Fig. 5: Rubicon’s localization to lysosomes in neurons is RAB7-dependent.
From: Mitochondrial damage triggers the concerted degradation of negative regulators of neuronal autophagy
A, B Rubicon expression is enhanced in neurons. A Representative western blot from lysates of HeLa cells or WT murine embryonic cortical neurons and probed for Rubicon. B Rubicon band intensity normalized to total protein in HeLa cells or WT neurons (N = 3 experiments, two-tailed unpaired t test). C–E Rubicon localizes to organelles distributed throughout the soma, dendrites, and axons of primary cortical neurons. C Representative max projection of the soma of a WT cortical neuron transfected with EGFP-Rubicon. D Representative max projection of the dendrites of a WT cortical neuron transfected with EGFP-Rubicon. E Representative max projection of an axon of a WT cortical neuron transfected with EGFP-Rubicon. F, G Rubicon colocalizes with the late endosome/lysosome marker LAMP1-Halo but not with the autophagosome marker mCherry-LC3. F Single z-plane confocal images of the soma of a WT cortical neuron transfected with mCherry-LC3, LAMP1-Halo and EGFP-Rubicon. Yellow boxes indicate EGFP-Rubicon colocalizing with either LAMP1-Halo or mCherry-LC3. G Fraction of the number of EGFP-Rubicon puncta in the soma of each neuron colocalizing with LAMP1-Halo or mCherry-LC3 (N = 3 experiments, two-tailed unpaired t test). H–K Rubicon localization to lysosomes is RAB7-dependent. H Schematics of the domain organization of RubiconWT and RubiconCGHL with the RUN, PI3K-binding domain (PIKBD) and Rubicon homology (RH) domains annotated (domains not drawn to scale). I Single z-plane confocal images of somas of neurons transfected with GFP-RAB7, LAMP1-Halo and either mCherry-RubiconWT or mCherry-RubiconCGHL. Yellow boxes indicate inset regions. J Fraction of total GFP-RAB7 area in each neuronal soma colocalizing with mCherry-RubiconWT or mCherry-RubiconCGHL. K Fraction of total Halo-LAMP1 area in each neuronal soma colocalizing with mCherry-RubiconWT or mCherry-RubiconCGHL (I–K: N = 3 experiments, two-tailed unpaired t test). All panels: Error bars indicate SEM, scale bars = 5μm. Source data are provided as a Source Data file.
