Fig 2: SNX27 silencing affects the pattern of p-LAT but not p-ZAP70 microclusters at the IS. (A, C) Representative confocal images of control and SNX27-silenced Jurkat T cells at the contact surface of anti-CD3 and ICAM-1-coated coverslips, immunostained for SNX27 (red), p-ZAP70 (Tyr 319) or p-LAT (Tyr 220) (green). DAPI was used to label the nuclei. Scale bar = 5 µm. (B, D) Quantification, at the cell-coverslip optical section, of p-LAT and p-ZAP70 microclusters density. Plot in (B) shows mean ± SEM of a representative experiment of two independent ones with similar results (shCtrl = 45 cells, shSNX27 = 47 cells). Plot in (D) shows mean ± SEM of a representative experiment out of three (shCtrl = 39 cells, shSNX27 = 21 cells). Significance was determined by unpaired t test (ns, not significant; ****p < 0.0001).

Fig 3: Positive feedback links Zap70 kinase activity and LAT substrate phosphorylation with further Zap70 activation(A) Images of kinase-dead tRFP-Zap70K369R (Zap70KD) in iLID-Drop cells. Images show cytosolic and membrane planes. Scale bars, 20 µm.(B) Western blot and quantification of pY319-Zap70 for the indicated cell lines.(C) Images of tRFP-Zap70 localization in LATFFF (LAT Y171F, Y191F, Y226F) iLID-Drop cells.(D) Western blot and quantification of pY319-Zap70 in the indicated cell lines.(E) Schematic of positive feedback loop between LAT and Zap70 in the iLID-Drop clusters. No increase in pZap70 is observed in Zap70KD or LATFFF cells, demonstrating that this upstream event depends on downstream steps.For (A)–(D), cells were incubated in the dark (gray) or after 20 min of light (blue). Graphs display mean ± SEM for all 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 S3.

Fig 4: Phosphorylation of Zap70 selectively requires iron supply via transferrin-TfR system. A–E Immunoblots with phosphorylation-specific antibodies against proteins involved in proximal TCR signalling. Jurkat T cell activation was accomplished with soluble anti-CD3e + anti-CD28 for the indicated times before cell lysis. Data from 4–5 independent experiments. A After blotting, the nitrocellulose membrane was probed with anti-phospho-Zap70 (Y319). Beta actin was used as loading control. Left: Band intensity quantification. Right: Relevant parts of one representative immunoblot. B Membrane was probed with anti-phospho-LAT (Y220). After membrane stripping, beta actin was used as loading control. Left: Quantification of band intensities. Right: Relevant parts of one representative immunoblot. C After blotting, the nitrocellulose membrane was probed with anti-phospho-SLP-76 (Y145). Beta actin was used as loading control. D Membrane was probed with anti-phospho-Vav1 (Y174). Beta actin was used as loading control. E After blotting, the nitrocellulose membrane was probed with anti-phospho-Src family (Y416). Beta actin was used as loading control. F Primary T cell activation was accomplished with soluble anti-CD3e + anti-CD28 for the indicated times before cell lysis and SDS PAGE. After blotting, the nitrocellulose membrane was probed with anti-phospho-Zap70 (Y319). Beta actin was used as loading control. Left: Quantification of reduction in Zap70 phosphorylation. Right: Relevant parts of one representative immunoblot. Error bars indicate mean ± SEM. Statistical significance determined with two-way ANOVA (A–E) or one-sample t-test (F). *p < 0.05; **p = 0.01; no indication—not significant

Fig 5: 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.