Fig. 6: Rubicon inhibits lysosomal function and autophagosomal maturation.
From: Mitochondrial damage triggers the concerted degradation of negative regulators of neuronal autophagy
A–D Rubicon knockdown increases the number of lysosomes and autolysosomes in cortical neurons. A Single z-plane confocal image of neurons nucleofected with control or Rubicon shRNA and treated with DAPRed and Lysotracker. Outline of the neuronal soma for quantification is indicated using dashed lines in cyan. Yellow boxes indicate inset regions. B Fold change in lysosome numbers (marked by Lysotracker punctae) per neuronal soma in control vs Rubicon knockdown neurons. C Fold change in autophagosome numbers (marked by DAPRed punctae) per neuronal soma in control vs Rubicon knockdown neurons. D Fold change in autolysosome numbers (marked by colocalizing DAPRed and Lysotracker punctae) per neuronal soma in control vs Rubicon knockdown neurons (A–D: N = 4 experiments, Mann–Whitney test). E Schematic depicting the steps of lysosomal pH detection assay in neurons using LysoPrime Green, pHLys Red to label total and acidic lysosomes, respectively, followed by treatment with 75 nM Baf A1. F–I Rubicon negatively influences lysosomal acidification in neurons. F Representative max projections of neurons nucleofected with Ctrl or Rubicon shRNA and assayed for lysosomal pH using the protocol represented in (E). Outline of the neuronal soma for quantification is indicated using dashed lines in cyan G Total no. of lysosomes labeled by LysoPrime Green in control or Rubicon knockdown neurons. H No. of acidic lysosomes labeled by pHLys Red in control or Rubicon knockdown neurons. I Ratio of acidic to total lysosomes in control or Rubicon knockdown neurons (F–I: N = 3 experiments, two-tailed unpaired t test). J, K Rubicon knockdown increases the number of proteolytically active lysosomes. J Single z-plane confocal images of neurons nucleofected with control or Rubicon shRNA and treated with Magic Red dye. Outline of the neuronal soma for quantification is indicated using dashed lines in cyan. K Fold change in the number of Magic Red puncta per neuronal soma in control vs Rubicon knockdown neurons (N = 5 experiments, Mann–Whitney test). (L, M) Rubicon is associated with lysosomes but not autolysosomes. L Single z-plane confocal images of a neuron transfected with EGFP-Rubicon, LAMP1-Halo and mCherry-LC3. Outline of the neuronal soma for quantification is indicated using dashed lines in cyan. Yellow boxes indicate inset regions. M Fraction of lysosomes (LAMP1-Halo) colocalizing with EGFP-Rubicon in the soma that do or do not colocalize with mCherry-LC3 (N = 3 experiments, two-tailed unpaired t test). N–Q Overexpression of Rubicon inhibits autophagosome maturation. N Single z-plane confocal images of neurons transfected with mCherry-LC3, LAMP1-Halo and either EGFP or EGFP-Rubicon. Yellow boxes indicate inset regions. O Number of autophagosomes (marked by mCherry-LC3 punctae) per neuronal soma, either expressing EGFP or EGFP-Rubicon. P Number of autolysosomes (marked by colocalizing mCherry-LC3 and LAMP1-Halo puncta) per neuronal soma, either expressing EGFP or EGFP-Rubicon. Q Fraction of autolysosomes (LAMP+LC3+) to autophagosomes (LC3+) per neuronal soma either expressing EGFP or EGFP-Rubicon (N–Q: N = 4 experiments, two-tailed unpaired t test). All panels: Error bars indicate SEM, scale bars = 5μm. Source data are provided as a Source Data file.
