Figure 7.
Maspardin deficiency causes delocalization of RAB7 molecules from late endosomes to lysosomes. (A–C) Colocalization analyses between mCherry-RAB7 WT and endogenous LAMTOR1 (green) (A), endogenous RAB7 (red) and VPS35 (green) (B), and endogenous RAB7 (red) and TBC1D5 (green) (C) in CTRL and SPG21 KO HeLa cells. Scale bar = 10 µm. The graphs show mean Manders’ coefficients ± SD. For RAB7-LAMTOR1 and RAB7-TBC1D5 colocalization analyses, n = 6 (three independent experiments with two CTRL and two KO clones); for RAB7-VPS35 analyses, n = 8 (four independent experiments with two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment (shown in light gray). Means of these values for each clone are indicated as follows: open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Two-tailed unpaired t test. ***P < 0.001; ****P < 0.0001. (D) Expression of WT mCherry RAB7 or mCherry alone in CTRL and SPG21 KO cells followed by their precipitation using RFP-trap beads. The presence of endogenous TBC1D5 proteins among the coprecipitated proteins was assessed by western blotting. n = 8 (four independent experiments, each including 2 CTRL and 2 KO clones). The graph shows mean fold changes ± SD of precipitated TBC1D5 signals normalized to the amount of precipitated bait proteins. Two-tailed unpaired t test. ****P < 0.0001. (E) Colocalization analysis between endogenous maspardin (green) and TBC1D5 (red) or between endogenous maspardin (blue) and VPS35 (red) in CTRL cells. The random overlap between signals was assessed as a negative control by rotating one of the images by 90°. Scale bar = 10 µm. Mean Manders’ coefficient ± SD. n = 6 (three independent experiments including two CTRL clones). 10 cells analyzed per biological replicate, as described above. Two-tailed unpaired t test. ****P < 0.0001. (F) Analysis of endogenous RAB7 distribution (blue) relative to endogenous VPS35 (red) 48 h after transfection of SPG21-myc-flag (green) or a mock construct in control cells (micrographies of individual channels are provided in Fig. S3 F). Scale bar = 5 µm. Mean Manders’ coefficient ± SD. n = 5 independent experiments. 10 cells analyzed per control clone (shown in light gray). The means for each CTRL clone are indicated with different white symbols. Two-tailed unpaired t test. **P < 0.01. RFP, red fluorescent protein. Source data are available for this figure: SourceData F7.

Maspardin deficiency causes delocalization of RAB7 molecules from late endosomes to lysosomes. (A–C) Colocalization analyses between mCherry-RAB7 WT and endogenous LAMTOR1 (green) (A), endogenous RAB7 (red) and VPS35 (green) (B), and endogenous RAB7 (red) and TBC1D5 (green) (C) in CTRL and SPG21 KO HeLa cells. Scale bar = 10 µm. The graphs show mean Manders’ coefficients ± SD. For RAB7-LAMTOR1 and RAB7-TBC1D5 colocalization analyses, n = 6 (three independent experiments with two CTRL and two KO clones); for RAB7-VPS35 analyses, n = 8 (four independent experiments with two CTRL and two KO clones). 10 cells were analyzed per clone in each experiment (shown in light gray). Means of these values for each clone are indicated as follows: open circles: CTRL1; open squares: CTRL2; black triangles: KO1; black diamonds: KO2. Two-tailed unpaired t test. ***P < 0.001; ****P < 0.0001. (D) Expression of WT mCherry RAB7 or mCherry alone in CTRL and SPG21 KO cells followed by their precipitation using RFP-trap beads. The presence of endogenous TBC1D5 proteins among the coprecipitated proteins was assessed by western blotting. n = 8 (four independent experiments, each including 2 CTRL and 2 KO clones). The graph shows mean fold changes ± SD of precipitated TBC1D5 signals normalized to the amount of precipitated bait proteins. Two-tailed unpaired t test. ****P < 0.0001. (E) Colocalization analysis between endogenous maspardin (green) and TBC1D5 (red) or between endogenous maspardin (blue) and VPS35 (red) in CTRL cells. The random overlap between signals was assessed as a negative control by rotating one of the images by 90°. Scale bar = 10 µm. Mean Manders’ coefficient ± SD. n = 6 (three independent experiments including two CTRL clones). 10 cells analyzed per biological replicate, as described above. Two-tailed unpaired t test. ****P < 0.0001. (F) Analysis of endogenous RAB7 distribution (blue) relative to endogenous VPS35 (red) 48 h after transfection of SPG21-myc-flag (green) or a mock construct in control cells (micrographies of individual channels are provided in Fig. S3 F). Scale bar = 5 µm. Mean Manders’ coefficient ± SD. n = 5 independent experiments. 10 cells analyzed per control clone (shown in light gray). The means for each CTRL clone are indicated with different white symbols. Two-tailed unpaired t test. **P < 0.01. RFP, red fluorescent protein. Source data are available for this figure: SourceData F7.

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