Figure 6.
Figure 6. APC-m4 alters autophagosome dynamics at FAs. All data are from live cell TIRF imaging of migrating MDA-MB-231 cells expressing APC constructs (APC-WT or APC-m4), along with markers for autophagosomes (GFP-LC3) and FAs (mCherry-zyxin). For all panels, data are averaged from at least three experiments. (A) Representative time-lapse imaging showing autophagosomes (GFP-LC3, cyan) and FAs (mCherry-zyxin, pink). Time = 0 corresponds to maximum mCherry-zyxin fluorescence intensity (peak FA growth). Scale bar, 3 µm. (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’s multiple comparisons test: n.s., not significant. (C) Histograms showing distributions of mature FAs targeted by autophagosomes in the 30-min observation window, from experiments as in A. n = 40 FAs from n > 5 cells per condition. (D) Scatter plot showing dwell times of autophagosomes at FAs, analyzed from experiments as in A. n ≥ 42 autophagosomes per condition from n > 10 cells per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: ****, P < 0.0001. (E) 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: ****, P < 0.0001. (F) Overlaid histograms showing time elapsed from peak FA maturity (time = 0) to arrival of autophagosome during the 40-min observation window. Negative numbers correspond to rare events in which autophagosomes arrive before FA peak maturation. Data are analyzed from experiments as in A. n = 20 FAs from n > 5 cells per condition. (G) Percentage of mature FAs targeted by GFP-NBR1 receptor, analyzed from live imaging experiments as in A, except using cells expressing mCherry-zyxin and GFP-NBR1. n = 100 FAs (from 15 cells) per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (H) Scatter plot showing dwell times of GFP-NBR1 interactions with FAs, analyzed from live imaging experiments as in G. n = 40 GFP-NBR1 visits to FAs (from n = 10 cells) per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (I) Coimmunoprecipitation of endogenous NBR1 with GFP-LC3, pulled down using GFP–Trap-A agarose beads. Cells transfected with empty vector (expressing GFP alone instead of GFP-LC3) serve as a negative control.

APC-m4 alters autophagosome dynamics at FAs. All data are from live cell TIRF imaging of migrating MDA-MB-231 cells expressing APC constructs (APC-WT or APC-m4), along with markers for autophagosomes (GFP-LC3) and FAs (mCherry-zyxin). For all panels, data are averaged from at least three experiments. (A) Representative time-lapse imaging showing autophagosomes (GFP-LC3, cyan) and FAs (mCherry-zyxin, pink). Time = 0 corresponds to maximum mCherry-zyxin fluorescence intensity (peak FA growth). Scale bar, 3 µm. (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’s multiple comparisons test: n.s., not significant. (C) Histograms showing distributions of mature FAs targeted by autophagosomes in the 30-min observation window, from experiments as in A. n = 40 FAs from n > 5 cells per condition. (D) Scatter plot showing dwell times of autophagosomes at FAs, analyzed from experiments as in A. n ≥ 42 autophagosomes per condition from n > 10 cells per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: ****, P < 0.0001. (E) 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: ****, P < 0.0001. (F) Overlaid histograms showing time elapsed from peak FA maturity (time = 0) to arrival of autophagosome during the 40-min observation window. Negative numbers correspond to rare events in which autophagosomes arrive before FA peak maturation. Data are analyzed from experiments as in A. n = 20 FAs from n > 5 cells per condition. (G) Percentage of mature FAs targeted by GFP-NBR1 receptor, analyzed from live imaging experiments as in A, except using cells expressing mCherry-zyxin and GFP-NBR1. n = 100 FAs (from 15 cells) per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (H) Scatter plot showing dwell times of GFP-NBR1 interactions with FAs, analyzed from live imaging experiments as in G. n = 40 GFP-NBR1 visits to FAs (from n = 10 cells) per condition. Error bars, SEM. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: n.s., not significant. (I) Coimmunoprecipitation of endogenous NBR1 with GFP-LC3, pulled down using GFP–Trap-A agarose beads. Cells transfected with empty vector (expressing GFP alone instead of GFP-LC3) serve as a negative control.

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