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Fig. 2. Perinuclear aggregates of GFP-xLBR bud off from the nuclear membrane and form vesicles with membrane stacks that do not contain lamin B or nucleoporins. (A) HeLa cells overexpressing GFP-xLBR were viewed by time-lapse microscopy. At the beginning, the vesicle was very small and was connected through its edges with the nuclear membrane (NE, arrows). Within several hours, the vesicle gradually and progressively pinched off from nuclear membrane. Also, the small vesicle aggregates could fuse to form a large one (arrowheads). The area indicated by the arrow in the top panels was enlarged and shown in the bottom panels. Bar, 10 µm. (B) HeLa cells overexpressing GFP-xLBR were visualized by fluorescence microscopy and then processed for TEM. The nuclear membrane in the transfected cells appeared normal (B1-B4; the two transfected and two nontransfected cells look similar). TEM examination of aggregates showed that vesicles had numerous bilayered stacks of NE-like membranes (indicated by arrows). In B4 the boxed areas a and b are shown at higher magnification in Ba and Bb. The insets in a and b are higher magnification of the boxed areas. N, nucleus. Bars, 10 µm in the upper panels and 1 µm in the lower panels. (C,D) HeLa cells overexpressing GFP-xLBR were fixed and stained with anti-nucleoporin monoclonal antibody mAb414 (C) or anti-lamin B (D). Neither the nuclear pore complex component nor Lamin B was observed on the membrane stacks. (E,F) Immunofluorescence of HeLa cells overexpressing GFP-xLBR using the antibody against a Golgi marker (E) or the ER marker (F). Bar, 10 µm.
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