Fig 1.
SUV39H1 associates with the architecture of the Golgi complex.
A. Confluent breast cancer cells, MDA-MB-231, which show active cell migration, were scratched with or without a histone methyltransferase inhibitor, chaetocin. After 5 h incubation, the cells were fixed and stained with anti-GM130 mAb, a cis-Golgi marker (green). The nucleus was counterstained with propidium iodide, PI (red). B. The cells with the Golgi complex located within the front quarter of the cell, facing the wound area, were considered “polarized”; the cells with the Golgi complex located in the rear three-quarters of the cell body, away from the wound, were considered “nonpolarized”. The ratio (%) ±SE of the cells containing the polarized Golgi complex is indicated (n > 100). ** P < 0.01, compared with the chaetocin-untreated cells. C. HeLa cells were transfected with siRNA against SUV39H1 (siSUV39H1) or negative control siRNA (siNC). After 48 h incubation, the cells were stained with DAPI (red) and anti-GM130 mAb (green). Bar, 10 μm. D. mRNA was obtained from the SUV39H1-depleted cells and analyzed by RT-PCR. ** P < 0.01, compared with the control cells. E. Quantification of GM130 labeled Golgi complex area per cell. ** P < 0.01, compared with control cells. F. Cells were transfected with siSUV39H1. Then, GFP-tagged mouse SUV39H1 was transfected. The cells were fixed and GFP and GM130 were detected. Asterisks show mouse_SUV39H1 untransfected cells. Bar, 10 μm. G. Cells were transfected with GFP, GFP-tagged SUV39H1 or GFP-tagged Y67A-SUV39H1. GFP and GM130 were then detected. Arrows show GFP-tagged Y67A-SUV39H1 transfected cells. H. Cells were transfected with siSUV39H1 or siNC. The cells were then stained with anti-GM130 pAb and anti-TGN46, a trans-Golgi marker protein, mAb. Bar, 10 μm.
Fig 2.
KIF20A is required for the Golgi dispersal observed in the SUV39H1-depleted cells.
A. Cells were transfected with siSUV39H1 and incubated for 36 h. After 12 h incubation with paprotrain, the cells were fixed and stained with anti-GM130 mAb. Bar, 10 μm. B. Cells were transfected with siRNA against KIF20A (siKIF20A) and/or siSUV39H1. After 48 h incubation, the cells were fixed and stained with anti-GM130 mAb and anti-SUN1 pAbs to show the nuclei. C. Quantification of GM130 labeled Golgi complex area per cell in the SUV39H1- and/or KIF20A- depleted cells. ** P < 0.01, compared with control cells.
Fig 3.
SUN2/nesprin-2 LINC complex functions in the Golgi dispersal caused by SUV39H1 depletion.
A. Cells were transfected with siSUV39H1. After 24 h incubation, the cells were again transfected with mCherry tagged DN-KASH. The cells were then fixed and stained with anti-GM130 mAb (green). Arrows and asterisks show mCherry tagged DN-KASH transfected and untransfected cells, respectively. B. Box-and-whiskers plots represent the GM130-labeled Golgi complex area. ** P < 0.01. C. Cells were transfected with siSUV39H1 and siSYN2 or siSUN2, which targets nesprin-2 and SUN2, respectively. The cells were then stained with anti-GM130 mAb. Bar, 10 μm. D. Box-and-whiskers plots represent the GM130-labeled Golgi complex area in the indicated cells. ** P < 0.01. E. To investigate SUN1 mobility, cells were transfected with siSUV39H1 or siNC; on the following day, the cells were transfected with GFP-tagged SUN2. FRAP analysis was then performed (n ≥ 10). Note that there are no significant differences in SUN2 mobility between siNC-transfected and -untransfected cells. The results are presented as means ± SEM. F. Cells were transfected with siSUV39H1, and SUN1 mRNA expression was analyzed by RT-PCR. G. Cells were transfected with siSUV39H1 and SUN1, and β-tubulin protein expression was analyzed by western blotting. SUN expression was represented as the amount of SUN1 in siNC-transfected cells as 1.
Fig 4.
H3K9me3 and SUN2 localize in close proximity.
A. Cells were fixed and incubated with the indicated antibodies, and signals were detected with PLA and DAPI. B. Cells were transfected with siSUV39H1 or siNC. Fixed cells were then incubated with anti-H3K9me3 mAb and anti-SUN2 pAb, and signals were detected with PLA and DAPI. C. The numbers of PLA signals between H3K9me3 and SUN2 were counted in the cells transfected with siSUV39H1 or in the siNC-transfected cells. The results are presented as the mean number in a nucleus ± SE. ** P < 0.01.
Fig 5.
The dispersed Golgi complex is responsible for the suppression of cell motility resulting from loss of SUV39H1.
A. MDA-MB-231 were transfected with the indicated siRNA. Confluent cells were scratched to create a model wound. Phase-contrast images 18 h after scratching to create the wound. B. Cell motility was quantified using phase-contrast images in A. Eighteen hours after wounding, the area of unclosed wounds was measured. The wound closures are shown relative to the area of unclosed wounds in siNC-transfected cells (means ± SE). Bar, 300 μm. ** P < 0.01, compared with siSUV39H1 transfected cells. C. A model in which SUV39H1 plays a critical role in maintaining the Golgi apparatus through the function of the LINC complex and KIF20A. In a steady state, i.e., in the presence of SUV39H1, SUV39H1 negatively regulates SUN2/nesprin-2 LINC complex formation (left), and the cells show the perinuclear localization of the Golgi complex. Upon the depletion of SUV39H1 function, the suppression of SUN2/nesprin-2 LINC complex formation is restored, and thus SUN2/nesprin-2/KIF20A function is activated, resulting in the scattered Golgi complex (right).