Figure 4. Autophagosomes associate with dynamic FAs during disassembly. (A) Spinning disk confocal microscopy of a migrating cell expressing GFP-LC3 (black) to label autophagosomes and paxillin-mCherry (magenta) to label FAs. Left panel shows maximum intensity projection (MIP) of a cell over 21 min, illustrating multiple associations between autophagosomes and FAs. Boxed inset areas are enlarged in right panel. Bar, 5 µm. Insets are magnified twofold. (B) Criteria for distinguishing GFP-LC3–targeted FAs versus nontargeted FAs. Left illustration depicts representations of targeted FAs (top and middle) and nontargeted FAs (bottom). Right images are examples of targeted and nontargeted FAs. Bar, 0.5 µm. (C) Quantification of the percentage of dynamic leading edge FAs per cell targeted by autophagosomes. FAs were randomly chosen independent of the GFP channel and then manually tracked from their appearance to disappearance for evidence of direct contact by GFP-LC3 vesicles. Scatter plot shows individual single cells (n = 12 total cells) and median (line), representing 129 total FAs analyzed from two independent experiments. (D) Analysis of GFP-LC3 vesicles in FA areas and non-FA areas at the leading edge of migrating cells. The total number of vesicles at FAs or in non-FA areas was counted and normalized to the total area for FA or non-FA regions, respectively. Scatter plot shows individual single cells (n = 12 total cells) and mean (line), representing 196 total leading edge GFP-LC3 vesicles analyzed from two independent experiments. P-value determined using unpaired t test. (E) Spinning disk confocal microscopy time-lapse sequences of representative targeted (box with dotted border, bottom) and nontargeted (box with solid border, top) FAs. Insets rotated such that leading edge is moving upward vertically. Arrows track single FAs over time, with autophagosome targeting indicated by arrowheads. Elapsed time (min) shown in top left of images. Right-most panels show MIP for each FA (arrow) shown in the corresponding time-lapse sequence. Bar, 5 µm. Insets are magnified 1.5-fold. (F) Representative paxillin-mCherry fluorescence intensity (y axis) plots over time (x axis) for the FAs shown in E. Frames in which GFP-LC3 was in direct contact with FAs are indicated by black data points and bracketing (right plot). (G) Temporal analysis of GFP-LC3 targeting to FAs. The phase during which GFP-LC3 associated with FAs was determined by counting the total number of GFP-LC3 targeting events in C and determining when during FA turnover each event occurred; if FAs were targeted multiple times during their lifetime, each event was independently counted. Scatter plot shows individual cells (n = 12 total cells) and median (line), representing 114 total targeting events analyzed from two independent experiments. P-values were calculated using a nonparametric Kruskall-Wallis test followed by Dunn post-hoc test. n.s., not significant. Images in this figure correspond to Videos 4–6.
Figure 4.

Autophagosomes associate with dynamic FAs during disassembly. (A) Spinning disk confocal microscopy of a migrating cell expressing GFP-LC3 (black) to label autophagosomes and paxillin-mCherry (magenta) to label FAs. Left panel shows maximum intensity projection (MIP) of a cell over 21 min, illustrating multiple associations between autophagosomes and FAs. Boxed inset areas are enlarged in right panel. Bar, 5 µm. Insets are magnified twofold. (B) Criteria for distinguishing GFP-LC3–targeted FAs versus nontargeted FAs. Left illustration depicts representations of targeted FAs (top and middle) and nontargeted FAs (bottom). Right images are examples of targeted and nontargeted FAs. Bar, 0.5 µm. (C) Quantification of the percentage of dynamic leading edge FAs per cell targeted by autophagosomes. FAs were randomly chosen independent of the GFP channel and then manually tracked from their appearance to disappearance for evidence of direct contact by GFP-LC3 vesicles. Scatter plot shows individual single cells (n = 12 total cells) and median (line), representing 129 total FAs analyzed from two independent experiments. (D) Analysis of GFP-LC3 vesicles in FA areas and non-FA areas at the leading edge of migrating cells. The total number of vesicles at FAs or in non-FA areas was counted and normalized to the total area for FA or non-FA regions, respectively. Scatter plot shows individual single cells (n = 12 total cells) and mean (line), representing 196 total leading edge GFP-LC3 vesicles analyzed from two independent experiments. P-value determined using unpaired t test. (E) Spinning disk confocal microscopy time-lapse sequences of representative targeted (box with dotted border, bottom) and nontargeted (box with solid border, top) FAs. Insets rotated such that leading edge is moving upward vertically. Arrows track single FAs over time, with autophagosome targeting indicated by arrowheads. Elapsed time (min) shown in top left of images. Right-most panels show MIP for each FA (arrow) shown in the corresponding time-lapse sequence. Bar, 5 µm. Insets are magnified 1.5-fold. (F) Representative paxillin-mCherry fluorescence intensity (y axis) plots over time (x axis) for the FAs shown in E. Frames in which GFP-LC3 was in direct contact with FAs are indicated by black data points and bracketing (right plot). (G) Temporal analysis of GFP-LC3 targeting to FAs. The phase during which GFP-LC3 associated with FAs was determined by counting the total number of GFP-LC3 targeting events in C and determining when during FA turnover each event occurred; if FAs were targeted multiple times during their lifetime, each event was independently counted. Scatter plot shows individual cells (n = 12 total cells) and median (line), representing 114 total targeting events analyzed from two independent experiments. P-values were calculated using a nonparametric Kruskall-Wallis test followed by Dunn post-hoc test. n.s., not significant. Images in this figure correspond to Videos 4–6.

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