Figure 4.
Figure 4. APC-m4 slows FA disassembly. All data are from live TIRF microscopy imaging of migrating MDA-MB-231 cells expressing APC-WT or APC-m4, and either control-RNAi treated (scramble) or Dia1-silenced. (A) Representative image of an APC-WT cell (left; scale bar, 25 µm) showing FAs marked with mCherry-zyxin. The four FAs marked by yellow boxes and numbered correspond to the montages (right). Montages show time points from time-lapse imaging of FA assembly and disassembly. Time = 0 represents FA maximum size (peak fluorescence intensity). Scale bar (in time-lapse montage), 3 µm. (B) Same as in A except APC-m4 cell and corresponding FAs. (C) Histograms showing the distributions of times for individual FAs to complete assembly (from the time of initial appearance of mCherry-zyxin signal to time of peak intensity). Bin width, 2 min. Data from live imaging experiments as in A and B. n = 100 FAs per condition from n = 10 cells per condition. (D) Overlaid histograms showing the distributions of times for individual FAs to complete disassembly (starting from the time of peak fluorescence intensity to complete disappearance of signal). Bin width, 2 min. Data from live imaging experiments as in A and B. n = 840 FAs (APC-WT) and n = 961 FAs (APC-m4), each from n > 20 cells per condition. (E) Box and whisker plots showing data points from all regions of the cell used to determine global FA assembly and disassembly rates. Data are from live imaging experiments as in A and B, and were analyzed with the webtool FAAS. APC-WT (n = 286 FAs for assembly rate and n = 312 FAs for disassembly rate) and APC-m4 (n = 208 FAs for assembly rate and n = 354 FAs for disassembly rate), from n ≥ 8 cells per condition. Statistical significance calculated by ordinary one-way ANOVA Holm–Sidak multiple comparisons test: ****, P < 0.0001; n.s., not significant. (F and G) Box and whisker plots showing data points used to determine FA assembly (F) and disassembly (G) rates at the leading edge (front) or trailing edge (rear) of cells. Data are from live imaging experiments as in A and B, and were analyzed with the webtool FAAS. n = 200 FAs from n ≥ 8 cells per condition. Statistical significance calculated by ordinary one-way ANOVA Holm–Sidak multiple comparisons test: ****, P < 0.0001; n.s., not significant. (H) Box and whisker plots showing data points from all regions of the cell used to determine global FA assembly and disassembly rates for mock (scramble)–treated or Dia1-silenced cells. Data from live imaging experiments were analyzed with the webtool FAAS. Scramble (n = 499 FAs for assembly rate, and n = 344 FAs for disassembly rate) and si-Dia (n = 667 FAs for assembly rate, and n = 628 FAs for disassembly rate), from n ≥ 12 cells per condition. Statistical significance calculated by ordinary one-way ANOVA Holm–Sidak multiple comparisons test: *, P < 0.05; n.s., not significant. (I) Total F-actin levels in cells determined by phalloidin staining. Data averaged from three independent experiments. n = 40–100 cells per condition. Error bars, SEM. Statistical significance calculated by ordinary one-way ANOVA Holm–Sidak multiple comparisons test: ****, P < 0.0001; n.s., not significant.

APC-m4 slows FA disassembly. All data are from live TIRF microscopy imaging of migrating MDA-MB-231 cells expressing APC-WT or APC-m4, and either control-RNAi treated (scramble) or Dia1-silenced. (A) Representative image of an APC-WT cell (left; scale bar, 25 µm) showing FAs marked with mCherry-zyxin. The four FAs marked by yellow boxes and numbered correspond to the montages (right). Montages show time points from time-lapse imaging of FA assembly and disassembly. Time = 0 represents FA maximum size (peak fluorescence intensity). Scale bar (in time-lapse montage), 3 µm. (B) Same as in A except APC-m4 cell and corresponding FAs. (C) Histograms showing the distributions of times for individual FAs to complete assembly (from the time of initial appearance of mCherry-zyxin signal to time of peak intensity). Bin width, 2 min. Data from live imaging experiments as in A and B. n = 100 FAs per condition from n = 10 cells per condition. (D) Overlaid histograms showing the distributions of times for individual FAs to complete disassembly (starting from the time of peak fluorescence intensity to complete disappearance of signal). Bin width, 2 min. Data from live imaging experiments as in A and B. n = 840 FAs (APC-WT) and n = 961 FAs (APC-m4), each from n > 20 cells per condition. (E) Box and whisker plots showing data points from all regions of the cell used to determine global FA assembly and disassembly rates. Data are from live imaging experiments as in A and B, and were analyzed with the webtool FAAS. APC-WT (n = 286 FAs for assembly rate and n = 312 FAs for disassembly rate) and APC-m4 (n = 208 FAs for assembly rate and n = 354 FAs for disassembly rate), from n ≥ 8 cells per condition. Statistical significance calculated by ordinary one-way ANOVA Holm–Sidak multiple comparisons test: ****, P < 0.0001; n.s., not significant. (F and G) Box and whisker plots showing data points used to determine FA assembly (F) and disassembly (G) rates at the leading edge (front) or trailing edge (rear) of cells. Data are from live imaging experiments as in A and B, and were analyzed with the webtool FAAS. n = 200 FAs from n ≥ 8 cells per condition. Statistical significance calculated by ordinary one-way ANOVA Holm–Sidak multiple comparisons test: ****, P < 0.0001; n.s., not significant. (H) Box and whisker plots showing data points from all regions of the cell used to determine global FA assembly and disassembly rates for mock (scramble)–treated or Dia1-silenced cells. Data from live imaging experiments were analyzed with the webtool FAAS. Scramble (n = 499 FAs for assembly rate, and n = 344 FAs for disassembly rate) and si-Dia (n = 667 FAs for assembly rate, and n = 628 FAs for disassembly rate), from n ≥ 12 cells per condition. Statistical significance calculated by ordinary one-way ANOVA Holm–Sidak multiple comparisons test: *, P < 0.05; n.s., not significant. (I) Total F-actin levels in cells determined by phalloidin staining. Data averaged from three independent experiments. n = 40–100 cells per condition. Error bars, SEM. Statistical significance calculated by ordinary one-way ANOVA Holm–Sidak multiple comparisons test: ****, P < 0.0001; n.s., not significant.

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