Figure 6. C9ORF72 localizes to lysosomes.

(A) Immunofluorescence of endogenous C9ORF72 stained with GTX632041 in U2OS cells expressing LAMP1-RFP. The large images on the left show a maximum projection to highlight intracellular structures. Insets 1 and 2 are single focal plane, higher magnification images of the boxed regions. Merge and grayscale images of C9ORF72 and LAMP1-RFP are shown. C9ORF72 localization was observed under basal conditions (top panels) or under starvation for 2 hr (bottom panels). Cells are outlined with white dashed lines. Scale bars = 20 µm for large images and 2 µm for insets. (B) Quantification of LAMP1 structures positive for C9ORF72 in basal growing condition (fed) or 2 hr starved condition (starved) in U2OS cells. Between 228–262 lysosomes were counted from at least nine different cells from three independent experiments. A Student’s t-test reveals that the percentage of LAMP1 structures positive for C9ORF72 is not significantly different between the fed and starved conditions (C) Lysates were prepared from HEK-293 cells expressing the Tmem192-3xHA (HA-Lyso cells) or the Tmem192-2xFlag (Control-Lyso cells). Lysosomes were immunoprecipitated using anti-HA magnetic beads. Quantitative immunoblotting for the indicated proteins was performed for starting material (SM), purified lysosomes and control immunoprecipitates (anti-HA IP). C9ORF72 was detected using GTX634482.

Figure 6—figure supplement 1. C9ORF72 co-staining with Rab.

GTPases Immunofluorescence of endogenous C9ORF72 stained with GTX632041 in U2OS cells expressing GFP-Rab5a, GFP-Rab7a, GFP-Rab9 and GFP-Rab11a as indicated. Insets are higher magnifications of the boxed regions. Merge and grayscale images of C9ORF72 and GFP-Rabs are shown. All images are the result of LIGHTNING applied to the confocal image. Cells are outlined with white dashed lines. For full-sized cells the bars = 10 µm, for insets the bars = 2 µm. White arrows point toward close proximity between the marker and C9ORF72. (B) Pearson’s correlation coefficient of C9ORF72 stained with GTX632041 and GFP-Rab5a, GFP-Rab11a, GFP-Rab7a and GFP-Rab9 in U2OS cells. Between 8 and 12 regions of interest were analyzed from at least five different cells for each construct. A Student’s t-test reveals that the means of the correlation coefficient between Rab5 and the other tested Rab is significantly different. ***=p value<0.01.

Figure 6—figure supplement 2. C9ORF72 subcellular fractionation.

(A) Lysates were prepared from HEK-293 cells non-transfected or expressing 3xHA-eGFP-OMP25 (MitoIP). Lysates were incubated with anti-HA magnetic beads to immunoprecipitate mitochondria. Immunoblotting for the indicated proteins was performed for starting material (SM), purified mitochondria and control immunoprecipitates (anti-HA IP). (B) Quantification of C9ORF72 fluorescent signal on LAMP1 structures or outside LAMP1 structures in basal growing condition (fed) or following 2 hr starvation (starved) in U2OS cells. The C9ORF72 fluorescent signal was quantified in nine individual cells for both conditions from three independent experiments. A Student’s t-test reveals that the C9ORF72 pool between fed and starved condition is not significantly different. (C) RAW264.7 cells treated with 1 µg/ml LPS and both parental and C9ORF72 KO U2OS cells were lysed in 20 mM HEPEs pH 7.4 without detergent. Lysates were spun at 200 000 g for 30 min. Supernatant (S) was collected and the pellet (P) was resuspended and conserved. The S and P fractions for each cell line were analyzed by quantitative immunoblot using GTX634482. The C9ORF72 protein signal in both the S and P fraction was determined, normalized to total protein, and presented as percentage (blue numbers) of the total S + P C9ORF72 signal for each cell line.