Table 1.
GM130 specific gRNA sequences.
Fig 1.
GM130 is required for normal Golgi organization in RPE-1 cells.
A. Clonal GM130 knockout (KO) cell lines were generated by targeting Cas9 to exon 1 (KO60) or exon 5 (KO2) of GOLGA2, the gene encoding for GM130. B. Western blotting with an antibody to the GM130 N-terminus (left panel) or C-terminus (right panel). GAPDH served as a loading control. C—E. Wild-type and GM130 KO cells were analyzed by immunofluorescence microscopy with antibodies to GM130 and Giantin (C.) GRASP65 and Giantin (D.) and Mannosidase II and Golgin-97 (E.). For each panel, merged images are also shown in which DNA is stained with DAPI. Scale 10μm.
Fig 2.
GM130 is not necessary for microtubule organization.
Wild-type and GM130 KO cells were stained with antibodies to α-tubulin and Golgin-84 to visualize microtubule organization and Golgi structure, respectively. Merged images in which DNA is stained with DAPI are also shown. The arrows highlight the perinuclear region of these cells. Scale bar, 10μM.
Fig 3.
GM130 controls Golgi-centrosome proximity.
A. Wild-type and GM130 KO cells were stained for γ-tubulin and Golgin-84 to visualize the centrosome and the Golgi, respectively. B. Left: Graph showing the percentage of cells with increased Golgi (GA)—centrosome distance. The data is from four independent experiments, with at least 50 cells per condition analyzed. p = 0.006 (KO2) and 0.0014 (KO60). Right: Graph showing the average distance between the centrosome and the nearest Golgi edge. p = 0.0029 (KO2) and 0.0031 (KO60). C. Wild-type and GM130 KO cells were transfected with a construct encoding for full length Flag-GM130 and stained for the presence of the Flag tag, γ-tubulin and Golgin-84 to visualize transfected cells, the centrosome and the Golgi, respectively. D. Graph showing the percentage of cells (untransfected and transfected) with increased Golgi-centrosome distance. The data is from four independent experiments with at least 40 cells analyzed per experiment. ns = not statistically significant. Scale 10μm.
Fig 4.
GM130 is necessary for AKAP450 recruitment to the Golgi.
A. Wild-type and GM130 KO cells were stained with antibodies to AKAP450, Golgin-84 and GM130. Boxes show AKAP450 staining in the area adjacent to Golgi and centrosome B. Immunofluorescence analysis of wild-type and KO cells with antibodies to AKAP450 and α-tubulin. Brightness was adjusted to visualize AKAP450 along microtubules. C. Western blot analysis of lysates from wild-type and GM130 KO cells with antibodies to AKAP450 and Golgin-84, which was used as a loading control. Scale 10μm.
Fig 5.
GM130 does not function in protein transport.
Wild-type and GM130 KO cells were transfected with tsO45-VSV-G-GFP. 18 hours post transfection, cells were transferred to 42°C for 5 hours to allow VSV-G accumulation in the ER (top panel), followed by a shift to the permissive temperature of 32°C. Cells were fixed after 0 min (top panel), 30 min (middle panel) and 120 min (bottom panel) post transfer. Scale 10μm. Representative immunofluorescence images for each time point are shown, with the GFP signal revealing the localization of the VSV-G protein.
Fig 6.
Golgi-centrosome proximity is dispensable for polarization and directional cell migration.
A scratch wound was introduced into confluent cell monolayers using a micropipet tip. A. Wounded monolayers were fixed 5 hours post wounding and stained for γ-tubulin and Golgin-84. Scale 10μm. B. Quantification of the cell polarization assay of Fig 6A. A centrosome was considered oriented if it was found in front of the nucleus, facing the wound. The data is from three independent experiments, with at least 100 cells analyzed per condition. ns = not statistically significant. C. Wounds were imaged at 0, 5 and 8 hours after wounding. Representative images for wild-type and KO2 cells are shown. Scale 50μm. D. Quantification of wound width for wild-type, KO2 and KO60 monolayers at 0 hours and 5 hours. The data is from three independent experiments, with at least ten measurements at different positions along the wound for each condition.
Fig 7.
GM130 and Golgi-centrosome proximity are not necessary for ciliogenesis.
A. Cilia formation was induced by serum withdrawal for 48 hours. Cells were fixed and stained with antibodies to polyglutamylated tubulin, γ-tubulin and Golgin-84 to visualize the ciliary axoneme, the basal body and the Golgi, respectively. Merged images with DNA stained with DAPI are also shown. Scale bar: 10μm. B. The percentage of cells with increased Golgi-centrosome distance that were ciliated was quantified. The data is from three independent experiments with at least 50 cells per condition analyzed. ns = not statistically significant.