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First published online 12 April 2007
doi: 10.1242/jcs.001776

Journal of Cell Science 120, 1663-1672 (2007)
Published by The Company of Biologists 2007
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Characterisation of IRTKS, a novel IRSp53/MIM family actin regulator with distinct filament bundling properties

Thomas H. Millard*, John Dawson and Laura M. Machesky{ddagger}

School of Biosciences, University of Birmingham, Birmingham, B15 2TT, UK


Figure 1
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  		Fig. 1. Alignment of the protein sequences of IRSp53 and IRTKS. (A) Human IRSp53 (splice variant S, accession no. BAC57948) aligned with human IRTKS (NP_061330). Identical residues are highlighted in black. The region boxed with a solid line is the IMD, that with a dash-dot line is the partial CRIB motif of IRSp53, that with a dashed line is the SH3 domain and that with a dotted line is the putative WW domain interacting motif. (B) Alignment of C-terminal region of IRTKS with the variable C-terminal regions of four splice variants of IRSp53; IRSp53-L (BAC57945), IRSp53-M (BAC57946), IRSp53-T (BAC57947) and IRSp53-S (BAC57948). Splice variant names are as defined by Miyahara et al. (Miyahara et al., 2003Go). Dashed box indicates region present in all splice variants of IRSp53.

 

Figure 2
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  		Fig. 2. GTPase binding of IRSp53 and IRTKS. GST-fused constitutively active (L61) or dominant negative (N17) Cdc42 and Rac bound to glutathione beads were incubated with extract from COS7 cells expressing Myc-tagged constructs (as indicated) or with untransfected C2C12 extract (endogenous). Bead-bound material was analysed by immunoblotting alongside an equivalent fraction of the original extracts. Blots were probed with anti-Myc (9E10). FL, full-length protein; aa, amino acid residues present in truncated constructs. IRTKS exhibits IMD-mediated binding to both Rac mutants.

 

Figure 3
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  		Fig. 3. Expression of IRSp53 and IRTKS in COS7 cells. COS7 cells were transfected with Myc-tagged IRSp53 and IRTKS constructs and 24 hours later fixed and stained with 9E10 and Alexa Fluor 488-anti mouse and TRITC-phalloidin. Left hand panels show staining of the Myc-tagged proteins and the right hand panels show the same field of view with F-actin stained. Cells expressing Myc-IRSp53 exhibit long filopodia-like extensions (A,B). Cells expressing low levels of Myc-IRTKS exhibit short actin microspikes (C,E), which at higher expression levels coalesce into clusters of short spikes (D,F). Confluent cells expressing Myc-IRTKS have a high concentration of clustered actin bundles at cell junctions (G,H). Cells expressing Myc-IRTKS with the Ct extension removed produce long wavy filopodia-like extensions and not actin clusters (I,J), whereas cells expressing Myc-IRSp53 with IRTKS Ct extension fused produce actin clusters (K,L). Boxed insets are magnifications of the indicated region of the image and highlight actin structures typically induced by the construct. Bars, 20 µm. The images shown are representative of more than three independent transfections per construct, where more than 100 cells have been viewed per transfection. For IRSp53, over 80% of cells have the described phenotype of long filopodial protrusions, and for IRSp60, 30-40% of cells show no change in shape, whereas 60-70% have short actin clusters and spikes. Bars, 20 µm.

 

Figure 4
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  		Fig. 4. Cell area changes in cells expressing IRSp53 or IRTKS Myc-tagged IRTKS, IRSp53-S, IRSp53-L, IRSp53+Ct or GFP (control) were expressed in COS7 cells as shown. IRSp53-S and IRSp53-L constructs decreased the area of the cell body in contrast to IRTKS and IRSp53+Ct. IRSp53-S and IRSp53-L have significantly reduced cell body areas compared with COS7 cells overexpressing only GFP. The graph shows the mean of three independent experiments in which 30 cells were measured for each construct per experiment ± s.e.m. *P<0.05 compared with GFP control cells. The cell body area is drawn in red on each image. Bar, 10 µm.

 

Figure 5
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  		Fig. 5. IRTKS co-localises with F-actin, cortactin, VASP and vinculin. COS7 cells were transfected with Myc-tagged IRTKS and labelled for Myc and F-actin, along with VASP, cortactin or vinculin. Myc-IRTKS co-localises with VASP (A-D, inset in D), cortactin (E-H, inset in H) and vinculin (I-L, inset in L) at sites of cell-cell contact where aggregates of F-actin (C,G,K) are observed. Bar, 20 µm.

 

Figure 6
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  		Fig. 6. In vitro filament bundling. (A-C) Fluorescently labelled actin filaments were incubated with IRTKS IMD (A), with IRTKS IMD-Ct (B) or with no additions (C) then imaged using a fluorescence microscope. IMD induces long bundles, whereas IMD-Ct induces short, clustered bundles. Bars, 20 µm. (D,E) 5 µM F-actin was incubated with the indicated concentrations of IRTKS IMD or IMD + Ct and then centrifuged at 10 000 g. Pellets and supernatants were analysed by SDS-PAGE after staining with Coomassie Blue. There is an IMD concentration-dependent increase in pelleting of actin, indicating filament bundling.

 

Figure 7
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  		Fig. 7. Actin binding by the IRTKS Ct extension. (A) Alignment of IRTKS C-terminal extension with the WH2 domains of five human proteins. Residues conserved in at least three of the displayed WH2 domains are highlighted in black. Residues in IRTKS Ct conserved in the WH2 domains are shown in grey. Residues critical for actin monomer binding by thymosin beta4 are indicated with asterisks. (B) Measurement of actin critical concentration by monitoring pyrene-actin fluorescence at a range of actin concentrations, alone or in the presence of 2.5 µM GST-Ct or GST-ScarWH2. Polymerisation of actin results in an increase in the gradient of fluorescence change with actin concentration and the critical concentration is derived from the intercept of the pre- and post-polymerisation gradients. Data shown are from three replicates of a single representative experiment. (C) Co-sedimentation assay at a range of F-actin concentrations showing binding of GST-Ct and GST-ScarWH2 to phalloidin stabilised F-actin. The quantification is described in the Materials and Methods. The binding curve fitted for GST-Ct gives a Kd of approximately 1 µM. GST-ScarWH2 data could not be fitted to a binding curve. Data shown are from three replicates of a single representative experiment.

 

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© The Company of Biologists Ltd 2007