Fig. 4: RUFY3 and RUFY4 localize to endolysosomes, and promote their juxtanuclear clustering.

a Co-localization of RUFY3-GFP and RUFY4-GFP with endogenous LAMP1. Immunofluorescence microscopy of HeLa cells transfected with plasmids expressing GFP (control), RUFY3-GFP or RUFY4-GFP (green), fixed and immunostained for endogenous LAMP1 (magenta). Nuclei were stained with DAPI (blue). Cells with low-to-moderate expression of RUFY3-GFP or RUFY4-GFP were selected for analysis of co-localization. Single channels are shown in grayscale. Cell edges are shown with dashed lines. Scale bars: 10 μm. Insets show 3-fold enlargements of the boxed areas. Arrows indicate vesicles where RUFY3/4-GFP proteins co-localize with LAMP1. b SuperPlot representation of the Pearson’s correlation coefficient for the co-localization of GFP, RUFY3-GFP or RUFY4-GFP with endogenous LAMP1 from experiments such as that shown in panel (a). Values were calculated and represented as described for Fig. 3c. Horizontal lines indicate the mean ± SD of the means from three independent experiments. Statistical significance was calculated using one-way ANOVA with multiple comparisons to the GFP control using Dunnett’s test. ***p < 0.001, ****p < 0.0001. c Overexpression of RUFY3-GFP or RUFY4-GFP causes juxtanuclear clustering of endolysosomes. This experiment was done as described for panel (a), except that highly overexpressing cells were chosen for analysis. Endogenous LAMP1 staining is shown in grayscale and GFP images in green (inset). The strong cytosolic staining of the GFP constructs is due to the overexpression. Nuclei were stained with DAPI (blue). Cell edges are highlighted with dashed lines. Scale bars: 10 μm. Insets show 2.85-fold reductions of the transfected cells. d SuperPlot representation of the ratio of juxtanuclear LAMP1 to total LAMP1 calculated by shell analysis from experiments such as those in panel (c). Values were calculated and represented as described for Fig. 3c, h. Horizontal lines indicate the mean ± SD of the means from three independent experiments. Statistical significance was calculated using one-way ANOVA with multiple comparison to the GFP control using Dunnett’s test. *p < 0.05, ***p < 0.001.