Fig 2: Intracellular distribution of LysoTracker red (LTR), acridine orange (AO), and late endosomal/lysosomal adaptor and MAPK and mammalian target of rapamycin activator 4 (Lamtor4) in polycystin-1 (Pkd1) KO and wt mouse embryonic fibroblasts (MEFs). Pkd1 KO (A) and Pkd1 wt (B) MEFs were incubated with LTR for 30 min before live-cell analysis. Pkd1 KO MEFs exhibited condensed perinuclear accumulation of LTR (A) compared with Pkd1 wt MEFs (B), in which these signals were more evenly distributed throughout the cytoplasm. Although quantification of representative acquired images revealed no difference in the number of LTR particles per cell (C), a significant increase in relative particle size (D; ****P < 0.0001) and mean fluorescence intensity [MFI, in arbitrary units (au); E; **P < 0.01] was observed in Pkd1 KO MEFs compared with Pkd1 wt MEFs. F: representative example of the results of the image processing procedure used for particle analysis, which is described in detail in materials and methods. Comparable results were obtained using AO, which exhibited a clustered perinuclear distribution in Pkd1 KO MEFs (G) compared with a more diffuse distribution in Pkd1 wt MEFs (H). Scale bars = 100 µm in A and B and 50 µm in G and H; scale bars in insets = 10 µm. Images are representative of six ×20 images acquired from three independent experiments and a total n > 500 cells. Pkd1 KO (I) and Pkd1 wt MEFs (J) were fixed and subjected to immunostaining with an antibody raised against Lamtor4. Quantification of representative acquired images revealed a significant decrease in the number of Lamtor4-positive particles (K; **P < 0.01) and an increase in relative particle size (L; ****P < 0.0001) and MFI (M; ****P < 0.0001) in Pkd1 KO MEFs compared with Pkd1 wt MEFs. N: representative example of the results of the image processing procedure used for particle analysis, which is described in detail in materials and methods. Scale bars = 100 µm; scale bars in insets = 20 µm. Images are representative of six ×20 images acquired from three independent experiments and a total n > 500 cells.

Fig 3: Golgi phenotype caused by Rab34-DN is distinct from the Golgi phenotype caused by shRILP.A–C, DIV9 rat hippocampal neuronal cultures were stained for GM130 (red, middle column) or LAMTOR4 (right column) after 48 h of expressing GFP (A), GFP-Rab34 (B), or high levels of GFP-Rab34-DN (C). The transfected cell is outlined in green. Untransfected cells in the same field are outlined in white. GFP-Rab34 localizes to similar cellular regions as GM130 as seen in the line scan in B’. LAMTOR4 is not clustered by GFP-Rab34 overexpression. (C′) High expression of GFP-Rab34-DN in about 20% of cells causes fragmented GM130 staining (right hand panel), whereas most cells show normal Golgi morphology (left hand panel). D, GM130 and LAMTOR4 intensity in the soma was quantified for RAB34- and RAB34-DN–expressing neurons and normalized to GFP controls. There were no statistically significant differences for GM130 (one-way ANOVA) or LAMTOR4 (Kruskal–Wallis test). N = number of cells for two independent experiments. N = 54 cells GFP, 39 cells RAB34, 46 cells RAB34-DN. DIV, days in vitro; sh, short hairpin.

Fig 4: Expression, activity, and self-association of PLEKHM2-FS.a. WT or ARL8-KO HeLa cells were transiently transfected with the indicated HA-tagged PLEKHM2 constructs (see scheme in Fig. 4a) for 48 h and analyzed by SDS-PAGE and immunoblot analysis with antibodies to the HA epitope. β-tubulin was used as a loading control. The positions of molecular mass markers (Mr, in kDa) are indicated on the left. b. WT HeLa cells like those shown in panel a were analyzed by confocal immunofluorescence microscopy for the HA epitope (green) and endogenous LAMTOR4 (lysosomes, red). Nuclei were labeled with DAPI (blue). Cell edges were outlined by staining of actin with fluorescent phalloidin (not shown) and indicated by dashed lines. Arrows indicate clusters of HA-PLEKHM2 together with lysosomes (yellow arrows) or without (green arrows) at the cell tips or more centrally (magenta arrows). Scale bars: 20 μm. Notice how all HA-PLEKHM2 constructs drive distribution of both HA-PLEKHM2 and lysosomes to cell tips. c. ARL8-KO HeLa cells were transfected and analyzed as described for panel b. Scale bars: 20 μm. Notice that HA-PLEKHM2 is unable to localize to and drive lysosomes to cell tips in ARL8-KO cells. In contrast, HA-PLEKHM2-FS can localize to, but cannot drive lysosomes to cell tips. HA-PLEKHM2–1-437 is incapable of moving either protein to cell tips. d. HEK293T cells were co-transfected with plasmids encoding the indicated HA-tagged constructs with myc-PLEKHM2-FS and subjected to immunoprecipitation with an antibody to the myc epitope. Immunoprecipitates (IP) and cell extracts (10%, Input) were analyzed by SDS-PAGE and immunoblotting (IB) for the HA and myc epitopes, or β-tubulin (loading control). Red arrows indicate the HA constructs that are co-immunoprecipitated with the myc-PLEKHM2-FS construct. The positions of molecular mass markers (Mr, in kDa) are indicated on the left. Notice the much greater co-immunoprecipitation of myc-PLEKHM2-FS with HA-PLEKHM2-FS relative to HA-PLEKHM2 and HA-PLEKHM2–1-437.

Fig 5: Ikarugamycin induces vacuolation and alters the intracellular distributions of Rab7 and LAMTOR4. (A) MDBK cells were transfected or not with clathrin-light chain A fused to GFP (LCA-GFP; first column), Rab5 fused to GFP (Rab5-GFP, second) or Rab7 fused to GFP (Rab7-GFP, third column). For these columns, at 12 h post-transfection, cells were treated (or not) with IKA (1 μM, 24 h). Following fixation-permeabilization, cells were stained with DAPI for visualization of nuclei and imaged by fluorescence microscopy. For the fourth column, untransfected cells were treated or not with IKA (1 µM, 24 h). Following fixation, the cells were consecutively stained against LAMTOR4, followed by DAPI staining. In the third and fourth columns, the dashed line delimits the boundaries of one cell in the field, the star (*) represents the nucleus, and the arrows point at the enlarged vacuoles induced by IKA. Micrographs depict typical fields of the different transfection/treatment conditions. Bars represent 10μm. (B) MDBK cells were pre-treated (or not) with IKA (2 μM, 24 h). Cells were subsequently incubated with lysotracker (30 μM, 25 min) and taken for live imaging. Lysotracker accumulates within acidic compartments (red). The dashed line, stars (*), arrows and bars are as in (A).