Functional role of syndecan-1 cytoplasmic V region in lamellipodial spreading, actin bundling, and cell migration

Abstract

Cell protrusions contribute to cell motility and migration by mediating the outward extension and initial adhesion of cell edges. In many cells, these extensions are supported by actin bundles assembled by the actin cross-linking protein, fascin. Multiple extracellular cues regulate fascin and here we focus on the mechanism by which the transmembrane proteoglycan, syndecan-1, specifically activates lamellipodial cell spreading and fascin-and-actin bundling when clustered either by thrombospondin-1, laminin, or antibody to the syndecan-1 extracellular domain. There is almost no knowledge of the signaling mechanisms of syndecan-1 cytoplasmic domain and we have tested the hypothesis that the unique V region of syndecan-1 cytoplasmic domain has a crucial role in these processes. By four criteria--the activities of N-cadherin/V region chimeras, syndecan-1 deletion mutants, or syndecan-1 point mutants, and specific inhibition by a membrane-permeable TAT-V peptide--we demonstrate that the V region is necessary and sufficient for these cell behaviors and map the molecular basis for its activity to multiple residues located across the V region. These activities correlate with a V-region-dependent incorporation of cell-surface syndecan-1 into a detergent-insoluble form. We also demonstrate functional roles of syndecan-1 V region in laminin-dependent C2C12 cell adhesion and three-dimensional cell migration. These data identify for the first time specific cell behaviors that depend on signaling through the V region of syndecan-1.

Figure 1.

Clustering of the VC2 region of syndecan-1 cytoplasmic domain is sufficient for cell spreading and fascin-and-actin bundling. (A) Schematic diagram of the chimeric constructs prepared. (B) Equivalent expression of the two proteins in COS-7 cells, as determined by immunoblot with antibody specific for chicken NCAD. Molecular mass markers are indicated in kDa. (C) Morphology and F-actin organization of COS-7 cells expressing either NCADt or NCADt/S1VC2 after plating for 1 h on surfaces coated with 50 μg/ml antibody to chick NCAD extracellular domain and staining with phalloidin. Bar, 10 μm. (D) Quantification of the assay from three independent experiments. Each column represents the mean; bars, SEM. Over 1000 cells were scored in total for each construct.

Figure 2.

The V region of syndecan-1 cytoplasmic domain is necessary for cell spreading and fascin-and-actin bundling. (A) Schematic diagram of the syndecan-1 deletion constructs prepared. (B) Quantification of transfected COS-7 cell spreading and fascin-and-actin bundling activities in syndecan-1 or NCAD adhesion assays. Each column shows the mean ± SEM for data from five independent experiments. One hundred cells were scored in each experiment.

Figure 3.

Identification of critical residues within the V region of syndecan-1. (A) ClustalW multiple sequence alignment of the cytoplasmic domains of vertebrate syndecans. Black shading shows identical amino acids; gray indicates conservative substitutions and no shading indicates unrelated amino acids. Cg, Cricetulus griseus; Dr, Danio rerio; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus muscularis; Rn, Rattus norvegicus; Xl, Xenopus laevis. Asterisk indicate conserved positions within the V region. (B) Morphometric scoring parameters used in the experiments with syndecan-1 point mutants transfectants. COS-7 cells expressing EGFP-fascin and mouse syndecan-1, adherent on antibody to mouse syndecan-1, were scored for cell area (white outline) and the number and length of fascin-containing bundles (white bar) using Openlab software. Bar, 10 μm.

Figure 4.

Identification of critical residues within the VC2 region of syndecan-1. Quantification of the effects of syndecan-1 point mutants on COS-7 cell spreading and fascin-and-actin bundling in the syndecan-1 adhesion assay. (A) cell area; (B) number of fascin- and-actin bundles per cell; (C) length of fascin-and-actin bundles. Each column shows the mean ± SEM for data pooled from at least three independent experiments. * Significantly different from wild type at p ≤ 0.002.

Figure 5.

Effect of a membrane-permeable TAT-S1V peptide on cell spreading and syndecan-1 extractability. Transfected COS-7 cells were preincubated with TAT peptides, as indicated, for 1 h before harvesting. Syndecan-1 adhesion assays were carried out for 1 h in the continued presence of peptide. (A–C) Quantification of the effects of TAT peptides on cell spreading and fascin-and-actin bundling. Each graph data point or column indicates the mean ± SEM for data from five independent experiments. In B and C, each peptide was used at 1200 nM. *Significantly different from untreated control at p ≤ 0.001. (D) Effects of TAT peptides on detergent extractability of syndecan-1. Cells were prepared either fixed or fixed and permeabilized. Mouse syndecan-1 was detected on the adherent cells with biotin-conjugated primary antibody and FITC-conjugated streptavidin, and cells were analyzed by confocal XY and XZ images. Arrow indicates example of alignment of syndecan-1 at sites of F-actin bundles. Each peptide was used at 1200 nM. Under control conditions, ligated syndecan-1 was specifically resistant to extraction (top row), and this property was specifically inhibited by TAT-S1V peptide (third row). Results shown are representative of five independent experiments. Bar, 10 μm.

Figure 6.

Adhesion to ECM ligands renders endogenous syndecan-1 nonextractable. (A) C2C12 cells were plated on surfaces coated with 50 μg/ml antibody to mouse syndecan-1; 50 nM EHS laminin, or 50 nM TSP-1 for 3 h. Syndecan-1 extractability was assessed by staining cells with biotin-conjugated primary antibody and FITC-conjugated streptavidin after fixation or after fixation and permeabilization. Cells were viewed as confocal XY or XZ sections. Bar, 10 μm. (B) Treatment with TAT-S1V peptide renders endogenous syndecan-1 detergent-extractable in laminin-adherent C2C12 cells. Cells were pretreated with 1200 nM TAT-S1V or control TAT-S1Vscr peptides for 2 h before adhesion on 50 nM laminin in the continued presence of peptides as described above. Bar, 10 μm.

Figure 7.

Syndecan-1 localizes to leading edge of migrating cells and has a functional role in 3-D cell migration. (A) C2C12 cells were grown to confluency and wounded with a micropipette tip. At different times after wounding, dishes were fixed, permeabilized, and stained for syndecan-1. At 4 h, syndecan-1 was most concentrated at the front of migrating cells, along with fascin and F-actin. β-tubulin was present behind the ruffling edges. Representative merged images are shown for actin and β-tubulin, and actin and syndecan-1. Bars, 10 μm. (B–D) Quantification of transfilter cell migration assays. Pore filters, 8-μm, were coated with 50 nM EHS laminin or 1 mg/ml BSA. TAT-S1V and TAT-S1Vscr peptides were added at 1200 nM. With the exception of syndecan-1 antibody, which was added only during the assay, C2C12 cells were preincubated with reagents at the concentrations indicated for 1 h and then harvested, and the experiments were carried out in the continued presence of each reagent. Each condition was carried out in triplicate in each experiment, and transfilter migration was scored after 4 h. DMSO, dimethyl sulfoxide; PcT, paclitaxel; VBS, vinblastine sulfate; CytoD, cytochalasin D. In D, C2C12 cells coexpressing wild-type or mutant syndecan-1 and EGFP or EGFP-fascin were sorted by flow cytometry, and 1500 EGFP-positive cells were added to each well coated with 50 nM EHS laminin. After 4 h at 37°C, cells that had migrated to the far side of the filter were fixed, stained, and counted under phase-contrast illumination. Each condition was carried out in triplicate in each experiment. Columns represent mean ± SEM for data from at least three independent experiments.