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First published online 15 March 2005
doi: 10.1242/jcs.01734

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Vinculin acts as a sensor in lipid regulation of adhesion-site turnover

Indra Chandrasekar¹, Theresia E. B. Stradal², Mark R. Holt³, Frank Entschladen⁴, Brigitte M. Jockusch¹ and Wolfgang H. Ziegler^1,*,

¹ Cell Biology, Zoological Institute, Technical University of Braunschweig, 38106 Braunschweig, Germany
² Cell Biology, German Research Centre for Biotechnology (GBF), 38124 Braunschweig, Germany
³ The Randall Division, King's College London, London SE1 1UL, UK
⁴ Institute of Immunology, Witten/Herdecke University, 58448, Witten, Germany

View larger version (44K): [in a new window]   Fig. 1. Mutagenesis of lipid binding sites in vinculin. (A,B) Ribbon and space-filling representations of the vinculin tail (Vt, amino acids 885-1066); data from Bakolitsa et al. (Bakolitsa et al., 1999). Solvent-accessible surfaces of wild-type (wt) in A and mutant vinculin tail in B are coloured according to electrostatic potential, with basic patches in blue and acidic patches in red. The two lipid-binding sites (basic collar and basic ladder) are indicated by dotted lines. In mutant vinculin tail (B), point mutations of the C-terminus (CT) and helix 3 (H3) are circled. Vinculin mutants are referred to as CT and H3, and as lipid-binding-deficient mutant (LD) for the combination of both, respectively. (C) Pull-down assay to analyse the lipid-binding capacity of Vt mutants Vt(CT, H3, LD) and Vt(1052), in comparison to the Vt wild type. Large unilamellar vesicles of the phospholipids PC, PS and PIP2 were incubated with Vt proteins and sedimented. Pairs of pellet (P) and supernatant (S) fractions were detected on Coomassie-stained gels and analysed by densitometry. Columns represent the proportions of vesicle-bound Vt protein (means±s.e.; n=3). Notice the decreased binding of Vt mutants to acidic phospholipids. Average residual binding of all Vt proteins to pure PC vesicles is indicated (dashed line). Identical molar amounts of Vt protein were used for all assays. (D) The interaction of Vt proteins with Vh was analysed by GST pull-down assay. GST-Vh(D1) fusion protein bound to glutathione-Sepharose beads was incubated with Vt protein. Pellet fractions were analysed on Coomassie-stained gels. (E) Interaction of Vt mutants with actin filaments was analysed in a high-speed cosedimentation assay. Pellet and supernatant fractions were analysed by densitometry. Differences in actin binding between the Vt mutants were observed at a molar ration of actin:Vt of 2:1. Columns represent the proportions of actin-bound Vt protein (means±s.e.; n=3). (F) Expression of full-length GFP-vinculin in stably transfected B16-F1 mouse melanoma cells was compared with endogenous vinculin levels by western blotting (WB) using anti-vinculin antibodies. (G) Proportions of exogenous vinculin (means±s.e.; n=3).

View larger version (101K): [in a new window]   Fig. 2. Localization of GFP-vinculin constructs in B16-F1 cells. B16-F1 populations were seeded on laminin and allowed to attach for 6 hours. Thereafter, cells were fixed and stained for paxillin and the actin cytoskeleton. Localization of GFP-vinculin constructs, paxillin and F-actin in exemplary cells are shown individually (greyscale) as well as on merged images of GFP-vinculin (green), paxillin (blue), F-actin (red) and enlargements of the regions indicated. Scale bars, 10 µm (overviews); 2 µm (insets).

View larger version (66K): [in a new window]   Fig. 3. Polarization of B16-F1 cells expressing vinculin variants. Populations expressing vinculin constructs were fixed 6 hours after seeding onto laminin and actin filaments stained using phalloidin. (A,B) Representative cells of populations expressing vinculin-wt (A) or vinculin-LD (B) are shown in fluorescence images (left) and as outlines of cells (right). Classification of cells into the categories polarized (P), unpolarized (N) and ambiguous (A) was based on cell shape and cytoskeletal staining. Scale bar, 10 µm. (C) Proportions of cells in each category taken from >600 cells (n=3 independent experiments) for each vinculin construct. Notice the significant difference between the numbers of polarized motile cells in the vinculin-wt-expressing B16-F1 and vinculin-LD-expressing cells.

View larger version (41K): [in a new window]   Fig. 4. Adhesion and spreading on different types of extracellular matrix. B16-F1 populations expressing different GFP-vinculin mutants were analysed on laminin, fibronectin and collagen. Identical numbers of cells were seeded onto 12-well plates coated with the matrix molecules. (A) Incubation times of 10 minutes on laminin and fibronectin, and 90 minutes on collagen, were chosen to obtain comparable cell adhesion. After washing and fixation, cells were counted and adhesion was quantified as a proportion of adherent cells (mean±s.d.; n=4-6) compared with adherent cells expressing the GFP-vinculin wild type. (B) To analyse spreading, cells were allowed to attach for 15 minutes on laminin or fibronectin and, after washing and fixation, stained with TRITC-phalloidin. Cells were classified into three groups: spread (S), non-spread (N) and ambiguous (A), as seen in cells expressing vinculin-wt or vinculin-LD. Depicted cells were scored as spread or non-spread, respectively, unless indicated otherwise. Spreading was quantified as a proportion of spread cells (mean±s.e.; n=3) compared with the total number of adherent cells.

View larger version (32K): [in a new window]   Fig. 5. Locomotory activity and velocity of B16-F1 populations in collagen gels. Migration of B16-F1 cells expressing vinculin variants was analysed by time-lapse video microscopy. The number and velocity of motile cells were calculated from tracking data (30 cells per vinculin variant, n=3 repeats). (A) Representative tracks of cells expressing vinculin-wt or vinculin-LD, respectively. Columns represent (B) average of motile cells (mean±s.d.) and (C) average velocity of active cells (mean±s.d.) of the population between 6-15 hours (constant phase).

View larger version (63K): [in a new window]   Fig. 6. Adhesion-site dynamics and PIP2 signals. In B16-F1 cells expressing GFP-vinculin variants, individual adhesions were analysed using confocal laser-scanning microscopy. (A) Representative images of adhesions before and after bleaching (red arrows) and kymographs from line scans parallel to the length axis of bleached adhesion. The half-time of fluorescence recovery was calculated based on the kymographs. Notice that vinculin variants wt and LD have comparable incorporation rates. Scale bar, 5 µm. (B) Movement of adhesions was followed over 20 minutes. Representative areas of cells expressing vinculin-wt or vinculin-LD are given, showing start position of adhesions (t=0 minutes) and tracks of adhesions tips marked in red (t=0-20 minutes). Tracks were used to calculate the average speed of retrograde movement. Scale bar, 5 µm. (C) B16-F1 cells were seeded onto fibronectin, transfected with murine PIP 5-kinase , wild type (wt) or kinase deficient (KD), and fixed 12 hours after transfection. Localization of GFP-vinculin variants (wt or LD) is shown individually (greyscale, left) and in merged images (green) together with PIP 5-kinase (blue) and F-actin (red). Scale bar, 10 µm. Notice that only cells expressing wild-type constructs of both PIP 5-kinase and vinculin show a localization of vinculin that is entirely perinuclear and start to detach.

View larger version (22K): [in a new window]   Fig. 7. Model of vinculin involvement in the regulation of adhesion turnover. In motile cells, areas with both high adhesion turnover and high actin dynamics under the control of the small GTPase Rac depend on elevated PIP2 levels, which leads to a weakening of the interaction of vinculin-containing adhesions with actin filaments. When acidic phospholipid binding to vinculin is defective, adhesion sites are stabilized and efficient progression of lamellipodia is impaired. This leads to a delay in cell spreading and an almost complete inhibition of cell motility in vinculin-LD-expressing cells.

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