Figure 1. Pathogenic mutations in SPG21... | Rockefeller University Press

Figure 1.

$Pathogenic mutations in SPG21 cause maspardin misfolding, and maspardin deficiency decreases pTFEB/TFEB ratio in HeLa cells. (A) HeLa cells were transfected for 48 h with either SPG21 WT-myc-flag, SPG21 A108P-myc-flag, or SPG21 601insA-myc-flag constructs and treated with DMSO (vehicle) or 1 µM of MG132 during the last 16 h. Maspardin was detected by western blotting using an anti-myc antibody (at ∼38 kDa for the WT and p.A108P mutants and at ∼28 kDa for the c.601insA mutant). α-Tubulin detection was used as a loading control. Fold changes of maspardin/α-tubulin signal are shown in the graph on the right. Mean ± SD. n = 3 independent experiments. Two-tailed unpaired t test. *P < 0.05; ***P < 0.001; ****P < 0.0001. (B) Western blotting detection of endogenous maspardin in two control (CTRL) and two newly-generated SPG21 KO HeLa clones. The graph shows fold changes of maspardin/α-tubulin signal. Mean ± SD. n = 3 independent experiments, including two CTRL and two KO clones (open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2). Two-tailed unpaired t test. ns, nonsignificant; ****P < 0.0001. (C) Immunofluorescence detection of endogenous maspardin (green) in CTRL and SPG21 KO clones. Nuclei were stained with DAPI. One representative set of n = 3 independent experiments, including two CTRL and two KO clones. Scale bar = 10 µm. (D) Western blotting analysis and quantification of the pTFEB(Ser211)/TFEB ratio in CTRL and SPG21 KO HeLa cells 48 h after transfection of a GFP-TFEB construct with an empty plasmid or a SPG21-myc-flag construct (WT, p.A108P, or c.601insA). Overexpressed proteins were detected with an anti-myc antibody (N.B. due to its extensive degradation, the 601insA mutant is not detected under these basal conditions). n = 6 for the KO cells (3 independent experiments, including two different KO clones) and n = 12 for the CTRL cells (each of the 6 blots contained the two CTRL clones co-transfected with GFP-TFEB and an empty plasmid. Only one is shown in the figure). Mean fold change of pTFEB/TFEB ratio ± SD. Two-tailed unpaired t test. *P < 0.05; **P < 0.01. (E) Analysis of GFP-TFEB distribution in CTRL and SPG21 KO HeLa by confocal microscopy 72 h after transfection with an empty vector or SPG21 WT-myc-flag. Scale bar = 10 µm. The graphs show the mean percentage of cells (± SD) with a predominant nuclear localization for TFEB (left) or TFEB nuclear/cytoplasmic signal ratio (right). n = 6 (three independent experiments, including each two different CTRL and two different KO clones). 10 cells were analyzed per clone in each experiment. These values are shown in light gray, while their averages are shown as follows: open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Two-tailed unpaired t test. **P < 0.01; ***P < 0.001; ****P < 0.0001. (F) Analysis of endogenous TFEB nuclear/cytoplasmic distribution in CTRL or SPG21 KO HeLa cells. The cells were homogenized and fractionated into a nuclear (N) fraction and a postnuclear supernatant (PNS) fraction. Equal amount of proteins from the cell homogenates (H), N, and PNS fractions were analyzed by western blotting using an anti-TFEB antibody. Histone H1 (nuclear marker) was used as a control. n = 16 (8 independent experiments, including two CTRL and two KO clones). The graph shows the mean percentage of TFEB in the N fraction relative to total cellular signal ± SD. Two-tailed paired t test. ***P < 0.001. Source data are available for this figure: SourceData F1.$

Pathogenic mutations in SPG21 cause maspardin misfolding, and maspardin deficiency decreases pTFEB/TFEB ratio in HeLa cells. (A) HeLa cells were transfected for 48 h with either SPG21 WT-myc-flag, SPG21 A108P-myc-flag, or SPG21 601insA-myc-flag constructs and treated with DMSO (vehicle) or 1 µM of MG132 during the last 16 h. Maspardin was detected by western blotting using an anti-myc antibody (at ∼38 kDa for the WT and p.A108P mutants and at ∼28 kDa for the c.601insA mutant). α-Tubulin detection was used as a loading control. Fold changes of maspardin/α-tubulin signal are shown in the graph on the right. Mean ± SD. n = 3 independent experiments. Two-tailed unpaired t test. *P < 0.05; ***P < 0.001; ****P < 0.0001. (B) Western blotting detection of endogenous maspardin in two control (CTRL) and two newly-generated SPG21 KO HeLa clones. The graph shows fold changes of maspardin/α-tubulin signal. Mean ± SD. n = 3 independent experiments, including two CTRL and two KO clones (open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2). Two-tailed unpaired t test. ns, nonsignificant; ****P < 0.0001. (C) Immunofluorescence detection of endogenous maspardin (green) in CTRL and SPG21 KO clones. Nuclei were stained with DAPI. One representative set of n = 3 independent experiments, including two CTRL and two KO clones. Scale bar = 10 µm. (D) Western blotting analysis and quantification of the pTFEB(Ser211)/TFEB ratio in CTRL and SPG21 KO HeLa cells 48 h after transfection of a GFP-TFEB construct with an empty plasmid or a SPG21-myc-flag construct (WT, p.A108P, or c.601insA). Overexpressed proteins were detected with an anti-myc antibody (N.B. due to its extensive degradation, the 601insA mutant is not detected under these basal conditions). n = 6 for the KO cells (3 independent experiments, including two different KO clones) and n = 12 for the CTRL cells (each of the 6 blots contained the two CTRL clones co-transfected with GFP-TFEB and an empty plasmid. Only one is shown in the figure). Mean fold change of pTFEB/TFEB ratio ± SD. Two-tailed unpaired t test. *P < 0.05; **P < 0.01. (E) Analysis of GFP-TFEB distribution in CTRL and SPG21 KO HeLa by confocal microscopy 72 h after transfection with an empty vector or SPG21 WT-myc-flag. Scale bar = 10 µm. The graphs show the mean percentage of cells (± SD) with a predominant nuclear localization for TFEB (left) or TFEB nuclear/cytoplasmic signal ratio (right). n = 6 (three independent experiments, including each two different CTRL and two different KO clones). 10 cells were analyzed per clone in each experiment. These values are shown in light gray, while their averages are shown as follows: open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Two-tailed unpaired t test. **P < 0.01; ***P < 0.001; ****P < 0.0001. (F) Analysis of endogenous TFEB nuclear/cytoplasmic distribution in CTRL or SPG21 KO HeLa cells. The cells were homogenized and fractionated into a nuclear (N) fraction and a postnuclear supernatant (PNS) fraction. Equal amount of proteins from the cell homogenates (H), N, and PNS fractions were analyzed by western blotting using an anti-TFEB antibody. Histone H1 (nuclear marker) was used as a control. n = 16 (8 independent experiments, including two CTRL and two KO clones). The graph shows the mean percentage of TFEB in the N fraction relative to total cellular signal ± SD. Two-tailed paired t test. ***P < 0.001. Source data are available for this figure: SourceData F1.