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B. and C. , Data averaged from three independent experiments. n ? 56 cells for phospho

. Src, White boxed regions correspond to zoom panels (right; scale bar, 2 ?m), highlighting the localization of phospho-Src and Paxillin at FAs. (F) Representative SIM images of cells immunostained for phospho-Paxillin(green) and Paxillin (pink). Scale bar, 5 ?m. White boxed regions correspond to zoom panels (right; scale bar, 2 ?m), highlighting the localization of phospho-Paxillin and Paxillin at FAs. (G) Representative SIM images of cells immunostained, = 35 cells for phospho-Paxillin, n ? 103 cells for phospho-FAK per condition. Error bars, SEM

, 2 ?m), highlighting the localization of phospho-Paxillin and Paxillin at FAs. (H) Density of phospho-Src, phospho-Paxillin, and phospho-FAK staining at individual FAs from cell images as in (E-G). Data averaged from two independent experiments. n = 50 FAs total from 15 cells per condition

. Mann-whitney, (I) Densities of signals at FAs for different components: GFP-Paxillin and mCherry-Zyxin densities were measured from cell images captured by TIRF microscopy; bars, SEM. Statistical significance calculated by non-parametric MannWhitney two-tailed student's t-test: **** P < 0.0001, *** P < 0.001. (APC-WT or APC-m4), along with markers for autophagosomes (GFP-LC3) and FAs (mCherry-Zyxin). For all panels

. Fas, Scale bar, 3 ?m. (B) Percentage of mature FAs targeted by autophagosomes, analyzed from experiments as in (A). n > 800 FAs per condition from n ? 20 cells per condition. Error bars, SEM. Statistical significance calculated by one-way Anova Dunn

, Scatter plot showing time after first appearance of an autophagosome at the FA to complete FA disassembly, analyzed from experiments as in (A). n = 31 FAs (APC-WT) or n = 50 FAs (APC-m4) from n > 10 cells per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann-Whitney two-tailed student's t-test: targeted by GFP-NBR1 receptor, analyzed from live imaging comparisons test: (compared with scramble, and in order from left to right): *, P < 0.05, n.s. (not significant). (B) Schematic of FA (grey) and stress fiber (green), highlighting regions of interest (ROI) analyzed for F-actin molecular organization by polarizationresolved microscopy. ROIs overlapping with FAs designated as 'inside', bars, SEM. Statistical significance calculated by non-parametric MannWhitney two-tailed student's t-test: **** P < 0.0001. (E)

, Remaining panels (left to right) show: the color-coded molecular order (?) superimposed on Alexa Fluor-488-phalloidin (F-actin, grey); intensity-thresholded image of FA (white) superimposed with stick representation of ? (encoded in stick color); and mean orientation, Cells were fixed and stained with Alexa Fluor-488-phalloidin (F-actin, grey) and phospho-Paxillin antibodies (pink)

, Histograms display ?? value distribution (in degrees) from inside and outside ROIs combined (<?> = 136°). Scale bar, 2 ?m. (E) Same as (D) except APC-m4 cells (<?> = 145°)

S. Figure, APC-m4 disrupts the molecular order of F-actin at focal adhesions. All data are from U2OS cells grown on collagen dishes, but not micropatterned as in Figures 2C-I. Left panels are data from cells depleted of endogenous APC (si-APC) and expressing APC-WT or APC-m4 rescue plasmids (silence and rescue), vol.1

S. D. Bars, Statistical significance calculated by non-parametric Mann-Whitney two-tailed student's t-test: *** P < 0.001, ** P < 0.01, * P < 0.05, n.s. (not significant). Data in right panels averaged from two independent experiments. n = 10 ROIs from n = 8 cells per condition. Error bars, SD. Statistical significance calculated by non-parametric MannWhitney two-tailed student

S. Figure, Error bars, SEM. Statistical significance calculated by non-parametric Mann-Whitney two-tailed student's t-test: * P < 0.05. (E) Average time to 50% maximal recovery for experiments in (B). Error bars, SEM. Statistical significance calculated by non-parametric Mann-Whitney two-tailed student's t-test: n.s. (not significant). (F) Average immobile fraction for experiments in (B). Error bars, SEM, Effects of APC depletion on actin dynamics in focal adhesions and stress fibers

S. Figure, Levels of APC and Dia1 in MDA-MB-231 cells. (A) Western blot analysis of whole cell extracts from MDA-MB-231 cells treated with scramble RNAi (control), depleted of endogenous APC (si-APC), depleted of endogenous APC and rescued with refractory APC-WT or APC-m4 (rescue), and cells expressing the same APC constructs without depleting endogenous APC (ectopic)

, Blots were probed with antibodies to APC and GAPDH (loading control). (B)

, Data averaged from two experiments. Error bars, SEM. Statistical significance calculated by ordinary oneway Anova Holm-Sidak's multiple comparisons test (compared with scramble, and in order from left to right): *** P < 0.001, n.s. (not significant). (C) Western blot analysis of whole cell extracts from MDA-MB, Quantification of the ratio of APC to GAPDH from blots as in (A)

. Fas-;-mcherry-zyxin, pink) in migrating MDA-MB-231 cells ectopically expressing APC-WT or APC-m4, as indicated. Yellow boxes that appear during the video highlight the same four FAs shown as examples in Figure 4 A and B, p.10

, Representative examples of time-lapse TIRF imaging of mCherry-Zyxin (FA marker) assembly and disassembly in migrating MDA-MB-231 cells ectopically expressing APC-WT or APC-m4, as indicated. The arrows that appear during the video indicate the point of FA peak growth or maturation (maximum intensity), which was set to time=0

, Related to Figure 5. Representative examples of time-lapse TIRF imaging of microtubules (3xGFP-EMDB, cyan) and FAs (mCherry-Zyxin, pink) in migrating MDA, vol.3

, MB-231 cells ectopically expressing full-length APC-WT or APC-m4, as indicated. Images were acquired every 5 sec. Video is shown at 7 frames per sec

, Representative examples of time-lapse TIRF imaging of autophagosomes (GFP-LC3, cyan) and FAs (mCherry-Zyxin, pink) in migrating MDA-MB-231 cells ectopically expressing APC-WT or APC-m4, as indicated