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Fig. 1. N-WASP deficiency causes decreased actin and Arp2/3 complex recruitment to internalizing CCS. (A-C) Internalization of individual CCS (red in merged images) in control (N-WASP^flox/flox) fibroblasts can coincide with transient recruitment to these sites (arrowheads) of actin (A), Arp2/3 complex (p16 subunit; B) or N-WASP (C) (each green in merged images) as revealed by TIRFM. As described previously (Merrifield, 2004), the transient recruitment of each component to preexisting clathrin-coated structures is followed by simultaneous disappearance of both clathrin and the respective component from the plane of TIRF illumination. Time is given in seconds; scale bar: 1 µm. (D) Quantification of recruitment of the respective components to internalizing clathrin-coated structures. All proteins were co-expressed as GFP-tagged proteins with mRFP-tagged clathrin light chain A in precursor (flox/flox) or N-WASP-defective (del/del) fibroblasts as indicated. Numbers give recruitment during CCS internalization (as a percentage) for each ectopically expressed protein. Note the significant reduction in recruitment frequencies of both actin (27.9±2.7%, n=15, 224 events) and Arp2/3 complex (p16: 24.1±4%, n=22, 151 events) in del/del cells as compared to parental controls (actin: 57.4±2.7%, n=14, 221 events; p16: 73.1±3%, n=24, 257 events). Differences between control and del/del cells were confirmed to be statistically significant using a non-parametric Mann-Whitney Rank Sum test (P<0.00001). The recruitment frequency of EGFP-N-WASP (flox/flox: 64±5.7%, n=8, 116 events; del/del: 58.8±5.4%, n=8, 107 events) was independent of the presence of endogenous protein and similar to those scored for actin and Arp2/3 complex in parental precursor cells (flox/flox).

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