JCS02444 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Arp2/3 complex recruitment in N-WASP knockout cells. Representative example of clathrin-coated structures (CCS) scored positive for Arp2/3 complex recruitment during internalization in the absence of functional N-WASP. N-WASP^del/del cells were co-transfected with mRFP-clathrin light chain A (red in merged images) and EGFP-tagged p16 (green in merged images) as indicated and subjected to time-lapse TIRF microscopy. Arrowheads indicate a CCS co-internalizing with Arp2/3 complex in the absence of functional N-WASP. Time is given in seconds; bar, 1 mm.

• Supplemental Figure 2 -

  Fig. S2. Co-localisation of N-WASP and actin in pit-like structures. TIRFM images of cells co-expressing GFP-tagged N-WASP and mRFP-actin. In cellular regions devoid of actin-rich stress fibres or focal adhesions (not shown), both actin and N-WASP accumulated at and co-localised in pit-like structures (arrows) in a pattern that was virtually identical. As expected, N-WASP was absent from focal adhesions (not shown). Bar, 2 mm.

• Supplemental Figure 3 -

  Fig. S3. Expression of WIP and Nck proteins in the presence and absence of functional N-WASP. Extracts from non-transfected N-WASP^flox/flox and N-WASP^del/del cells or from both cell lines transfected with EGFP-tagged murine WIP were subjected to western blotting employing a polyclonal anti-WIP antiserum, followed by reprobing of the membrane with monoclonal anti-Nck antibodies and staining with Coomassie Blue as indicated. The polyclonal anti-WIP antiserum was raised in rabbits against a synthetic peptide derived from the rat sequence 453-VPTTKTYPSKVARSESRSGSNRRERGA-479, and affinity-purified using the same peptide immobilized on CNBr-Sepharose 4B (Amersham Biosciences). The specificity of the antiserum for WIP recognizing a polypeptide of approx. 59 kDa in non-transfected extracts was confirmed by detection of ectopically expressed EGFP-tagged murine WIP (asterisk). Monoclonal anti-Nck antibody was purchased from Transduction Laboratories. Molecular masses (in kDa) are given on the left.

• Supplemental Figure 4A
• Supplemental Figure 4B
• Supplemental Figure 4C -

  Fig. S4. WIP and Nck dynamics at disappearing CCS. (A,B) TIRFM images of control (A, flox/flox) and N-WASP^del/del (B) fibroblasts co-expressing EGFP-tagged WIP or Nck2 (green in merged images) with mRFP-tagged clathrin light chain A (red in merged images). Similar to actin and N-WASP, CCS (arrowheads) internalization is frequently preceded by accumulation of the WASP interacting protein family member WIP as well as the SH2/SH3-adaptor protein Nck2. Time is given in seconds; bars, 1 mm. (C) Quantification of WIP and Nck2 recruitment to internalizing CCS. The EGFP-tagged proteins were co-expressed with mRFP-tagged clathrin light chain A in precursor (flox/flox) or N-WASP-defective (del/del) fibroblasts as indicated. Numbers are percentage recruitments during CCS internalization for each ectopically expressed protein. Note the robust recruitment frequencies of both WIP (80.8±6.1%, n=11, 115 events) and Nck2 (79.6±4.5%, n=11, 134 events) in flox/flox cells and the slight reduction in N-WASP^del/del cells (WIP: 71.3±3.4%, n=15, 166 events; Nck2: 65.2±4.1%, n=20, 180 events). For Nck2, the difference was found to be statistically significant (Mann-Whitney Rank Sum test; P<0.03).

• Supplemental Figure 5 -

  Fig. S5. Accumulation of haematopoietic WASP at CCS. TIRFM images of N-WASP^flox/flox fibroblast co-expressing mRFP-tagged clathrin light chain A (red in merged images) and EGFP-tagged WASP (green in merged images). As observed for N-WASP, the haematopoietic WASP displayed transient accumulations at CCS (arrowheads) shortly before their disappearance. Time is given in seconds; bar, 1 mm.

• Supplemental Figure 6 -

  Fig. S6. Co-internalization of EGFR and N-WASP. TIRFM images of cells co-expressing mRFP-EGFR and EGFP-N-WASP. Representative examples of EGFR-containing pit-like structures, the internalization of which coincided with N-WASP accumulation. Time is given in seconds; bar, 1 mm.

• Supplemental Figure 7 -

  Fig. S7. EGFR, Grb2 and Sos accumulation in the absence of functional N-WASP. TIRFM images of mRFP-clathrin light chain A (red in merged images) and EGFP-tagged EGFR, Grb2 or Sos (green in merged images) were co-expressed in N-WASP-defective (del/del) cells. In spite of the lack of functional N-WASP, these EGFR signalling components robustly targeted to CCS, in a manner highly reminiscent of control cells. Time is given in seconds; bar, 2 mm. For quantification of those CCS, the internalization of which coincided with EGFR, Grb2 or Sos accumulation, see Fig. 6D.

• Movie 1 -

  Movie 1. Representative example of clathrin-coated structures (CCS; arrow), the internalization of which coincided with transient actin accumulation (arrow), observed by total internal reflection microscopy (TIRFM) of N-WASP^flox/flox fibroblasts co-expressing mRFP-clathrin light chain A and EGFP-actin as indicated. Movie represents a time period of 103 seconds; display rate is 123, bar equals 1 mm.

• Movie 2 -

  Movie 2. N-WASP recruitment to internalizing clathrin-coated structure (arrows) as visualised by co-expression of EGFP-N-WASP and mRFP-tagged clathrin light chain A and observation by TIRFM. Intensity and duration of this type of N-WASP accumulation were highly reminiscent of actin (see Movie S1). Movie length is 108 seconds, display rate is 123. Bar, 1 mm.

• Movie 3 -

  Movie 3. Actin and clathrin dynamics as revealed by TIRFM in N-WASP^flox/flox fibroblasts either upon Arp2/3 complex inhibition (WA) or in W-expresser (control) as indicated. Asterisks mark disappearing CCS without detectable actin polymerisation (WA). Neither formation nor internalization of CCS appear to coincide with detectable actin recruitment upon sequestration of Arp2/3 complex (WA). Arrows indicate examples of actin assembly at CCS in the W-expressing control. For both cells, movie length is 484 seconds, display rate is 603. Bar, 1 mm.