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Intranuclear endoplasmic reticulum induced by Nopp140 mimics the nucleolar channel system of human endometrium

¹ Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
² Departments of Developmental and Molecular Biology, and Obstetrics and Gynecology and Women’s Health, Center for Study of Reproductive Biology and Women’s Health, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

View larger version (170K): [in a new window]   Fig. 1. R-rings are membranous structures. Fluorescence of COS cells transfected with the HA-tagged full-length Nopp140 (HA-Nopp140) (a and b) or the Nopp140 repeat domain (c). The cells were stained for the transfected protein with HA-antibodies (a,b) or for membranes with the lipophilic dye, DiOC6 (c). Note the varied appearance of the R-rings in different cells by indirect immunofluorescence (a,b) and by phase contrast microscopy (a’-c’). Two R-rings are pointed out in each panel (arrows). Note that the phase dense particles of the R-rings (arrows in a’ and c’) are distinct from nucleoli (asterisks). An overlay of the fluorescence with the phase contrast images shows the location of the R-rings in the nucleoplasm and in the nucleoli (a"-c"). Bar, 10 µm.

View larger version (188K): [in a new window]   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.

View larger version (115K): [in a new window]   Fig. 3. The ultrastructure of the R-rings is indistinguishable from that of the nucleolar channel system. (A) Immunoelectron micrograph of an R-ring induced by NoppR in COS cells and labeled with Nopp140 antibodies as in Fig. 2G. (B) Electron micrograph of a nucleolar channel system (NCS) induced by progesterone in a human endometrial cell in the secretory phase. Reproduced from The Journal of Cell Biology (Terzakis, 1965) by copyright permission of The Rockefeller University Press. A section of the nuclear envelope (nuclear membrane, NM) is visible. Note the granular matrix that embeds both tubular membrane systems and the halo-like appearance of their surrounding nucleoplasm. Bars, 0.2 µm.

View larger version (126K): [in a new window]   Fig. 4. R-rings are distinct from the nuclear envelope but contain an ER-specific membrane protein. Double immunofluorescence of HA-NoppR-transfected COS cells stained for the inner nuclear membrane protein, LAP2 (a), the nuclear pore complex protein, p62 (b), the nuclear lamina protein, lamin B (c) and the ER transmembrane protein, calnexin (d). To identify the R-rings, the cells were double labeled with HA-antibodies for the transfected HA-NoppR (a’-d’) and imaged by phase contrast (a"-d"). One R-ring is pointed out (arrow). Bar, 10 µm.

View larger version (123K): [in a new window]   Fig. 5. R-rings consist of intranuclear ER. Merged images of double immunofluorescence of HA-NoppR-transfected COS cells stained for HA-NoppR (red) and the ER marker proteins (green), Sec61 (A), SRP receptor (B), TRAM (C), TRAP (D), BiP (E) PDI (F), HMG-CoA reductase (G), cytochrome P450 (H) and the IP3 receptor (I). Note the overlap in yellow and orange identifying the R-rings (two are pointed out by arrows in each panel). Bar, 10 µm.

View larger version (105K): [in a new window]   Fig. 6. The NCS, like the R-rings, contains calnexin, BiP and Nopp140. (A–C) Paraffin sections of human endometrium in the secretory phase, stained by double immunofluorescence for the ER transmembrane marker calnexin (A) and for Nopp140 (B and C, red panels). (A) and (B) were double labeled for the nucleolus with nucleolin and (C) for ER with BiP antibodies (green panels). The two images in each were merged. Some putative NCSs are indicated by arrows and two nucleoli are pointed out by arrowheads in (C). Bars, 10 µm. (D) Transmission electron micrograph of a deparaffinized section as in (A-C). The nucleolus (No), an adjacent NCS and the nuclear envelope (NE) are labeled. Note, the white holes in the nucleoplasm reflect the poor preservation of the paraffin sections. Bar, 0.5 µm.

View larger version (33K): [in a new window]   Fig. 7. Models of R-ring/NCS biogenesis from the nuclear envelope. (A) R-rings/NCS form by the invagination of the entire nuclear envelope, the outer and inner nuclear membranes, lined by an electron-dense Nopp140 matrix (gray sprinkled layer). (B) R-rings/NCS form by the exclusive invagination of the inner nuclear membrane. Integral membrane proteins of the rough (1) and the smooth ER (2) and synthesized on the outer nuclear membrane (3) can laterally diffuse through the pore membrane domain (4) and the inner nuclear membrane (5) into the R-rings. The boxed regions show how a close-up of the repeat patterns of Nopp140 matrix and intranuclear membrane cisternae would look for each model.