Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Jun;18(6):2296-304.
doi: 10.1091/mbc.e07-02-0154. Epub 2007 Apr 11.

The nucleolar channel system of human endometrium is related to endoplasmic reticulum and R-rings

Nupur Kittur  1 , Gregory Zapantis, Mira Aubuchon, Nanette Santoro, David P Bazett-Jones, U Thomas Meier
Affiliations

The nucleolar channel system of human endometrium is related to endoplasmic reticulum and R-rings

Nupur Kittur et al. Mol Biol Cell. 2007 Jun.

Abstract

The nucleolar channel system (NCS) is a well-established ultrastructural hallmark of the postovulation endometrium. Its transient presence has been associated with human fertility. Nevertheless, the biogenesis, composition, and function of these intranuclear membrane cisternae are unknown. Membrane systems with a striking ultrastructural resemblance to the NCS, termed R-rings, are induced in nuclei of tissue culture cells by overexpression of the central repeat domain of the nucleolar protein Nopp140. Here we provide a first molecular characterization of the NCS and compare the biogenesis of these two enigmatic organelles. Like the R-rings, the NCS consists of endoplasmic reticulum harboring the marker glucose-6-phosphatase. R-ring formation initiates at the nuclear envelope, apparently by a calcium-mediated Nopp140-membrane interaction, as supported by the calcium-binding ability of Nopp140, the inhibition of R-ring formation by calcium chelators, and the concentration of Nopp140 and complexed calcium in R-rings. Although biogenesis of the NCS may initiate similarly, the reduced presence of complexed calcium and Nopp140 suggests the involvement of additional factors.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Transmission electron micrograph of a human endometrial gland from a biopsy of cycle day 18. (A) Low magnification illustrates the hallmarks of the epithelial cells from this stage of the cycle, subnuclear vacuoles (single asterisks), pinopodes (double asterisks), giant mitochondria (mito), and NCSs (boxed). (B) and (C) Higher magnifications of the respective NCSs.
Figure 2.
Figure 2.
Transmission electron micrographs of histochemical labeling of glucose-6-phosphatase activity in R-rings (A and B) and in NCSs (C and D). (A) Black lead-phosphate precipitates in the ER, the nuclear envelope (NE), and R-rings of Nopp140 repeat domain transfected COS-1 cells after fixation and incubation with glucose-6-phosphate and lead nitrate. (B) Higher magnification of an R-ring identifies the precipitates in the lumen of R-ring cisternae. (C) As in A but on an endometrial biopsy from the secretory phase. Note the specific labeling of the NCS and the ER, but not the mitochondria (mito), which are surrounded by a single layer of ER in these cells. (D) Higher magnification of an NCS demonstrating the precipitates in the lumen of the NCS cisternae.
Figure 3.
Figure 3.
Induction of R-rings and intranuclear aggregates by the transfection of HA-tagged, full-length human Nopp140 into U2OS cells. (A) Double immunofluorescence of a transfected cell stained for the HA-epitope (I) and the integral ER membrane protein calnexin (II); and a phase-contrast image of the same cell (III). Note that some HA-, i.e., Nopp140-positive structures are devoid of calnexin (some are marked by asterisks). (B) Transmission electron micrograph of a transfected cell with an R-ring (arrow) and intranuclear aggregates that are apparently membrane free (asterisks).
Figure 4.
Figure 4.
Initiation of R-ring formation at the nuclear envelope. (A) HA immunofluorescence of a COS-1 cell transfected with human Nopp140 lacking its amino terminus, which only induces R-rings but no aggregates, (I) and the corresponding phase contrast image (II). Note the Nopp140 accumulation at the nuclear envelope (arrows) in addition to the intranuclear R-rings. (B) R-ring formation in live cells visualized by cotransfection of the Nopp140 repeat domain and the GFP-tagged ER integral membrane marker, Sec61γ. GFP fluorescence of the same cell observed 30 min apart (II and III, deconvolved images) and phase contrast image at time zero (I). Note the stalklike connections to the nuclear envelope of some of the intranuclear membrane structures (arrows). Insets document the growth of an R-ring over time. (C) Transmission electron micrograph of an example of an R-ring attached to the inner membrane of the nuclear envelope through a stalk in a Nopp140-transfected cell.
Figure 5.
Figure 5.
A role for calcium in R-ring formation. (A) Nopp140 is a calcium-binding protein. Autoradiogram of a nitrocellulose membrane overlaid with 45Ca (lanes 1–5) and subsequently stained with amido black (lanes 6–10). Nopp140 was immunoprecipitated from low-salt nuclear extracts with peptide antibodies (lanes 2, 4, 7, and 9) in the absence (lanes 1, 2, 6, and 7) or presence of competing peptide (lanes 3, 4, 8, and 9). Half of each supernatant after precipitation was analyzed (lanes 1, 3, 6, and 8). Purified translocon associated protein (TRAP) was loaded as a control (lanes 5 and 10). (B) Calcium overlay (top panel) and amido black stain (bottom panel) as in A of purified phosphorylated (lane 1) and unphosphorylated recombinant Nopp140 (lane 2). Note the difference in mobility due to the difference of ∼80 phosphates between the two Nopp140 forms. (C) Effect of the membrane permeable calcium chelator BAPTA-AM on R-ring formation. Cells were transfected with Nopp140 lacking its amino terminus as in Figure 4A and treated with 10 μM BAPTA-AM. The transfected cells containing R-rings were counted 30 h later. The average of 11 independent transfections with SE is depicted as the percentage of cells with R-rings. Approximately 200 transfected cells were counted in each case; p < 0.001.
Figure 6.
Figure 6.
Electron spectroscopic imaging reveals enrichment of complexed calcium between the membrane cisternae of R-rings and NCS. (A) Electron spectroscopic micrographs of complexed calcium (I) and phosphorus (II) of a cell containing an R-ring. The nuclear envelope (NE) and heterochromatin (HC) are indicated. III and IV show enlarged images of the boxed sections of the R-ring in I and II, respectively. Note the enrichment of complexed calcium and phosphorus in the matrix but not the membrane cisternae of the R-ring. (B) The same as A but of an NCS in a human endometrial biopsy. III and IV represent histograms of the pixel intensities (based on the originally collected data) along the dashed line between the two dots in I and II, respectively. Note how calcium is enriched to a much lesser extent in the NCS (gray background) relative to the central NCS cavity and surrounding chromatin (white background) compared with phosphorus.
Figure 7.
Figure 7.
Immunoelectron micrographs with Nopp140 antibodies on ultrathin cryosections. (A–C) Nopp140 is enriched in the dense fibrillar component of nucleoli (No) but only moderately present in NCSs (A and C) or not at all (B). All gold particles are pointed out by arrows. Bars, 0.5 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Areas J. A., Grobner G., Glaubitz C., Watts A. Interaction of a type II myosin with biological membranes studied by 2H solid state NMR. Biochemistry. 1998;37:5582–5588. - PubMed
    1. Azadian-Boulanger G., Secchi J., Laraque F., Raynaud J. P., Sakiz E. Action of midcycle contraceptive (R 2323) on the human endometrium. Am. J. Obstet. Gynecol. 1976;125:1049–1056. - PubMed
    1. Bentin-Ley U., Sjogren A., Nilsson L., Hamberger L., Larsen J. F., Horn T. Presence of uterine pinopodes at the embryo-endometrial interface during human implantation in vitro. Hum. Reprod. 1999;14:515–520. - PubMed
    1. Chen H. K., Pai C. Y., Huang J. Y., Yeh N. H. Human Nopp140, which interacts with RNA polymerase I: implications for rRNA gene transcription and nucleolar structural organization. Mol. Cell. Biol. 1999;19:8536–8546. - PMC - PubMed
    1. Chiu C. M., Tsay Y. G., Chang C. J., Lee S. C. Nopp140 is a mediator of the protein kinase A signaling pathway that activates the acute phase response alpha1-acid glycoprotein gene. J. Biol. Chem. 2002;277:39102–39111. - PubMed

Publication types

LinkOut - more resources