Fig. 2: TKS4/5 are recruited to endosomes by coincidence binding of their PX domain to MT1-MMP and PtdIns3P.

a HeLa cells were transfected with mCh or mCh-MT1-MMP wt or ΔC and cotransfected with the indicated TKS5 constructs. The localisation of TKS5 was scored by fluorescence microscopy and example images with and without endosomal recruitment are displayed. Graph shows the percentage of cells with a dot-like localisation of the respective TKS5 construct as mean +/− SEM from n = 3 independent experiments. At least 100 cells were counted per condition and experiment. One-way ANOVA of each individual construct set of 3 conditions with Tukey’s multiple comparisons test. b Predicted 3D protein structure of the PX domains from human TKS5 and TKS4 using AlphaFold2 via ColabFold. Arginine residues R42/R43 and R93/94 are highlighted. c Co-immunoprecipitation experiment using HeLa cells transfected with TKS5-GFP and mCh-MT1-MMP wt or ΔC. mCh-MT1-MMP wt, but not ΔC can be co-immunoprecipitated with TKS4- or TKS5-GFP. Representative of three independent experiments. d Co-immunoprecipitation experiment using HeLa cells transfected with mCh-MT1-MMP wt and TKS5-GFP wt, R42A, R93A or the double mutant R42A/R93A (“µµ”). mCh-MT1-MMP wt can be co-immunoprecipitated with TKS5-GFP wt and the R42A mutant, but not when R93 is mutated. Representative of three independent experiments. e Mannose-binding-protein (MBP) pulldown experiment. Purified MBP-TKS5 PX wt, but not MBP-TKS5 PX µµ binds to MT1-MMP from MDA-MB-231 cell lysate. Purified MBP alone serves as a negative control. Graph shows the relative mean intensity of the MT1-MMP band quantified from n = 3 independent experiments +/− SEM. One-sample two-sided t-test. f HeLa cells were co-transfected with mCh-MT1-MMP wt and TKS4- or TKS5-GFP and treated with DMSO or 6 µM SAR405 for 15 min, stained with antibodies against GFP and mCh and analysed by confocal microscopy. Graphs represent the mean fluorescence intensity of TKS4-GFP or TKS5-GFP in mCh-MT1-MMP dots, measured automatically by NIS-Elements. Error bars denote mean +/− SEM from n = 3 independent experiments indicated by coloured dots. TKS4-GFP: In total 60 (DMSO) and 53 (SAR405) cells were analysed. TKS5-GFP: In total, 60 cells were analysed per condition, unpaired two-sided t-test.