OF SIGNIFICANCEWe report geometric requirements for potent T cell activation by synthetic TCR ligands that mimic biophysical properties of the native pMHC ligand, but have the additional ability to activate polyclonal T cell populations. We find that increasing the space between apposed membranes at TCR binding events decreases ligand potency, but that changing the ligands binding epitope on the TCR has essentially no effect. The observed decrease in potency with increased ligand height is attributed to the longer ligands attenuated ability to trigger TCR at binding events.

biophysics↗