Fig. 8: TFEB subcellular localization under α-synuclein overexpression.

a Representative single-plane images of HeLa cells expressing TFEB-GFP or co-expressing TFEB-GFP and α-Syn WT. b Quantification of nuclear TFEB-GFP intensity signal, mean ± SEM: Ctrl 0 ± 0 n = 7, TFEB-GFP 79.76 ± 8.07 n = 8 cells from 2 independent experiments. c Representative single-plane images of HeLa cells stably expressing TFEB-GFP and transiently transfected with pcDNA3 (Mock) or α-Syn WT. d Quantification of nuclear TFEB-GFP intensity signal. TFEB was represented by GFP fluorescence upon excitation at 488 nm (green) and α-synuclein was detected by anti-α-synuclein upon excitation at 633 nm (red); mean ± SEM: Ctrl 0 ± 0 n = 7, TFEB-GFP 69.44 ± 9.52 n = 9 cells from 2 independent experiments. e Representative confocal images of HeLa cells stably expressing TFEB-GFP, transfected with α-Syn WT and left untreated or treated with BAPTA-AM (20μM, 6 h), quantification of the nuclear TFEB signal is shown in (f) mean ± SEM: Untreated 88.33 ± 7.86 n = 10, BAPTA-AM 0 ± 0 n = 11 cells from 3 independent experiments. g Proposed model: under basal conditions Ca²⁺ released from lysosomes is taken up by mitochondria in close proximity, thus reducing calcineurin activation and TFEB nuclear translocation. The untethering of the Ly-Mt contacts by α-synuclein potentially increases the local Ca²⁺ concentration due to reduced buffering action by mitochondria, thus leading to increased local activation of calcineurin- and Rag GTPase-dependent pathways and consequent TFEB nuclear translocation. Scale bar 10 μm. The data were obtained from at least 2 independent transfections. ***p ≤ 0.01, unpaired two-tailed t test). Source data are provided as a Source Data file.