Fig. 3: UBAP1 mediates lysosomal recruitment of mTOR and regulates the activity of mTORC1.
From: Lysosomal and mTORC1 signaling dysregulation underpin the pathology of spastic paraplegia type 80
a Diagram illustrating lysosomes as platforms for the recruitment, assembly, and activation of mTOR signaling. b COS-7 cells were transfected with siCtrl/siUBAP1 and LAMP1-mYH for 48 h. Cells were then fixed and immunostained with an antibody against mTOR. Colocalization analysis of mTOR with LAMP1 in COS-7 cells that were transfected with siCtrl or siUBAP1. siCtrl n = 28, siUBAP1 n = 29 cells. Immunoblots with lysates from COS-7 cells that were transfected with siUBAP1 or siCtrl for 48 (c) or 96 (d) hours, and probed with the indicated antibodies. Relative expression was calculated from three independent experiments. (n = 3 independent experiments) e Left: COS-7 cells transfected with siCtrl/siUBAP1 for 48 h and stained with TFEB antibody. Right: Quantification of ratio of cells with TFEB in nucleus. Both siCtrl and siUBAP1 n = 6 micrographs. f Immunoblots with lysates from COS-7 cells that were transfected with siCtrl/siUBAP1 and siTFEB for 48 h, and probed with the indicated antibodies. Relative expression was calculated from three independent experiments. g Primary fibroblasts that were derived from SPG80 patients and healthy controls. Cells were fixed and immunostained with an antibody against TFEB. The relative fluorescence intensity of TFEB in the nucleus was quantified by measuring the mean TFEB signal intensity within DAPI-defined nuclear regions, normalized to the cytoplasmic TFEB intensity. Ctrl n = 121, Patient 1 n = 114, Patient 2 n = 45, Patient 3 n = 99 cells. h Immunoblot analysis of lysates from SPG80 patient-derived and control fibroblasts with indicated antibodies. Relative expression was calculated from three independent experiments. All scale bars: 10 μm. All data represent the mean ± SEM; Student’s t test (b–e and h), One-way ANOVA followed by Tukey’s multiple comparisons test (f), Kruskal–Wallis test followed by Dunn’s multiple comparisons test (g); ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.0001. Exact p-values and test statistics are provided as a Source Data file.
