Figure 1.
Cell-cell interaction improves the efficiency of PC3 cell migration in 3D Matrigel.
(A) Morphology of prostate cancer cell lines in 3D Matrigel. PC3 cells form multi-cellular invasive cluster but 22Rv1, LNCaP, RWPE-1 and RWPE-2 cells form multi-cellular spheroids and are not invasive. Arrowheads indicate single, non-migratory PC3 cells. Right panel shows immunoblots for N-cadherin (Ncad), E-cadherin (Ecad), α-catenin (α-cat) and tubulin (Tub) of prostate cancer cell lines. (B) Cell migration on 2D surface. Red star and black arrow separately mark and track two neighboring cells passing each other without forming and maintaining cell-cell contact. Time in minutes. (C) Time-lapse images of collective cell migration of PC3 cells in 3D Matrigel. Time in hours. (D) Representative trajectories (left), average speed (middle) and end-to-end displacement (right) of single cells (N = 33) and multicellular clusters (N = 43) in 3D Matrigel (**, p<0.01). (E) EDTA addition disrupts cell-cell interactions. EDTA was added at a final concentration of 1.4 mM. Time in minutes. (F) Immunostaining for N-cadherin in PC3 cells seeded in 3D Matrigel. N-cadherin (clone 6G11) co-localizes with actin (phalloidin) at cell-cell contacts. Arrow indicates cell-cell contact. All scale bars are 10 µm.
Figure 2.
N-cadherin expression levels correspond with increased cell migration in 3D Matrigel.
(A) Immunostaining of N-cadherin with phalloidin (top panel) and E-cadherin with phalloidin (bottom panel) of parental PC3 cells plated on coverslips. (B) Two representative subclones from parental PC3 cells, PC3e and PC3n, were immunoblotted for E-cadherin and N-cadherin. Tubulin is shown as a loading control. The original blots are shown in Supplementary Figure S1. (C) Immuno-staining of PC3 subclones for E-cadherin and N-cadherin (clone 32). (D) Cell morphologies, cell speed and directional persistency of PC3e (N = 19) and PC3n (N = 20) clones in 3D Matrigel (**, P<0.01; N = 11–42). All scale bars are 20 µm. (E) Heat map of microarray analysis for all cadherin expression levels in parental PC3, PC3e and PC3n cells.
Figure 3.
Overexpression of the cytoplasmic domain of N-cadherin reduces 3D cell migration.
(A) Schematic of the full-length N-cadherin and the cytoplasmic N-cadherin (N-cyto) constructs. N-cadherin cytoplasmic domain is fused with membrane targeting domain from Lyn kinase (black) at the N-terminus and tandem dimer-tomato (red) at the C-terminus. (B) Immunoblots for N-cadherin extracellular domain (clone 8C11), N-cadherin cytoplasmic domain (clone 32), and E-cadherin (clone 36) in N-cyto expressing PC3 cell subclones. #1, #2, #3, #4 are four populations of N-cyto cells with gradually increased N-cyto levels. (C) Heat map of microarray analysis for all cadherin levels in PC3, N-cyto #1 and N-cyto #4 cells. (D) Immuno-staining of extracellular domain of N-cadherin (clone 8C11), with phalloidin in parental PC3 cells (top panel) and N-cyto #4 cells (bottom panel). N-cyto localization was detected by the tandem-tomato signal (bottom panel). Scale bar 10 µm. (E) Statistical analysis of N-cyto expressing PC3 cell 3D migration. Average speed and end to end distance are shown (**, P<0.01; N = 42 for parental cells and N = 11 for N-cyto cells). Error bars are standard error of the mean. Scale bar, 40 µm.
Figure 4.
N-cadherin deficient cells are non-migratory in 3D Matrigel.
(A) Immunoblots for N-cadherin, E-cadherin and cadherin 11 in N-cadherin knockdown cells and cells transfected with scrambled sequences. Tubulin is shown as a loading control. KD1, KD2 are two knockdown clones. Scr1, Scr2 are two clones transfected with corresponding scrambled sequences. (B) Immunostaining of N-cadherin in control cells (top panel) and knockdown cells (bottom panel). (C) Cell aggregate formation of knockdown cells and control cells over 3 hours in suspension. At hour 0, 1, 2, 3 in suspension, the numbers of cell aggregates analyzed were 144, 101, 133, 62 for parental PC3 cells, 147, 117, 107, 81 for KD1 cells, 142, 121, 107, 73 for KD2 cells, 146, 115, 87, 88 for Scr1 cells, 131, 147, 107, 68 for Scr2 cells, respectively. (D) 3D cell migration analyses of parental (N = 17), scramble shRNA (N = 17 for Scr1, N = 20 for Scr2) and knockdown cells (N = 37 for KD1, N = 33 for KD2). Statistical analysis of average speed is shown (**, P<0.01). All error bars are standard error of the mean. All scale bars are 20 μm.
Figure 5.
Expression of α-catenin strengthens cell-cell adhesion and decreases 3D cell migration.
(A) Immunoblots for α-catenin, N-cadherin and E-cadherin in α-catenin expressing cells. Tubulin is shown as a loading control. LNCaP was used as a positive control for α-catenin and E-cadherin. (B) Localization of α-catenin in parental PC3 cells and GFP-tagged α-catenin expressing cells in bottom panels. (C) Co-localization of GFP-tagged α-catenin with N-cadherin. (D) Cell aggregation analysis of parental and α-catenin expressing cells in suspension. Inset: enlarged views of cell clusters. GFP signal is overlayed onto bright-field image for GFP-tagged α -catenin expressing cells. Average aggregation sizes at each time points are shown in the bar graph. (E) Cell migration analyses in 3D Matrigel. GFP channel of GFP-tagged α-catenin expressing cells in the white box (bottom left panel) is shown in bottom right panel. Statistical analysis of cell migration speed is shown (**, P<0.01; N = 35 for parental cells, N = 31 for GFP-α-catenin expressing cells). All scale bars are 20 μm.
Figure 6.
The N-terminal half of α-catenin is sufficient to suppress invasive phenotype.
(A) Schematic of full-length α-catenin and its C-terminal truncated mutants. GFP tag, Vinculin binding (Vin), its inhibitory (Inhibit), F-actin binding (Actin) domains are shown. (B) Immunoblots for α-catenin, N-cadherin and E-cadherin in various α-catenin expressing cells. Tubulin is shown as a loading control. 22Rv1 was used as a positive control for α-catenin and E-cadherin. The original blots are shown in supplementary Figure S2. (C) Cell morphology (top panel) and localization of α-catenin (bottom panel) in various α-catenin expressing PC3 cells. (D) Cell aggregation analysis of parental, various α-catenin expressing cells, and 10 μM cytochalasin D (CD) treated cells. Inset: enlarged views of cell clusters. GFP signal is overlayed onto bright-field image for GFP-tagged α–catenin/truncation mutant expressing cells. Aggregation size of the cells at each time points are shown in the mean ± standard error of the mean; ANOVA analysis combined with posthoc test Tukey HSD were used between groups at timepoint hour 3 (**, P<0.01). (E) Cell migration analyses in 3D Matrigel for parental (N = 35), the full-length α-catenin (N = 31), α-catenin 1–509 (N = 73), α-catenin 1–670 (N = 39), and α-catenin 1-848 (N = 52) expressing cells. Average speed and endpoint distance of migration are shown in mean ± standard error of the mean; ANOVA and posthoc test Tukey HSD were used (***, P<0.001). All scale bars are 20 μm.