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First published online 1 March 2005
doi: 10.1242/jcs.01698

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Actin-filament cross-linking protein T-plastin increases Arp2/3-mediated actin-based movement

Adeline Giganti¹, Julie Plastino^2,*, Bassam Janji^1,*, Marleen Van Troys³, Delphine Lentz¹, Christophe Ampe³, Cécile Sykes² and Evelyne Friederich^1,

¹ Laboratoire de Biologie Moléculaire, d'Analyse Génique et de Modélisation, Centre de Recherche Public-Santé, 42, rue du Laboratoire, L-1911, Luxembourg
² Laboratoire Physicochimie `Curie', UMR168 CNRS/Institut Curie, 11, rue Pierre et Marie Curie, 75231 Paris Cedex 05, France
³ Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, and Medical Protein Research, Flanders Interuniversity Institute for Biotechnology (VIB09), Belgium

View larger version (25K): [in a new window]   Fig. 1. T-Plastin affects the movement of VCA beads in a dose-dependent manner. (A) Time-lapse phase-contrast microscopy of bead movement. Beads were incubated in HeLa-cell extracts in the absence (control, left) or presence (1.1 µM, middle, and 2.2 µM, right) of endogenous T-plastin, observed from 3 minutes to 7 minutes (top to bottom). Little depolymerization of the actin tail is observed when T-plastin is added (black arrow). (B) Bead velocity as a function of T-plastin concentration. Bead velocity shows a bell-shaped dependence on T-plastin concentration with a maximum velocity at 1.1 µM T-plastin. Each bar represents the mean±s.d. of 20 measurements. An asterisk (*) indicates results that differ significantly from the results obtained with the addition of 1.1 µM T-plastin (P<0.0001).

View larger version (45K): [in a new window]   Fig. 2. The first actin-binding domain of T-plastin (ABD1), which binds but does not bundle actin filaments, has a similar effect on bead movement to wild-type T-plastin. (A) Protein domain scheme of T-plastin and ABD1 variant. (B) Cosedimentation of T-plastin or the ABD1 variant with F-actin in vitro. G-Actin was polymerized in the presence of T-plastin (+ T-plastin) or ABD1 (+ ABD1), or their absence (control). The molar ratio of actin:plastin or actin:ABD1 was 4:1. Proteins were centrifuged at high speed to sediment actin filaments (top) or at low speed to sediment actin bundles (bottom). Proteins in supernatants (S) and pellets (P) were separated by SDS-PAGE under reducing conditions. Protein bands were stained with Coomassie Brilliant Blue. (C) Time-lapse phase-contrast microscopy of bead movement. Beads were incubated in HeLa extracts supplemented with 1.1 µM or 2.2 µM ABD1. No depolymerization of the actin tail is observed when ABD1 is added (black arrow). (D) Bead velocity as a function of ABD1 concentration Bead velocity shows a bell-shaped dependence on ABD1 concentration, with a maximum velocity for 1.1 µM ABD1. Each bar represents the mean±s.d. of ten measurements. An asterisk (*) indicates results that differ significantly from the result obtained with the addition of 1.1 µM ABD1 (P<0.0006).

View larger version (42K): [in a new window]   Fig. 3. T-Plastin and ABD1 increase in actin tails concomitant with a significant increase in actin content and partial displacement of cofilin. (A) Quantification of actin and T-plastin or ABD1 in actin comets. Beads were incubated in HeLa-cell extracts supplemented with 1 µM Alexa-568-labeled actin in the presence of 1.1 µM T-plastin (middle) or ABD1 (bottom), or their absence (control, top); actin is shown on the left. Samples were immunostained with anti-plastin primary antibody and Alexa-488-coupled secondary antibody (right). Graphs indicate the integrated fluorescence intensities of actin and T-plastin in the comets, measured at 2 µm from the bead. Each bar represents the mean±s.d. of 25 measurements. Asterisks (**) indicate results that differ significantly from the results obtained with the control (*, P<0.0031; **, P<0.0063). (B) Quantification of cofilin/actin ratio in actin comets. Beads were incubated in extracts and processed as described in A, with the exception that anti-cofilin antibody was used for staining (middle). Representative fluorescence images are shown. Graphs indicate the ratios of the integrated fluorescence intensities of cofilin/actin in the comets, measured at 2 µm distance from the bead.

View larger version (29K): [in a new window]   Fig. 4. T-plastin or ABD1 stabilize actin filaments and protect them against cofilin-mediated depolymerization. G-Actin (4 µM, 25% pyrene labeled) was co-polymerized with various concentrations of T-plastin or ABD1. To induce depolymerization, T-plastin- or ABD1-decorated pyrene-labeled -F-actin was diluted under the critical concentration of the filament minus (–) end. Depolymerization of F-actin as a function of time was measured as a decrease in fluorescence because of the rapid depolymerization of actin filaments from their pointed ends. The fluorescence scale was adjusted so that the fluorescence of F-actin was 100 and that of G-actin was 0. (A) F-Actin (4 µM) co-polymerized with T-plastin (actin:T-plastin ratio of 1:1) was diluted to a concentration of 400 nM. Control (actin) was the depolymerization of F-actin in the absence of protein addition. (B) F-Actin decorated with ABD1 (actin:ABD1 ratios of 1:1, 1:2 or 1:4) was diluted to a concentration of 400 nM. Control (actin) was the depolymerization of F-actin in the absence of protein addition. (C) F-Actin decorated with T-plastin (4 µM) was diluted to a concentration of 400 nM in F buffer in the presence of 400 nM of GST-cofilin or its absence. Controls were the depolymerization of F-actin in the absence of T-plastin or GST-cofilin (Actin) or in the presence of 400 nM of GST-cofilin (Actin + cofilin). (D) F-Actin decorated with ABD1 (actin:ABD1 ratios of 1:2 or 1:4) was diluted to a concentration of 400 nM in the absence or presence of 400 nM of GST-cofilin. Controls as described in C.

View larger version (35K): [in a new window]   Fig. 5. Wild-type T-plastin stabilizes actin filaments in a Ca2+-independent manner. (A) Depolymerization of T-plastin-decorated pyrene-labeled F-actin in the presence of increasing free Ca2+ concentrations. G-Actin (4 µM, 25% pyrene labeled) was co-polymerized with T-plastin (4 µM) in the presence of increasing concentrations of free Ca2+ (4.6 nM to 1.6 µM). F-Actin was diluted and depolymerization was measured as described in Fig. 4. Control (actin) was the depolymerization of F-actin in the absence of T-plastin. (B) Cosedimentation of T-plastin with F-actin in the presence of increasing free Ca2+ concentrations. G-Actin (7 µM) was co-polymerized with T-plastin (3 µM) in the presence of increasing concentrations of free Ca2+. Proteins were centrifuged at high speed to sediment actin filaments. Proteins in supernatants (S) and pellets (P) were separated by SDS-PAGE under reducing conditions. Protein bands were stained with Coomassie Brilliant Blue. The migration positions of plastin and actin are indicated. Notice that, owing to the presence of Ca2+, more actin is present in the supernatant than observed in the experiment in Fig. 2.

View larger version (100K): [in a new window]   Fig. 6. The C-terminal F-actin-binding domain of villin causes the elongation and densification of F-actin comets. Beads were incubated in HeLa-cell extracts in the presence of villin headpiece domain (VHP, 1 µM) or its absence (control). Phase-contrast images are shown.

View larger version (90K): [in a new window]   Fig. 7. ABD1 stabilizes F-actin structures in transfected cells. (A) ABD1 induces the formation of long actin cables in cells to which the actin-binding proteins -actinin and VASP are recruited. Transfected Vero cells transiently expressing epitope-tagged ABD1-vsv were processed for triple staining with Alexa-320/phalloidin as a probe for F-actin (left), anti-vsv tag primary antibody and Cy2-coupled secondary antibody for immunofluorescence staining of ABD1-vsv (middle), anti--actinin antibody and Texas-Red-coupled secondary antibody (right, top) or anti-VASP antibody and Texas-Red-coupled secondary antibody (right, bottom). Bar, 10 µm. Notice that cells expressing ABD1 exhibit much-increased F-actin staining and many actin cables, which are not detected in neighboring untransfected cells. (B) F-Actin cables induced by ABD1 are more resistant to latrunculin-A treatment than those of non-transfected cells. Transfected Vero cells were treated with 100 nM latrunculin A for 60 minutes and double stained for F-actin (left) and vsv-tag (right). Bar, 15 µm. Notice that the actin cytoskeleton of untransfected cells (arrows) is disrupted compared with that of cells expressing ABD1.

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