Fig 2: Clustering is required for light-induced Zap70 and LAT phosphorylation(A) Schematic of the Zap70:LAT interaction. Zap70 is phosphorylated on Tyr319 and then phosphorylates LAT rapidly at Tyr171, 191, and 226 and slowly at Tyr132.(B and C) Western blots and quantification of phospho-LAT Y132 and Y191 (B) and pY319-Zap70 (C) in the dark and after 20 min of blue light stimulation.(D) Schematic showing predicted responses to optogenetic clustering of LAT and Zap70 versus dimerization between LAT and Zap70K362E, where the Zap70 mutation could potentially enable LAT phosphorylation from dimers alone.(E) Cartoon of iLID-Only Zap70 K362E.(F) Quantification of LAT pY191 and pY132 in cells expressing iLID-Only Zap70 K362E.(G) Quantification of the integrated signaling response from ErkKTR-irFP (C/N ratio) and GCaMP (fold-change).Boxes represent 25th–75th percentile, center line shows the mean, and whiskers show minimum and maximum values. n = 30 data points are shown from three different experiments. Statistical comparisons were performed using Student’s t test across all independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also Figure S2 and Video S5.

Fig 3: Analog amplifier model for the mechanism of coagonism. Nonstimulatory pMHC molecules recruit CD8-Lck into the immunological synapse. Preconcentrated Lck further phosphorylate the ITAMs of TCR-CD3? which are engaged with specific antigenic pMHC. Enhanced pCD3? provide more docking sites for Zap70 to be phosphorylated. Enhanced pZap70 signal amplifies the downstream TCR signaling pathway including the phosphorylation of Erk. pErk translocates into the nucleus and enhances the effector functional readouts of T-cell activation

Fig 4: Development of optogenetic systems to compare Zap70:LAT oligomerization states(A) Schematic of TCR signaling and how optogenetic approaches can plug in at the step of Zap70:LAT clustering; see Abraham and Weiss (2004).(B) Design of the optogenetic constructs to compare dimerization and clustering of Zap70 and LAT. tRFP, TagRFP.(C) Schematic of protein complexes formed by light stimulation for the optogenetic constructs in (B).(D) tRFP-SSPB-Zap70 localization in NIH 3T3 cells. Images were taken prior to blue light illumination (gray border) or 5 min after illumination (blue border). Scale bars, 20 µm.(E) Quantification of cytosolic tRFP-SSPB-Zap70 fluorescence after illumination in both iLID-Only and iLID-Drop cells.(F) Quantification in change of the coefficient of variation (CV) of tRFP intensity for images taken in the membrane plane during 15 min of blue light illumination.For (E) and (F), envelope shows mean + SEM for at least 20 cells in each condition. See also Figure S1 and Videos S1 and S2.

Fig 5: Positive-feedback-driven Zap70 activation depends on SFK activity(A) Schematic SFK inhibitor treatment in iLID-Drop cells.(B) Western blot and quantification of phospho-Zap70 after 20-min treatment with PP1 and PP2 versus DMSO.(C) Schematic of experiments in iLID-Drop SYF cells.(D) Western blot and quantification of pY319-Zap70 in indicated cell lines.(E and F) AUC of ErkKTR-irFP (E) or GCaMP (F) responses for iLID-Drop SYF cells. Boxes represent 25th–75th percentile, center line shows the mean, and whiskers show minimum and maximum values. n = 20 cells from two different experiments.(G) Schematic of potential SFK inputs into the LAT-Zap70 feedback circuit.(H) Schematic of experiments in Src?SH2-3 iLID-Drop SYF cells.(I) Western blot and quantification of pY319-Zap70 in the indicated SYF cell lines. Bar graphs display mean ± SEM and independent biological replicates (points).All statistical comparisons were performed using Student’s t test using all independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also Figure S4.