Fig 2: 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 3: 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 4: 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 5: 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.