Fig 1.
Expression of α-actinin-1 (ACTN1) and ACTN4 in human colon cancer cell lines.
(A) Western blot analysis of ACTN1, ACTN4, and actin in human colon cancer cell lines. ACTN1 and ACTN4 protein levels were measured in comparison with recombinant His-tag ACTN1 and ACTN4 proteins used as external standards. Actin was used as a loading control and purified rabbit skeletal muscle actin was used as an external standard. (B) Band intensities were quantified, and the molar ratios of ACTNs were calculated and represented as mol% against actin. The data represent the mean ± SD of three independent experiments.
Fig 2.
Effect of ACTN1 and ACTN4 on DLD-1 cell morphology.
(A) ACTN1 and ACTN4 expression was silenced by siGENOME SMARTpool siRNA in DLD-1 cells. Efficient knockdown was confirmed by western blotting using ACTN isoform-specific antibodies. The arrowhead indicates endogenous ACTN1. The asterisk indicates a non-specific crossreacting band. (B) Cells transfected with the indicated siRNAs were stained for vinculin (VCL) and F-actin and observed by confocal microscopy. The arrows and arrowheads indicate focal adhesions and stress fibers, respectively. Scale bar = 10 μm. (C) The percentages of siRNA-treated cells forming stress fibers are graphed. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01. (D) The focal adhesion numbers per cell of siRNA-treated cells were graphed. Data represent the mean ± SD of 71 cells for control, 73 cells for si-ACTN1 cells, and 55 cells for si-ACTN4 cells. *P < 0.05. (E) Transient expression of ACTN1-GFP and ACTN4-GFP in DLD-1 cells was confirmed by western blotting using ACTN isoform-specific antibodies. (F) GFP, ACTN1-GFP, and ACTN4-GFP were expressed in DLD-1 cells, which were then stained for VCL and F-actin and observed by confocal microscopy. The arrows and arrowheads indicate focal adhesions and stress fibers, respectively. Scale bar = 10 μm. (G) The percentages of transfected cells forming stress fibers are graphed. Data represent the mean ± SD of three independent experiments. Tfx indicates transfection. **P < 0.01. (H) The focal adhesion numbers per cell of cells expressing GFP, ACTN1-GFP, and ACTN4-GFP were graphed. Data represent the mean ± SD of 130 cells for GFP, 52 cells for ACTN1-GFP, and 73 cells for ACTN4-GFP. *P < 0.05.
Fig 3.
Turnover rate of focal adhesions in mCherry-, ACTN1-mCherry-, or ACTN4-mCherry-overexpressing DLD-1 cells.
(A) mCherry, ACTN1-mCherry, or ACTN4-mCherry were co-expressed with GFP-VCL and VCL time lapse image was observed by total internal reflection fluorescence (TIRF) microscopy to trace the assembly and disassembly of focal adhesions. Upper panels show representative kymographs of GFP-VCL fluorescence along 5-μm long lines drawn in regions of extending lamellipodia. Fluorescence intensity in the upper panel was plotted against time and shown as a graph to the bottom of respective kymographs. The turnover phases of focal adhesion assembly, stability, and disassembly are presented as red dashed lines in the mCherry vector graph. (B–D) Duration of each turnover phase exemplified in the left graph in A was analyzed and quantified from 51 adhesions for mCherry-expressing cells, 57 adhesions for ACTN1-mCherry-expressing cells, and 64 adhesions for ACTN4-mCherry-expressing cells. Data are presented as box and whisker plots with boxes representing 25th–75th percentile range and whiskers representing 10th–90th percentile range. n.s., not significant, **P < 0.01.
Fig 4.
ZYX recruitment to focal complexes in ACTN1- or ACTN4-expressing DLD-1 cells.
(A) GFP, ACTN1-GFP, and ACTN4-GFP (shown in green) were expressed in DLD-1 cells, which were stained for PAX (blue) and ZYX (red) proteins and imaged under a confocal fluorescence microscope. Boxed regions in the leftmost column indicate the peripheral lamellae used for PAX and ZYX plaque quantification. A series of enlarged images of the boxed regions are shown to the right. The arrows and arrowheads indicate PAX-positive and PAX/ZYX-double-positive plaques, respectively. Scale bar = 30 μm. (B–C) Numbers of PAX and ZYX plaques per unit area of peripheral lamellae were counted in GFP-, ACTN1-, or ACTN4-expressing cells. Eight to sixteen cells were analyzed per group. Error bars refer to standard deviations between cells. n.s., not significant, *P < 0.05, **P < 0.01. (D) Ratio of ZYX-positive to PAX-positive plaques, i.e., total plaque adhesions. Error bars refer to standard deviations between cells. n.s., not significant, **P < 0.01. (E) PAX plaque size in GFP-, ACTN1- or ACTN4-expressing cells was measured in at least 171 adhesions from 8–11 cells per group. Error bars refer to standard deviations between cells. n.s., not significant, *P < 0.05, **P < 0.01.
Fig 5.
ZYX binding to ACTN1 and ACTN4.
(A) Pull-down assay showing that a putative ACTN-binding site, GST-ZYX-N (aa 1–51), binds specifically to His-ACTN1, but not to His-ACTN4. Bound His-ACTN1 or His-ACTN4 was analyzed by western blotting using anti-His-tag antibody. Equal amounts of input proteins were confirmed by SDS-PAGE followed by Coomassie blue staining (CBB). (B) FLAG-ZYX-full (aa 1–572) or FLAG-ZYX-C (aa 52–572) bound to anti-FLAG M2 beads were mixed either with His-ACTN1 or His-ACTN4 proteins, or without ACTN proteins (denoted by “-”). Bound His-ACTN1 or His-ACTN4 was analyzed by western blotting using anti-pan-actinin antibody. Note that the faint bands detected in lanes 1 and 4 of the pull-down blot represent non-specific background signals of pan-actinin antibody. Equal amounts of ZYX proteins were confirmed by western blotting using anti-FLAG antibody.
Fig 6.
Invasion of DLD-1 cells expressing GFP, ACTN1-GFP, and ACTN4-GFP.
(A) Stable expression of GFP, ACTN1-GFP, and ACTN4-GFP in DLD-1 cells was examined by western blotting using the antibodies indicated to the left of each blot. (B) The invasion assay was performed using GFP-, ACTN1-GFP-, or ACTN4-GFP-expressing stable cell lines. Cells were seeded into Matrigel invasion chambers and allowed to invade into the Matrigel for 26 h. Then, the invaded cells were fixed and imaged by examining the GFP signals on the undersurface of the chamber using fluorescence confocal microscopy. Scale bar = 100 μm. (C) The GFP-positive cells shown in (B) were quantified. At least six microscopic fields obtained from duplicated chambers were used for quantification in each experiment. The results represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.
Fig 7.
ACTN1 expression suppresses SW480 cell invasion.
(A) SW480 cells expressing GFP or ACTN1-GFP (shown in green) were stained for PAX (blue) and ZYX (red) proteins and imaged as described for Fig 4A. Boxed regions in the leftmost column are enlarged and shown to the right. The arrows and arrowheads indicate PAX-positive and PAX/ZYX-double-positive plaques, respectively. Scale bar = 20 μm. (B–C) Numbers of PAX and ZYX plaques per cell were counted in GFP- or ACTN1-GFP-expressing cells. At least 66 cells were analyzed per group. Error bars refer to standard deviations between cells. n.s., not significant, *P < 0.05. (D) Ratio of ZYX-positive to PAX-positive plaques. Error bars refer to standard deviations between cells. **P < 0.01. (E) SW480 cells were transiently transfected with GFP or ACTN1-GFP, and protein expression was analyzed by western blotting using the antibodies indicated to the left of each blot. Tfx indicates transfection. The asterisk indicates a non-specific crossreacting band. (F) The invasion assay was performed using GFP- or ACTN1-GFP-expressing SW480 cells as described in the legend to Fig 6B. Invaded cells were fixed and imaged by GFP fluorescence. Scale bar = 100 μm. (G) The GFP-positive cells shown in (F) were quantified as described in the legend to Fig 6C. **P < 0.01.