The link between CD44v6 and the cytoskeleton through Ezrin is required for Met internalization.

<p>(<b>A</b>) HeLa cells were transfected with a rat CD44v4-7 construct deleted from the cytoplasmic domain (CD44v4-7Δcyt) or a control vector. The kinetic of Met internalization upon HGF induction was measured in a MESNA experiment (Material and Methods). In the first sample cells were kept at 4°C. –M refers to a sample obtained from cells that were not treated with MESNA. The Western Blot analysis was performed with a Met specific antibody and the rat specific CD44v6 antibody 1.1ASML (Material and Methods). (<b>B</b>) HeLa cells were transfected either with human CD44v6 wild type or with human CD44v6 tailless constructs as indicated. After 24 hours cells were serum starved, treated with 50 µM cycloheximide for 2 hours prior to HGF-induction, stimulated with 25 ng/ml HGF for 1 hour on ice (cold start) and incubated at 37°C for the indicated time periods. Cells were then fixed, stained, and imaged using a confocal microscope (Leica SPE) with a 63× objective. Met (red), CD44v6 wild type (green), CD44v6 tailless (green), Dapi (blue). Scale bar = 15 µm. The quantification of three independent experiments (n = 20) is shown. The percentage of transfected cells with Met exclusively located on endosomes or exclusively at the plasma membrane or on both was calculated for each time point. Values are the means of three independent experiments. Student´s t test: *p<0,05 and **p<0,01. (<b>C</b>) HeLa cells were transfected with an Ezrin construct deleted from the actin-binding domain (EzrinΔABD) or a control vector. The cells were serum starved for 24 hours followed by biotinylation (0,5 mg/ml), induced with HGF (50 ng/ml) and treated with MESNA. Upon cell lysis proteins were pulled down by NeutrAvidin beads. The precipitates were subjected to Western Blot analysis for Met and CD44v6. For the first sample the cell were kept at 4°C. –M: cells were not treated with MESNA. The experiment was repeated at least three times.