Fig. 2. The R-rings are multilamellar intranuclear membrane cisternae. Transmission electron microscopy for ultrastructural analysis (A-E and H) and postembedding immunodetection (F,G). (A) Low-power overview of a NoppR-transfected COS cell. Several R-rings (arrows) are detectable in the nucleoplasm (N), the nucleoli (No) and at the nuclear envelope (NE). A mitochondrion (M) can be seen in the cytoplasm (C). Bar, 2 µm. (B) High-power magnification of the framed section of the central R-ring in (A). Note the two electron-dense lines characteristic for biological membranes (arrowhead) that separate the individual lumina (white dots) of the cisternae from the electron-dense material between the membranes, and the regular structure separating them from the nucleoplasm (arrow). Bar, 0.1 µm. (C-E) Close-up of a serial section through the central R-ring in (A). Note the rounding off on either side (C,E) of the central section (D). Bars, 1 µm. (F) R-ring immunogold labeled with anti-HA antibodies for the transfected HA-NoppR. Note the specific labeling of the electron-dense matrix between the membrane cisternae, which were often disrupted during preparation because they presented the point of least resistance during sectioning. Bar, 1 µm. (G) Close-up of the nuclear envelope, where the outer (ONM) and inner nuclear membrane (INM) were separated during sectioning on the left side and held together by two nuclear pore complexes (arrowheads) on the right side. The section was immunogold labeled with anti-Nopp140 antibodies, which only recognize the transfected NoppR but not the endogenous Nopp140. Note the gold decoration of specifically the inner membrane and of a bud (arrow) forming a putative R-ring. The dark material attached at the inner membrane is a nucleolus based on the comparable appearance of other nucleoli in the same section (not shown). In this section, the nucleus is in close proximity with the plasma membrane (PM). Bar, 0.5 µm. (H) High magnification of a bleb off the inner nuclear membrane (INM, arrow) forming a putative R-ring (RR). A section of endoplasmic reticulum (ER) coming off the outer membrane (ONM) of the nuclear envelope (NE) is visible in the cytoplasm. Bar, 0.2 µm.