Journal: Nature Communications
Article Title: Mitochondrial damage triggers the concerted degradation of negative regulators of neuronal autophagy
doi: 10.1038/s41467-025-62379-5
Figure Lengend Snippet: 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 Rubicon WT and Rubicon CGHL 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-Rubicon WT or mCherry-Rubicon CGHL . Yellow boxes indicate inset regions. J Fraction of total GFP-RAB7 area in each neuronal soma colocalizing with mCherry-Rubicon WT or mCherry-Rubicon CGHL . K Fraction of total Halo-LAMP1 area in each neuronal soma colocalizing with mCherry-Rubicon WT or mCherry-Rubicon CGHL ( 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.
Article Snippet: The following plasmids were used for transfections: EGFP-Rubicon (RRID: Addgene_221659), LAMP1-Halo (RRID: Addgene_221655), mCherry-LC3 (RRID: Addgene_221656), mCherry-Rubicon WT (RRID: Addgene_221657), mCherry-Rubicon CGHL (RRID: Addgene_221658) and EGFP-Rab7A (RRID: Addgene_28047), EGFP-N1 (RRID: Addgene_6085-1).
Techniques: Expressing, Western Blot, Two Tailed Test, Transfection, Marker, Binding Assay
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