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First published online November 9, 2005
doi: 10.1242/10.1242/jcs.02640

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Involvement of Rac in actin cytoskeleton rearrangements induced by MIM-B

Guillaume Bompard^1,*, Stewart J. Sharp¹, Gilles Freiss² and Laura M. Machesky^1,

¹ School of Biosciences, Division of Molecular Cell Biology, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
² Inserm U540, Endocrinologie Moléculaire et Cellulaire des Cancers, 60, rue de Navacelles, 34090 Montpellier, France

View larger version (10K): [in a new window]   Fig. 1. Schematic representation of the MIM-B expression constructs used in this study. Protein segments are indicated within parentheses. IMD, IRS/MIM domain; PRR, proline-rich region; W, WASP homology 2 domain (WH2).

View larger version (22K): [in a new window]   Fig. 2. MIM-B protein is widely expressed and is not downregulated in metastatic cell lines. (A) Characterisation of anti-MIM-B antibody. (Left) Lysates from mouse brain (L) were used to immunoprecipitate endogenous MIM-B using pre-immune serum (PI) or affinity-purified anti-MIM-B antibody (Pur) and analysed by immunoblotting with anti-MIM-B antibody. (Right) Lysates from COS-7 cells transfected with constructs expressing myc-MIM-B, myc-MIM-B IMD (IMD) or myc-IMD (see Fig. 1) were analysed by immunoblotting with anti-MIM-B antibody and reprobed with anti-myc antibody. Positions of molecular size markers are indicated in kDa. (B) Tissue distribution of MIM-B. 50 µg lysates from various mouse tissues were separated on SDS-PAGE and analysed by immunoblotting with anti-MIM-B antibody. SK, skeletal. (C) MIM-B expression is not downregulated in metastatic cell lines. Expression of endogenous MIM-B was studied by immunoblotting with anti-MIM-B antibody in non-metastatic (-) or metastatic (+) cell lines from bladder (top), prostate or breast (bottom). Tubulin was subsequently probed as a loading control. This result is representative of three independent experiments.

View larger version (118K): [in a new window]   Fig. 3. The IMD of MIM-B is required for MIM-B-induced actin-rich membrane protrusions in serum-starved Swiss 3T3 cells. Constructs (see Fig. 1) encoding myc-MIM-B (A-C), myc-MIM-B 234 (D-F), myc-MIM-B WH2 (G-I) or myc-IMD (J-L) were microinjected into serum-starved quiescent Swiss 3T3 cells. Cells were treated for indirect FITC (green) localisation of MIM-B constructs with anti-myc antibody (A,D,G,J) and F-actin with TRITC-coupled (red) phalloidin (B,E,H,K). Merged images (C,F,I,L) are presented. Boxed image in C is an enlargement of the merged image. Bar, 20 µm.

View larger version (68K): [in a new window]   Fig. 4. Involvement of Rac but not Cdc42 in MIM-B-induced actin cytoskeletal reorganisation. A construct encoding myc-MIM-B was co-microinjected with constructs expressing either the FLAG-tagged dominant negative form of Rac (N17 Rac, A-C) or Cdc42 (N17 Cdc42, D-F). Cells were treated as described in Fig. 3. Bar, 20 µm.

View larger version (14K): [in a new window]   Fig. 5. MIM-B activates Rac through its IMD. (A) MIM-B overexpression activates Rac. Activity of endogenous Rac was determined by pull-down from lysates of COS-7 cells transfected with an empty vector (Ø) or constructs encoding myc-MIM-B, myc-MIM or an HA-tagged constitutively active form (DH-PH) of mouse Vav-1 (Vav) using GST-PAK-CRIB. Rac activation was revealed by immunoblotting with anti-Rac antibody. Expression of tagged proteins was subsequently determined by immunoblotting with anti-myc and anti-HA antibodies. (B) The IMD of MIM-B is necessary and sufficient to activate Rac. Rac activity from COS-7 cells overexpressing myc-MIM-B IMD (IMD), myc-MIM-B, myc-IMD or myc-MIM-B K4D (K4D) was determined as described above. (C) IRSp53 does not activate Rac. Rac activity of COS-7 cells expressing myc-IRSp53 (IRS) or myc-MIM-B was determined as above. These results are representative of at least three different experiments.

View larger version (19K): [in a new window]   Fig. 6. MIM-B binds to Rac. (A) MIM-B binds Rac through its IMD. Binding to GTPase mutants was determined by GST pull down. Lysates of COS-7 overexpressing myc-MIM-B, myc-MIM-B IMD, myc-MIM-B K4D, myc-IMD or myc-IMD K4D were incubated with recombinant GST-N17 or -L61-Rac or -Cdc42 in low-salt conditions and binding was determined by immunoblotting with anti-myc antibody. L, lysate. (B) Endogenous MIM-B binds to Rac. Representative pull down experiments performed as described above in normal salt conditions using mouse brain extract as protein source. (C) The MIM-B IMD directly binds to Rac mutants. (Left) Recombinant His-tagged IMDs used for pull-downs with recombinant GTPases were separated on SDS-PAGE and stained with Coomassie Blue. (Right) Recombinant GST-GTPases were incubated with either purified His-IMD wild-type (wt) or K4D and binding was revealed by SDS-PAGE and Coomassie Blue staining.

View larger version (47K): [in a new window]   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.

View larger version (70K): [in a new window]   Fig. 8. K4D mutations inhibits MIM-B activity towards the actin cytoskeleton. COS-7 cells transfected with constructs expressing myc-MIM-B wt (A-C), K4D (D-F) or IMD (G-I) were treated for indirect immunofluorescence as described in Fig. 3. Bar, 10 µm.

View larger version (17K): [in a new window]   Fig. 9. The F-actin bundling activity of the IMD of MIM-B is inhibited by Rac. (A) Protein used in this assay. Recombinant His-tagged IMD and GTPase mutants were purified to homogeneity and analysed on SDS-PAGE followed by Coomassie Blue staining. Position of molecular size marker in kDa is indicated. (B) In this representative experiment, 5 µM His-tagged IMD (see Fig. 9A) were pre-incubated with various concentrations of recombinant N17-Rac or L61-Cdc42 (see Fig. 9A) before addition of 5 µM F-actin. F-actin bundling was determined as described in Fig. 7. Pellet fractions were analysed by SDS-PAGE and the band intensity of actin was measured densitometrically. Percentage of F-actin bundled was normalised by F-actin bundled in the absence of IMD and expressed as percentage of bundling inhibition.