Progressive enhancement of kinetic proofreading in T cell antigen discrimination from receptor activation to DAG generation
- Cardiovascular Research Institute and Department of Biochemistry and Biophysics, University of California, San Francisco, United States
Figures
Figure 1
Kinetic proofreading discriminates binding events by half-life.
(A) The kinetic proofreading model consists of a chain of slow sequential events (Ci with rates ki) that begin once the ligand binds the receptor (kon). The chain of events must be completed before …
Figure 2
ICAM-1 adhesion improves robustness of proximal signaling live-cell biosensors.
(A) With our optogenetic system, we can control the concentration and lifetime of ligand-receptor interactions with blue light. A supported lipid bilayer (SLB) functionalized with the …
Figure 3 with 1 supplement
Measurement of receptor occupancy, binding half-life, and signaling output for evaluating proofreading strength.
(A) After adhering the cells to the functionalized supported lipid bilayers (SLBs), we expose them to a series of 3 min blue-light illuminations generating defined average ligand-binding half-lives. …
Figure 3—figure supplement 1
Simulations of ligand discrimination with and without kinetic proofreading reset pathways.
Using the architecture and parameterization of the kinetic proofreading model described in Ganti et al., 2020, we simulated the signaling output of non-proofreading and kinetic proofreading systems …
Figure 4 with 5 supplements
Kinetic proofreading starts upstream of Zap70 recruitment and progressively increases in strength at LAT clustering and diacylglycerol (DAG) generation.
(A) Collected data of Zap70 recruitment as a function of receptor occupancy (y-axis) and ligand half-life (x-axis) (left). Zap70 recruitment best fits a model of kinetic proofreading with a strength …
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Figure 4—source data 1
Data tables (.csv) of the biological replicates used to fit the degree of kinetic proofreading (n) for each biosensor.
Each row is one cell in one condition of a time course. Column ‘response_data’ is the reporter output (zap, lat, or dag) labeled in the title of the file. Column ‘resonse_lov’ is the occupancy measurement, and the column ‘half-life’ is the average ligand-binding half-life of the condition. We generated these data tables from raw images and plotted heatmaps using custom Python scripts available at https://github.com/dbritain/KP-paper, Britain, 2022.
- https://cdn.elifesciences.org/articles/75263/elife-75263-fig4-data1-v2.zip
Figure 4—figure supplement 1
Correlation of signaling output with receptor occupancy and ligand-binding half-life.
Mean reporter output (±1 std) plotted as a function of receptor occupancy (left) and ligand-binding half-life (right) for Zap70 recruitment (A), LAT clustering (B), and diacylglycerol (DAG) …
Figure 4—figure supplement 2
Zap70 data subset replicates previously reported kinetic proofreading value.
In our previous work, we failed to detect kinetic proofreading in Zap70 recruitment with supplementary bilayer adhesion. In our previous studies lacking light-independent bilayer adhesion, it is …
Figure 4—figure supplement 3
Residuals of kinetic proofreading model fits.
(A–C) Residual plots of the kinetic proofreading model fit for each reporter dataset. Heatmaps show the model fit subtracted from the underlying data, highlighting areas where the model over …
Figure 4—figure supplement 4
Output of steady-state model from Ganti et al., 2020.
This model predicts the minimum number of proofreading steps needed to achieve a desired Hopfield error rate at given ratios of cognate-ligand to self-ligand concentration and half-life ratios (Hopfi…
Figure 4—figure supplement 5
Kinetic proofreading model selection using Akaike information criterion (AIC).
We used AIC to select between models where Zap70, LAT, and diacylglycerol (DAG) each have their own unique value for the number of upstream proofreading steps (N value), models where two steps share …
Figure 5 with 1 supplement
Downstream signaling complexes reset slower than active receptors upon ligand unbinding.
(A) Schematic of reset experiment. After synchronously unbinding all receptors with intense blue light, we measure the reset rate of LOV2 unbinding, Zap70 loss, and LAT cluster dissociation. (B) …
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Figure 5—source data 1
Data tables (.csv) for each cell used for the lifetime distributions of each reporter.
Each data table is the TrackMate ‘spot-statistics’ output of one cell over four cycles of blue-light illumination (frames 0–40, 100–140, 200–240, and 300–340, with 3 s interval between frames). We calculated the lifetime of TrackIDs that existed at the start of each period of blue-light illumination that did not last for the full period and fit the resulting lifetime distributions using custom Python scripts available at https://github.com/dbritain/KP-paper (copy archived at swh:1:rev:167fa456901903740f0f3ee6a3e5324bc45207ea, Britain, 2022).
- https://cdn.elifesciences.org/articles/75263/elife-75263-fig5-data1-v2.zip
Figure 5—figure supplement 1
Bi-exponential fit of biosensor intensity reset curve is comparable to lifetime analysis from Figure 5.
As an alternative to tracking each reporter’s lifetime distribution, we tracked the intensity of identified cluster regions following blue-light inactivation. We fit the median curve of each …
Videos
Video 1
ICAM-based adhesion yields reversible LOV2 binding for optogenetically stimulated cells.
Time course of LOV2 on a supported lipid bilayer reversibly binding zdk-CARs expressed in Jurkat cells in the presence of ICAM-1 adhesion to the bilayer (light-independent cellular interaction). …
Video 2
ICAM-based adhesion yields reversible Zap70 recruitment for optogenetically stimulated cells.
Time course of reversible Zap70 recruitment in Jurkat cells in the presence of ICAM-1 adhesion to the bilayer (light-independent cellular interaction). ZAP70 is recruited to phosphorylated zdk-CAR …
Video 3
ICAM-based adhesion yields reversible LAT clustering for optogenetically stimulated cells.
Time course of reversible LAT clustering in Jurkat cells in the presence of ICAM-1 adhesion to the bilayer (light-independent cellular interaction). LAT forms clusters during active antigen …
Video 4
ICAM-based adhesion yields reversible diacylglycerol (DAG) generation for optogenetically stimulated cells.
Time course of reversible DAG generation in Jurkat cells in the presence of ICAM-1 adhesion to the bilayer (light-independent cellular interaction). DAG is generated during active antigen signaling …
Video 5
Lifetime of LOV2 and Zap70 after optogenetic signal termination.
Example cell from time course of bound LOV2 (left) and recruited Zap70 (right) loss after inactivation of antigen signaling with strong blue light. Jurkat cells expressing zdk-CAR and Zap70 …
Video 6
Lifetime of LOV2 and LAT after optogenetic signal termination.
Example cell from time course of bound LOV2 (left) and LAT clusters (right) loss after inactivation of antigen signaling with strong blue light. Jurkat cells expressing zdk-CAR and LAT biosensor …
