Fig. 7. Characterization of K4D mutant. (A) Alignment of IMD sequences. IMD sequences of IRSp53 (NP_059344), IRTKS (NP_061330), FLJ22582 (NP_079321), MIM-B (AK027015) and ABBA-1 (NP_61239) were aligned using MultAlign (http://prodes.toulouse.inra.fr/multalin/). Symbols above the sequence alignment refer to the secondary structure assignment of IRSp53. Critical basic region of IRSp53 IMD involved in F-actin binding is indicated by a bold bar. Corresponding residues in MIM-B sequence, lysines 149, 150, 152 and 153 mutated to glutamic acids (K4D mutant), are indicated by asterisks. (B) K4D mutations abrogate F-actin binding. Representative high-speed cosedimentation assay of the interaction of 5 µM His-tagged wt or K4D IMD with F-actin (2.5 µM). Results were analysed by SDS-PAGE and Coomassie Blue staining. P, pellet; S, supernatant. (C) K4D mutations prevent F-actin bundling. Various concentrations of His-tagged IMD wt or K4D were incubated with 5 µM F-actin and F-actin bundling was determined by low-speed co-sedimentation assay. F-actin present in the supernatant (S) and pellet (P) fractions was revealed by SDS-PAGE and Coomassie Blue staining. Similar results were independently obtained at least three times. (D) Bundling defect of His-tagged IMD K4D revealed by fluorescence-microscopy-based F-actin-bundling assay. Cy3-labelled F-actin (1 µM) was incubated with 10 µM His-tagged IMD wt or K4D and imaged using a fluorescence microscope. (E) K4D mutations do not affect IMD dimerisation. COS-7 cells were co-transfected with constructs expressing myc-IMD wt or K4D and HA-MIM-B. Myc-tagged proteins were immunoprecipitated with anti-myc antibody and binding to HA-MIM-B was determined by immunoblotting with anti-HA antibody. Expression and immunoprecipitation of myc-tagged protein was subsequently analysed with anti-myc antibody. Bar, 20 µm.