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. 2013 Sep 5;93(3):579–586. doi: 10.1016/j.ajhg.2013.07.023

Figure 4.

Figure 4

The Golgi Apparatus Is Disorganized in a Large Proportion of BAP31-Deficient Fibroblasts

(A) Representative images show the abnormal structure of the Golgi apparatus in BAP31-deficient fibroblasts. The typical bean-shaped perinuclear Golgi structure observed in control fibroblasts was lost in a large proportion of BAP31-deficient fibroblasts, where it was replaced by small patches of GOLGA2-positive structures scattered in the cytoplasm. Immunohistochemistry was performed on fibroblasts from two control individuals (C1 and C2), individual III.2 from family 1 (F1-III.2), individual IV.1 from family 2 (F2-IV.1), and individual III.1 from family 3 (F3-III.1) with an antibody specific to GOLGA2 (green) (1:200, Sigma G7295). The fibroblasts were stained with DAPI for visualization of nuclear DNA. Scale bars represent 25 μm.

(B) Percentage of fibroblasts showing a severely perturbed Golgi apparatus morphology in control samples and in the fibroblasts of the three affected individuals (∗p < 0.01, Chi-square test; 100 cells were counted in each of two experiments for each condition).

(C) Representative electron-microscopy images reveal the ultrastructure of control fibroblasts (i and ii) and BAP31-deficient fibroblasts in individuals F1-III.2 (iii and iv), F2-IV.1 (v and vi), and F3-III.1 (vii and viii). The cells were washed, fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), postfixed in 1% osmium tetroxide, scraped, and centrifuged, and the pellet was dehydrated in graded alcohol solutions and embedded in SPURR low-viscosity medium. Ultrathin sections (50–60 nm) were counterstained with uranyl acetate and lead citrate before observation with a JEOL JEM1400 electron microscope at 80 kV. Images were obtained with a MegaView III camera (Soft Imaging System). The affected individuals’ fibroblasts contained an enlarged ER (black arrows) and numerous vesicles containing electron-dense material (asterisks) and lost the normal structure of their Golgi apparatus (white arrows in control fibroblasts). N = nucleus. Scale bars represent 2 μm (iv, v, and vi) or 1 μm (i, ii, iii, vii, and viii).