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First published online 28 September 2004
doi: 10.1242/jcs.01406

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Xin repeats define a novel actin-binding motif

Dirk Pacholsky^1,*, Padmanabhan Vakeel^1,*, Mirko Himmel¹, Thomas Löwe¹, Theresia Stradal², Klemens Rottner², Dieter O. Fürst¹ and Peter F. M. van der Ven^1,

¹ Department of Cell Biology, Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm, Germany
² Department of Cell Biology, German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany

View larger version (37K): [in a new window]   Fig. 1. Characterization of human Xin. (A) The structure of human Xin and recombinant constructs used for actin-binding and transfection assays. Xin repeats (XR) are indicated by blue boxes. The constructs are named according to the numbers of the repeats they encode. Amino acid residues for the respective constructs are indicated. (B) The sequence of the 16 human Xin repeats and their consensus sequence (lower line; in italic). Identical amino acids at each position are shaded in different colours and conserved residues are grey. (C,D) Circular dichroism spectra of native and/or denatured recombinant Xin repeats in the near (C) and far (D) UV ranges.

View larger version (74K): [in a new window]   Fig. 9. The repeat region of XIRP2 has Xin-repeat-like properties. (A) The putative structure of XIRP2. Grey boxes indicate the 28 Xin repeats within the repeat region of the protein; their sequences and consensus sequence (italic) are given in (B). Identical amino acids at each position are shaded dark grey and conserved residues light grey. In actin-binding (C) and actin-bundling (D) assays, these repeats (arrow) are pelleted together with skeletal-muscle actin (asterisk). Upon expression in A7r5 cells, the full-length EGFP-tagged XIRP2 repeat region (E) is targeted to structures known to contain F-actin, such as stress fibres (arrows) and focal adhesions (arrowheads) that are counterstained by TRITC-phalloidin (F).

View larger version (112K): [in a new window]   Fig. 2. Full-length Xin is targeted to actin containing structures upon transient transfection. PtK2 cells were transfected with Xin-EGFP. Cells were fixed 48 hours after transfection, and stained with CPITC-phalloidin to visualize F-actin. The Xin-EGFP polypeptide (A,C-E, green) is targeted to F-actin-containing structures (B-E, blue), including cell-cell contacts (C, arrows), focal adhesions (D, arrows) and stress fibres (E, arrows). Scale bar, 10 µm.

View larger version (78K): [in a new window]   Fig. 3. The Xin-repeat region and truncated variants are targeted to actin-containing structures. A7r5 cells were transfected with XR1-16 (A,B), XR1-6 (C,D), XR 4-6 (E,F), XR3-4 (G,H) or XR10-11 (I,J), fixed and stained with CPITC-phalloidin (A-F) or analysed using live imaging to exclude fixation-induced association of EGFP with stress fibres (G-J). For the latter images, localization of the EGFP-tagged construct is shown together with the phase-contrast image. Scale bar, 10 µm.

View larger version (85K): [in a new window]   Fig. 4. Xin repeats are excluded from lamellipodia. B16F1 cells were transfected with XR1-16-EGFP, plated on laminin and analysed using phase-contrast and fluorescence video microscopy. (A-H) Representative frames derived from a movie recorded over 15 minutes as indicated. The black lines in B,D,F,H outline the border of the distribution of XR1-16-EGFP within the cell. In I,J, the distribution of Xin repeats (I) is compared with that of actin filaments (J) in the same cell stained with phalloidin. Xin repeats bind to the contractile actin filament bundles and focal adhesions in the body of the cell (G, arrowheads). By contrast, the repeats are virtually absent from the dynamic actin structures in the lamellipodium (I,J, arrows). Scale bar, 20 µm.

View larger version (61K): [in a new window]   Fig. 5. Xin repeats bind directly to actin filaments. Bacterially expressed XR1-16 (A-C) and its truncated variants XR1-6 (D-F), XR1-3 (G,H) and XR3-4 (I) were purified and their abilities to bind skeletal-muscle F-actin were investigated using high-speed co-sedimentation assays. The positions of actin and recombinant truncated Xin are marked with an asterisk and an arrow, respectively. The numbers on top of each lane indicate the amount of recombinant protein that was added to 5 µM actin. In H, the pellet was dissolved in less sample buffer (25%) to be able to visualize precipitated XR1-3 clearly. XR1-16, XR1-6 and XR1-3 are precipitated together with F-actin, whereas the two-repeat construct XR3-4 remains in the supernatant. Binding of the full Xin-repeat region to 1 µM F-actin is saturated with approximately 0.25 µM XR1-16 (C), whereas complete saturation of binding of XR1-6 to 1 µM F-actin is reached with approximately 0.5 µM recombinant protein (F).

View larger version (86K): [in a new window]   Fig. 6. Ultrastructural analysis of F-actin/Xin-repeat complexes. Bacterially expressed XR1-11 was purified and its capability to cross link skeletal-muscle F-actin was investigated using low-speed co-sedimentation assays. (A,B) Lanes 1 show the supernatant or pellet fractions of mixtures of XR1-11 and F-actin upon centrifugation. The numbers on top of each lane indicate the amount of recombinant protein that was added to 5 µM actin. The positions of actin and XR1-11 are marked with an asterisk and an arrow, respectively. To investigate the organization of the cross-linked actin filaments, F-actin alone (C,D) or mixtures of actin and XR1-11 (E,F) were applied to carbon-coated Formvar grids and analysed by electron microscopy after negative stain. The Xin-repeat construct does not bundle actin filaments but, instead, seems to cross-link F-actin into a meshwork (E,F). Scale bars, 500 nm (CE), 100 nm (D,F).

View larger version (58K): [in a new window]   Fig. 7. Xin repeats inhibit binding of tropomyosin to actin filaments. Actin was mixed under polymerization conditions with a stable amount of tropomyosin and the indicated quantities of XR1-11. Actin filaments and associated proteins were precipitated under low-speed sedimentation conditions. In the absence of XR1-11 (X), only trace amounts of actin (A) were found in the pellet (control lane). The addition of only 0.5 µM XR1-11 resulted in precipitation of actin filaments together with the associated tropomyosin (T); larger quantities of recombinant Xin cause a dose-dependent increase of F-actin and a dose-dependent reduction of tropomyosin in the pellet.

View larger version (167K): [in a new window]   Fig. 8. Stress fibres are stabilized upon interaction with Xin repeats. A7r5 cells were transfected with XR1-16-EGFP and incubated with 0.5 µM Latrunculin A (LatA) for the time indicated at the top of each panel. Cells were fixed, permeabilized and counterstained for F-actin with CPITC-phalloidin and with an antibody specific for vinculin as indicated on the left of each row, in order to compare the localization of Xin repeats with that of F-actin and focal adhesions, respectively. Without LatA treatment (0 minutes, A-E), untransfected cells contain many stress fibres (A) and focal adhesions (B). Cells transfected with XR1-16 show extensive binding of the EGFP-tagged construct to stress fibres and focal adhesions. Stress fibres have a thickened appearance (D) and focal adhesions (E, arrow) are very prominent in transfected cells compared with untransfected controls. After 5 minutes of exposure to LatA, untransfected cells show the first focal breakdown of stress fibres (F, arrow), whereas focal adhesions still have their normal appearance (G), and transfected cells do not seem to show any effects (H-K). Notice the conspicuous elongated focal adhesions in transfected LatA-treated cells (K,P,U, arrows). After a 10-minute incubation, the actin cytoskeleton is heavily disordered in untransfected cells (L) but focal adhesions show a normal appearance (M). In transfected cells, stress fibres and focal adhesions are generally well preserved (N-P). A 15 minute exposure to LatA leads to a total breakdown of the actin cytoskeleton in untransfected cells. Detectable quantities of filamentous actin are only observed close to focal adhesions (Q,R). Transfected cells show the first lesions (S,T, arrows) but stress fibres and focal adhesions are still relatively well preserved (S-U). Scale bar, 10 µm. (For a representative time-lapse movie showing the effect of LatA on A7r5 cells with or without expression of Xin repeats, see Movie 1 in supplementary material.)