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The interaction of plectin with actin: evidence for cross-linking of actin filaments by dimerization of the actin-binding domain of plectin

Lionel Fontao*,§, Dirk Geerts{ddagger},§, Ingrid Kuikman, Jan Koster, Duco Kramer and Arnoud Sonnenberg

The Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
* Present address: Department of Dermatology, Geneva University Hospital, 24 rue Micheli-Ducrest, 1211 Geneva 4, Switzerland
{ddagger} Present address: Department of Human Genetics M1-159, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
§ L. Fontao and D. Geerts contributed equally to this work



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  		Fig. 1. Comparison of the ABD of plectin, dystonin and dystrophin and interaction of plectin with different isoforms of actin. (A) Alignment of the N termini of human plectin (AAB05427), mouse dystonin (P11277) and dystrophin (P11532). Alignment was performed with the CLUSTAL-W program. Black boxes, identical amino acids; gray boxes, amino acid similarity. The bars above the sequence indicate the ABS1, ABS2 and ABS3 sequences. The CH1 and CH2 domains, identified by sequence homology among the ß-spectrin family of proteins, are delineated by dotted boxes. (B) Two-hybrid interaction between the N-terminal part of plectin and different actin isoforms. (Top) Schematic representation of the largest N-terminal construct of plectin (residues 1-339) with its ABD used in this study. (Bottom) Different actin isoforms, {alpha}-skeletal muscle ({alpha}-actin), ß-cytoplasmic (ß-actin) and {gamma}-cytoplasmic ({gamma}-actin) actin, were used to determine plating efficiency following cotransformation of yeast host strain PJ69-4A with each of the pAS2-plectin subclones listed together with pACT2-actins. Transformation mixtures were spread on SC-LT and SC-LTHA plates and grown at 30°C. Plating efficiency on selective SC-LTHA plates is expressed as a percentage of plating efficiency on non-selective SC-LT plates of the same transformation, thus: ++, >50%; +, >= 50% (slowly growing colonies); ±, 5-25%; -, 0%. ND, not determined. Plates were scored after 6 and 12 days of growth; slowly growing colonies could only be scored after 12 days of growth. Plating efficiencies of <25% always represented slowly growing colonies. All efficiencies listed represent an average of multiple independent transformations on at least two separate occasions.

 


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  		Fig. 2. Biochemical analysis of plectin/actin interactions. (A) Copolymerization assays were performed using bovine {alpha}-skeletal muscle actin at 2.5 µM together with different concentrations of MBP-plectin-ABD1-339 (lanes 1-8 correspond to 0.2, 0.37, 0.5, 0.6, 1, 1.5, 1.6 and 2.2 µM, respectively). Actin filaments and bound proteins were sedimented by centrifugation and equivalent samples of pellets (upper panel) and supernatants (lower panel) were resolved by SDS-PAGE. (B) Scatchard plot of plectin binding to actin filaments (values are means differing by less than 5% of duplicate experiments). Scatchard plotting indicates that plectin binds to filamentous actin with an apparent Kd of 0.3 µM and a molecular ratio of 1 plectin molecule per actin monomer. (C) Pull-down assay of plectin-ABD1-339/G-actin binding. Immobilized MBP-plectin-ABD1-339 in suspension (3 µM) was incubated with soluble {alpha}-skeletal muscle G-actin (2.5 µM) in the absence (lanes 1 and 2) or presence of a fivefold excess of soluble MBP-plectin-ABD1-339 (lanes 3 and 4). After incubation, the beads were pelleted by centrifugation and the supernatants removed. The beads were then washed and the proteins eluted by boiling in sample buffer. Equivalent samples of supernatant (unbound (UB) actin, lanes 1 and 3) and bead eluates (bound (B) actin, lanes 2 and 4) were analyzed by SDS-PAGE. Proteins were visualized by Coomassie Brilliant Blue staining.

 


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  		Fig. 3. Effect of plectin-ABD on actin polymerization. (A) {alpha}-skeletal muscle actin (4 µM) containing 10% of pyrene-labeled actin was polymerized in the presence of increasing concentrations of MBP-plectin-ABD1-339 (squares, 0 nM; triangles, 30 nM; diamonds, 150 nM; circles, 300 nM). Actin polymerization was initiated at time 0 by the addition of 0.1 volume of 10x initiation mix and fluorescence was recorded at 407 nm using an excitation wavelength of 365 nm. (B) Actin polymerization was monitored by light scattering at 300 nm. Polymerization of 2 µM {alpha}-skeletal muscle actin in the absence (squares) and presence of varying concentrations of MBP-plectin-ABD, 0.5 µM (triangles), 1 µM (diamonds) was initiated at time 0 by the addition of 0.1 volume of 10x initiation mix.

 


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  		Fig. 4. Plectin-ABD cross-links F-actin into bundles. Electron microscopy examination of polymerized actin mixtures, described in Fig. 3. F-actin alone (A) or in combination with 0.5 µM of purified plectin-ABD (B). Note the numerous bundles of F-actin in the presence of plectin-ABD. Bar, 0.5 µm.

 


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  		Fig. 5. The plectin-ABD interacts with other plectin-ABD molecules in vitro and bundles actin filaments. (A) Low-speed sedimentation assay. Polymeric actin (4 µM) was incubated with MBP (2 µM) or MBP-plectin (0.5, 1 or 2 µM) for 1 hour at room temperature. Samples were centrifuged at 14,000 g for 1 minute and equal amounts of pellet (P) and supernatant (S) were subjected to SDS-PAGE and visualized by Coomassie Blue staining. (B) MBP (lanes 1 and 2) and MBP-plectin1-339 (lanes 3 and 4) bound to amylose-agarose beads were incubated with 35S-labeled plectin-ABD1-339 (lanes 2 and 4) or ß4R1281W (lanes 1 and 3), obtained by in vitro translation. After incubation and washing, the beads were boiled in sample buffer and bound proteins were subjected to SDS-PAGE and visualized by autoradiography. Lanes at left show 35S-labeled in vitro-translated plectin-ABD1-339 and ß4R1281W. (C) Lysates of COS-7 cells, untransfected (lanes 1, 5, 9) or transiently transfected with HA-plectin-ABD1-339 (lanes 2, 6, 10), HA-plectin-ABD65-339 (lanes 3, 7, 11) or HA-ß4R1281W (lanes 4, 8, 12), were incubated with GST (lanes 5-8) or GST-plectin1-339 (lanes 9-12), immobilized on glutathione-Sepharose beads. After washing, beads were boiled in sample buffer and associated proteins were visualized by immunoblotting using anti-HA antibody. Lanes 1-4, total COS-7 cell lysates probed by immunoblotting with anti-HA antibody to verify the expression of the HA-tagged proteins. The upper band in lanes 1-4 and 9-10 corresponds to an unidentified protein that is non-specifically recognized by anti-HA antibody.

 


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  		Fig. 6. Mapping of the binding site in the N-terminal part of plectin that is involved in dimerization. Plating efficiency following cotransformation of yeast host strain PJ69-4A with one of each of the pAS2-plectin subclones listed together with pACT2-plectin1-339, pACT2-plectin36-181 or pACT2-ß cytoplasmic actin are shown. Details are as for Fig. 1B.

 


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  		Fig. 7. Yeast two-hybrid analysis of the interactions between the ABD of plectin, dystonin and dystrophin. Plating efficiency following cotransformation of yeast host strain PJ69-4A with the pAS2- and pACT2-plectin, dystonin and dystrophin subclones listed are shown. Details are as for Fig. 1B.

 


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  		Fig. 8. Codistribution of F-actin and plectin N terminus protein fragments in transfected cells. Rat embryo fibroblasts were transiently transfected with HA-tagged plectin1-339 (A-C) or HA-tagged plectin65-339 (D-F). The cells were fixed, permeabilized and processed for double labeling using rhodamine-phalloidin to visualize F-actin (A,D) and anti-HA antibodies, followed by FITC-conjugated secondary antibodies to detect the HA-tagged proteins (B,E). Images were obtained using a Leica confocal microscope. (C,F) Composite images. Colocalization appears as yellow. Bar, 10 µm.

 

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© The Company of Biologists Ltd 2001