Fig 2: Characterization of p18 in hippocampal neurons.(A) Images of cultured hippocampal neurons co-immunostained for lysosomal protein LAMP2 (green) and p18 (red). Insets are enlarged images of LAMP2- and p18-immunoreactive puncta along the dendrites. Arrowheads indicate co-localized puncta. Scale bar: top, 20 µm; inset, 10 µm. (B) Left, p18 forms a complex with p14 and MP1 in hippocampal neurons. Lysates from cultured hippocampal neurons were immunoprecipitated with an anti-p18 antibody or control IgG and probed with the indicated antibodies. Right, RagA co-immunoprecipitates RagB, RagC, p18, and p14. Lysates from mouse hippocampi were immunoprecipitated with an anti-RagA antibody or control IgG and probed with the indicated antibodies. (C) Images of cultured hippocampal neurons co-immunostained for p18 (magenta) and LAMP2 (red). Neurons were infected with shRNA AAV directed against p18 with GFP co-expression or scrambled shRNA control before processing for immunofluorescence assay and imaging. Scale bar, 20 µm. (D) Images of hippocampal neurons stained for LAMTOR4 (red). Cells were infected and processed as in (C). Scale bar, 10 µm; inset, 5 µm. (E) Images of hippocampal neurons stained for RagA (red). Cells were infected and processed as in (C). Scale bar, 10 µm; inset, 5 µm. (F) Quantification of fluorescent signals for p18 (n = 13, p<0.001), LAMTOR4 (n = 11, p<0.001), RagA (n = 6, p=0.002), and LAMP2 (n = 6, p=0.356) in control shRNA and p18 shRNA-infected neurons shown in C– E. Student’s t-test. Note that n refers to the number of culture dishes analyzed. (G) Left, Western blot analysis of p18, p14, LAMTOR4, RagA, RagB, and LAMP2 in enriched lysosomal fractions prepared from WT neurons transfected with Accell control or p18 siRNA. Right, quantitative analysis of blots. Results are expressed as % of values in control siRNA-transfected WT neurons and shown as means ± SEM N = 3 independent experiments, p<0.001 for p18, p14, LAMTOR4, and RagA, p=0.001 for RagB (unpaired, two-tailed Student's t-test). See also Figure 2—figure supplement 1 and Figure 2—source data 1.10.7554/eLife.37993.007Figure 2—source data 1.Quantitative analyses of images and Western blots used for Figure 2 and Figure 2—figure supplement 1.

Fig 3: Generation of Ragulator-deficient clone with total mTORC1 activity.A, schematic representation of the findings of this study, depicting the phosphorylation of the TFEB substrate by mTORC1 bound to the Ragulator-Rag scaffold. B, the results of Western blotting studies on ?Ragulator and parental RAW264.7 cells. Lamtor1, Lamtor2, Lamtor3, Lamtor4, and Lamtor5 are components of the Ragulator complex; ß-actin served as the internal control; mTOR and Raptor are components of mTORC1; RagC is necessary for the docking of TFEB to Ragulator. C, representative result of Western blotting studies for determining the activity of mTORC1. The control RAW264.7 cells and ?Ragulator cells were cultured in nutrient-sufficient and serum-supplemented DMEM. The phosphorylation of S6K in these cells was abolished following treatment with the mTOR kinase inhibitor, Torin1, at the indicated concentrations for 1 h. D, the densities of the p-S6K and S6K bands in panel C were quantified. The ratio of densities of the p-S6K band to S6K band is shown. Four independent experiments were performed. Bars indicate the median. Statistical significance was calculated using Mann–Whitney U test. E, the results of Upstream Regulator Analysis, as determined using the web-based software, Ingenuity Pathway Analysis. The pathways with z scores less than -2 are significantly inactivated in the ?Ragulator cells, compared to the RAW264.7 cells. The z score for the TFEB pathway was >2, which indicates that TFEB is significantly activated in the ?Ragulator cells than in the control RAW264.7 cells. F, the sizes of the ?Ragulator cells were larger than those of the parental RAW264.7 cells. The cell sizes of 1 × 104 cells were estimated using flow cytometry. Statistical significance was calculated using Welch’s t test. G, Western blotting for determining the phosphorylation of TSC2. Control RAW264.7 cells and ?Ragulator cells were cultured in nutrient-sufficient and serum-supplemented DMEM. Three biological triplicates were examined. H, the densities of the p-TSC2 and the TSC2 bands in panel G were quantified. The ratio of densities of the p-TSC2 band to TSC2 band is shown. Statistical significance was calculated using the Mann–Whitney U test. mTORC1, mammalian target of rapamycin complex 1; TFEB transcription factor EB.