Fig. 4: FCHO1 deficiency impairs TCR internalisation.

a FCHO1-sufficient and -deficient clones of Jurkat cells were used in the heterologous system, in which ko clones were left either non-transduced or stably transduced with wt or FCHo1 construct carrying one of the patient-associated mutations, as indicated. Cells were mock-treated (0’) or stimulated with anti-CD3 Ab (60’) at 37 °C, fixed and stained for CD3 and DAPI. Representative confocal microscopy pictures show that only wt FCHO1 facilitates the formation of CD3 puncta upon stimulation. Scale bar 5 µm. White arrowheads indicate CD3 puncta. The chart summarises data of three independent experiments in which an average number of CD3 puncta per cell is shown. Each point indicates an average number of puncta per cell that could be found in one field of view. Statistical analysis of significance was performed using ANOVA test followed by Sidak’s multiple comparison test to assess differences between groups, whiskers indicate the range (5–95 percentile). b FACS analysis of TCR internalisation in wt or FCHO1 ko Jurkat clones. Jurkat cells were stained with anti-CD3 Ab in cold and then TCR internalisation was assessed over time at 37 °C in the presence of anti-mouse F(ab’)₂ fragments labelled with Ax647. At indicated time points remaining surface TCRs were stripped, thus fluorescent signal corresponds to the internalised TCR only. The chart summarises data of one representative experiment out of three, n = 3 clones per genotype. c Intracellular Ca²⁺ flux upon TCR stimulation. Wt or FCHO1 ko Jurkat clones were loaded with Ca²⁺-sensitive FuraRed and Fluo-4 dyes and stimulated with anti-CD3 Ab and then Ca²⁺ flux was recorded flow cytometrically over time. α-CD3 and Iono indicate time points of respective stimulations. Data are representative of three independent experiments in which minimum three different clones of each genotype were analysed. d Ca²⁺ flux of FCHO1-deficient clones upon reconstitution with either wt or indicated mutants of FCHO1. GFP histogram indicates transduction efficiency. Intracellular Ca²⁺ flux was assessed as in c, representative data of two independent experiments are shown. Two FCHO1-deficient clones were analysed. b, e Statistical analysis was performed using two-way ANOVA (p-values for the effect of the genotype). Source data are provided as a Source Data file.