Fig. 1: TKS4/5 are recruited to endosomes by MT1-MMP.

a MDA-MB-231 cells stably expressing TKS5-GFP and mCh-MT1-MMP (MDA-MB-231-TKS5-GFP-mCh-MT1-MMP) were seeded on a thin layer of Alexa-647 labelled collagen-I, fixed and examined by confocal microscopy. Shown is a maximum intensity projection of a z-stack. TKS5-GFP and mCh-MT1-MMP co-localise both in linear invadopodia (left) and on endosome-like structures (right). Representative of 27 images from three experiments. b MDA-MB-231 cells were transiently transfected with mCh-MT1-MMP, fixed and immunostained for TKS5, mCh-MT1-MMP and LAMP1. Shown is a maximum intensity projection of a confocal z-stack. The mCh-MT1-MMP expressing cell (indicated by *) shows increased recruitment of endogenous TKS5 to LAMP1-positive endosomes. Representative of 17 images from two experiments. c Confocal images of HeLa cells transiently transfected with TKS5-GFP and mCh or mCh-MT1-MMP showing co-occurrence of MT1-MMP and TKS5-GFP at early (EEA1) and late (LAMP1) endosomes. Representative of 24 (left), 10 (middle) and 24 (right) transfected cells from three experiments. d Confocal images of HeLa cells transiently transfected with TKS4-GFP and mCh or mCh-MT1-MMP showing co-occurrence of MT1-MMP and TKS4-GFP at early (EEA1) endosomes. Representative of 9 (left) and 11 (right) transfected cells from three experiments. e MDA-MB-231-TKS5-GFP-mCh-MT1-MMP wt/ΔC cells were transfected with siRNA targeting endogenous MT1-MMP, stained with antibodies against GFP, mCh and a combination of EEA1 and LAMP1 (Endosomes), and analysed by confocal microscopy. Graphs represent Manders’ overlap coefficient showing mean +/− SEM from three independent experiments indicated by coloured dots. In total, 15 images with 5–7 cells were analysed per condition. Unpaired two-sided t-test.