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Fig. 6. Dynamics of EGFR signalling components during clathrin pit internalization. (A-C) Recruitment of EGFR (A), Grb2 (B) and Sos (C) to internalizing CCS (red in merged images) as visualised by TIRFM in N-WASP^flox/flox cells. All EGFR signalling components were visualised by co-expression of the GFP-tagged variants (green in merged images) with mRFP-tagged clathrin light chain A (red in merged images) as indicated. Time is given in seconds; scale bar: 2 µm. (D) Quantification of the recruitment frequencies of these proteins during CCS internalization in N-WASP^flox/flox (EGFR: 87.1±2.6%, n=13, 230 events; Grb2: 82.5±3.1%, n=13, 226 events; Sos: 79.4±2.1%, n=21, 298 events) versus N-WASP^del/del (EGFR: 87.5±2.3%, n=10, 181 events; Grb2: 77.5±3.2%, n=12, 196 events; Sos: 88.2±3%, n=10, 142 events) fibroblasts as indicated. The reduction of actin assembly in N-WASP knockout cells (see Fig. 1D) does not influence the frequency of association of the EGFR signalling components tested here with those CCS capable of internalization.

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