Revealing the unseen: the organizer region of the nucleolus
Marco Biggiogera1,2,*,
Manuela Malatesta1,3,*,
Sousan Abolhassani-Dadras1,
François Amalric4,
Lawrence I. Rothblum5 and
Stanislav Fakan1,
1 Centre of Electron Microscopy, University of Lausanne, 1005 Lausanne, Switzerland
2 Dipartimento di Biologia Animale, Laboratorio di Istologia, and Centro di Studio per l’Istochimica del CNR, University of Pavia, 27100 Pavia, Italy
3 Istituto di Istologia ed Analisi di Laboratorio, University of Urbino, 61029 Urbino, Italy
4 Institut de Pharmacologie et de Biologie Structurale, CNRS, 31077 Toulouse, France
5 Weis Center for Research, Geisinger Clinic, Danville, PA 17822, USA
* These authors contributed equally to this work
View larger version (151K):
[in a new window]
|
Fig. 1. Nucleolus of a mouse P815 cell. Glutaraldehyde-osmium fixation, Epon embedding, uranyl-lead staining. The DFC (arrows) appears as a strongly electron-dense structure surrounding the FCs. Most of the nucleolus is formed by the GC (asterisk). Bar, 0.5 µm.
|
|
View larger version (100K):
[in a new window]
|
Fig. 2. (a) Immunolabeling for fibrillarin and specific DNA staining with osmium ammine. The labeling for fibrillarin, a marker of the DFC, is present at the periphery of a stained area formed by thin fibers of DNA. Clumps of intranucleolar chromatin are also stained (arrow). Notice that the different nucleolar components shown in Fig. 1 are no more recognizable. Bar, 0.2 µm. (b) Control sample submitted to anti-fibrillarin labeling, hydrolysis with 5 N HCl for 20 minutes and staining with uranyl acetate. The DFC surrounding the FC (asterisk) is specifically labeled (arrows). Bar, 0.2 µm. (c) After immunolabeling with an anti-RNA-polymerase-I antibody, the gold particles are located over the DFC (arrows). The FCs (asterisks) and the GC (G) are devoid of labeling. Bar, 0.5 µm.
|
|
View larger version (204K):
[in a new window]
|
Fig. 3. (a) Ultrathin (25 nm) section through a nucleolus immunolabeled as in Fig. 2 and stained for DNA. Observation at 0 eV loss. Under these conditions, the nucleolus (Nu) appears as an ‘empty’ area in which DNA fibers are present as clumps scattered in the nucleolar body. A cloud of thin DNA fibers, the periphery of which overlaps the anti-fibrillarin labeling, can be seen in the center (arrowhead). Abbreviation: N, nucleoplasm. Bar, 0.5 µm. (b) At a higher magnification, it is clearly evident that the gold grains localizing fibrillarin are present at the periphery of the cloud (asterisk) and superimposed on the thinner fibers within the external part of the DNA cloud (arrows). (c) The same area as in b, observation at 250 eV energy loss. At this energy loss, it is clear that the inner core of the DNA cloud (asterisk) is formed by fibers thinner than chromatin clumps but thicker than those overlapping the anti-fibrillarin labeling (arrows). (d) The same area as in b and c, after phosphorus mapping. Phosphorus is present in all the locations that were positive in the DNA staining. Even the thin filaments at the periphery of the cloud are visible (arrows). Bar, 0.2 µm.
|
|
View larger version (83K):
[in a new window]
|
Fig. 4. Schematic interpretation of Fig. 3b-d. The DNA cloud originates in the peripheral clumps of condensed chromatin, forms the FC (intermediate step of uncoiling) and expands to form the DFC (thinnest DNA fibers, active genes). The gray areas represent the zone labeled by the anti-fibrillarin antibody. The DNA cloud and the condensed chromatin from which it originates correspond to the interphase NOR.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2001
