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First published online 28 June 2005
doi: 10.1242/jcs.02444

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N-WASP deficiency impairs EGF internalization and actin assembly at clathrin-coated pits

Stefanie Benesch^1,*, Simona Polo², Frank P. L. Lai¹, Kurt I. Anderson³, Theresia E. B. Stradal⁴, Juergen Wehland⁴ and Klemens Rottner^1,

¹ Cytoskeleton Dynamics Group, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany
² IFOM, the FIRC Institute for Molecular Oncology Foundation, Via Adamello 16, 20139, Milan, Italy
³ Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
⁴ Department of Cell Biology, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany

View larger version (74K): [in a new window]   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-WASPflox/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).

View larger version (79K): [in a new window]   Fig. 2. Clathrin pit internalization in N-WASPdel/del cells. (A,B) Images show representative examples of CCS (red in merged images) internalizing with (B) or without (A) detectable actin (green in merged images) assembly. In these cells, the majority of pit-like structures (72.1%, see also Fig. 1D) disappeared without detectable actin recruitment (A). The remaining pits (B) recruited actin in a manner that was indistinguishable in intensity to parental control cells (C). Time is given in seconds; scale bar: 2 µm. (C) Columns represent average peak fluorescence intensities shortly before internalization of the components indicated, which were assessed in those CCS recruiting actin in both cells types. For neither component, significant differences between the two cell types could be scored.

View larger version (72K): [in a new window]   Fig. 3. Actin assembly at CCS is Arp2/3 complex dependent. TIRFM images of N-WASPflox/flox fibroblasts transiently co-expressing mRFP-clathrin light chain A (red in merges), EGFP-actin (green in merged images), and either WA-Scar1 or W-Scar1. Note the disappearance of CCS in cells expressing WA without any detectable coincident actin polymerization (asterisks) and the apparent actin assembly at disappearing CCS in the W-expressing control (arrowheads). Time is given in seconds; scale bar: 1 µm. The quantifications (bottom) of actin recruitments during CCS internalization revealed a complete abolition upon WA over-expression (0%, n=16, 110 events) and levels comparable to wild-type cells (Fig. 1D) in W-expressers (69.9±2.7%, n=16, 223 events).

View larger version (149K): [in a new window]   Fig. 4. N-WASP pits do not incorporate WAVE2. (A,B) Representative examples of cells co-expressing EGFP-tagged N-WASP (green in merged images) and mRFP-WAVE2 (red in merged images) analysed by TIRFM. (A) N-WASP readily accumulates in pit-like structures, which were entirely devoid of detectable WAVE2. Arrowheads indicate examples for pit-like N-WASP-containing structures disappearing in the course of the experiment. Arrow indicates pit-like structure that has appeared in the same time period. None of these structures displayed detectable WAVE2 enrichment. (B) Episodes of N-WASP accumulation and disappearance with high time resolution. Time is given in seconds. Scale bars: 3 µm (A) and 1 µm (B).

View larger version (54K): [in a new window]   Fig. 5. N-WASP deficiency causes a reduction of EGF internalization. (A) After 3 hours of starvation, cells were incubated with rhodamine-EGF at 37°C for 10 minutes and directly processed for fluorescence microscopy. Red, rhodamine-EGF; blue, DAPI nuclear staining. (B) After 3 hours of starvation, cells were incubated with 1.5 ng/ml of 125I-EGF at 37°C for the time points indicated. The amount of surface and internalized radioactivity was determined at the end of the incubation. After correction for non-specific binding, the rate of internalization was expressed as the ratio between internalized/surface radioactivity (mean ± s.e.m. from three independent experiments). The differences were confirmed to be statistically significant (P<0.032 by linear regression analysis).

View larger version (68K): [in a new window]   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-WASPflox/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-WASPflox/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-WASPdel/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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