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. 2019 Jul 16;8:e44219. doi: 10.7554/eLife.44219

Figure 3. Stalled AVs predominate in neurons from aged mice.

(A–B) Time series of merge micrographs of mCh-ATG13 and GFP-LC3B from live cell imaging of the distal neurite of DRGs from aged mice depicting a productive (A) or a stalled (B) autophagosome biogenesis event. Yellow arrowheads denote colocalization of mCh-ATG13 and GFP-LC3B; green arrowheads denote a GFP-LC3B-positive punctum from which mCh-ATG13 has dissociated; red arrowheads denote mCh-ATG13-positive puncta that fail to recruit GFP-LC3B; solid arrowheads track one punctum, hollow arrowheads follow a second punctum. Magnified views of denoted puncta are shown below full micrograph; border color represents channel or colocalization state in merge. Retrograde is to the right. Scale bars, 2 μm. (C–D) Individual intensity profiles were averaged to improve signal-to-noise of mCh-ATG13 (red) and GFP-LC3B (green) for productive (C) and stalled (D) AVs (mean ± SEM; n ≥ 5 biogenesis events from five neurons from three biological replicates). (E) Quantification of the proportion of total mCh-ATG13-positive AV biogenesis events (both productive and stalled events) in DRG neurons from young adult (light gray) and aged (dark gray) mice (mean ± 95% confidence interval; n ≥ 62 AVs from three biological replicates for each condition). ****p<0.0001 by Fisher’s exact test. (F–G) Time series of merge micrographs of mCh-ATG5 and GFP-LC3B in the distal neurite of DRGs from aged mice depicting a productive (F) or stalled (G) autophagosome biogenesis event. Arrowheads point to puncta magnified below micrograph; colors denote channel or colocalization state in merge. Scale bars, 2 μm. (H–J) Mean intensity profiles of mCh-ATG5 (red) and GFP-LC3B (green) for productive (H) and stalled (I) AVs (mean ± SEM; n = 5 productive biogenesis events from five neurons or n = 4 stalled biogenesis events from four neurons from three biological replicates). Vertical dashed line in (C and H) indicates the half-maximum of GFP-LC3B intensity, which was used to align the traces. (J) Quantification of the proportion of total mCh-ATG5-positive AV biogenesis events (both productive and stalled events) in DRG neurons from young adult and aged mice (mean ± 95% confidence interval; n ≥ 62 AVs from three biological replicates for each condition). ****p<0.0001 by Fisher’s exact test. See also Videos 1–6.

Figure 3.

Figure 3—figure supplement 1. Productive and stalled AVs occur in the same axonal tips.

Figure 3—figure supplement 1.

(A) Time series of GFP-LC3B, mCh-ATG13, and SNAP-ATG9 in the distal neurite of a DRG neuron from a young adult mouse depicting a productive autophagosome biogenesis event (solid arrowhead, #2) and a stalled biogenesis event (outlined arrowhead, #1). (B) Time series of GFP-LC3B and mCh-ATG5 in the distal neurite of a DRG neuron from an aged mouse depicting a productive autophagosome biogenesis event (solid arrowhead, #2) and a stalled biogenesis event (outlined arrowhead, #1). Magnified views of denoted puncta are shown below full micrograph; border color represents channel or colocalization state in merge. Retrograde is to the right. Scale bars, 2 μm.