First published online 5 May 2004
doi: 10.1242/jcs.01098
Journal of Cell Science 117, 2481-2490 (2004)
Published by The Company of Biologists 2004
Actin- and protein-4.1-containing filaments link nuclear pore complexes to subnuclear organelles in Xenopus oocyte nuclei
Elena Kiseleva1,2,
Sheona P. Drummond1,
Martin W. Goldberg3,
Sandra A. Rutherford1,
Terence D. Allen1 and
Katherine L. Wilson4,*
1 Cancer Research UK, Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Manchester, M20 9BX, UK
2 Department of Morphology and Function of Cell Structure, Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk-90, 630090, Russia
3 Department of Biological and Biomedical Sciences, Science Laboratories, University of Durham, South Road, Durham, DH1 3LE, UK
4 Department of Cell Biology, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA
View larger version (134K):
[in a new window]
|
Fig. 1. Xenopus oocyte nuclei contain an extensive filamentous network associated with spherical bodies, a subset of which is recognized by antibodies specific for coilin. (A-E) Nuclear contents fixed and visualized by scanning electron microscopy (feSEM) after `peeling' away the nuclear envelope (A,B) and then applying either moderate (C) or strong (E) manual `spreading' forces. (D) Imaging by field emission electron microscopy (feSEM) of a gently opened oocyte nucleus; the folded-back nuclear envelope is visible at left, and intranuclear filaments associated with spherical bodies are seen at right. (F) Quantification of the diameters of spherical bodies (n=212 bodies). (G) Western blot of isolated Xenopus oocyte nuclei probed with anti-coilin antibodies. (H,I) Representative feSEM images of spherical bodies, fixed and labeled with anti-coilin antibodies (H) or no primary antibody (I), followed by incubation with secondary antibodies conjugated to 10 nm gold-particles (pseudo-colored yellow).
|
|
View larger version (156K):
[in a new window]
|
Fig. 5. Adjacent nuclear pore complexes are connected by branching filaments that exhibit a right-handed periodicity. (A,B) Filaments of  40 nm diameter are seen to interconnect groups of neighboring nuclear pore complexes (circled). (C) Higher magnification view of a pore-linked filament with pronounced periodicity (arrows indicate repeat elements). (D) Filaments attached to the nuclear envelope extend deep into the nucleus, branching and forming junctions (arrows).
|
|
View larger version (125K):
[in a new window]
|
Fig. 6. Immuno-gold SEM of nuclear filaments with anti-actin antibodies. (A) Western blot of isolated Xenopus oocyte nuclei probed with anti-actin antibodies. (B,C) The filamentous network is specifically decorated by antibodies against actin, visualized using gold-conjugated secondary antibodies (pseudo-colored yellow). (D) Only the distal regions of NPC baskets (the filaments of which are marked by dotted lines), at and beyond the terminal ring, are labeled by anti-actin antibodies (arrows). (E) Control filaments treated with secondary gold-conjugated antibody only. Arrow indicates a single gold particle, seen in higher magnification in the inset.
|
|
View larger version (156K):
[in a new window]
|
Fig. 2. Filaments of various diameters are embedded directly into the surface of a spherical organelle. (A) FeSEM imaging of gently disrupted oocyte nuclear contents showing characteristic filaments attached to the surface of a large spherical body. (B,C) Higher magnification to show structural heterogeneity of the `filament' at and near its points of attachment.
|
|
View larger version (160K):
[in a new window]
|
Fig. 3. Chromatin is associated with filaments and spherical bodies. FeSEM of meiotic chromosomes (A, arrows) within the oocyte nucleus. Chromosomes were also associated with intranuclear filaments (A,B) and small spherical bodies (B).
|
|
View larger version (121K):
[in a new window]
|
Fig. 4. Filaments link spherical bodies to the inner membrane of Xenopus oocyte nuclei. (A,C) Low and high magnification, respectively, of filaments. Small diameter (12 nm) filaments (arrows in C) bridge large filaments and also connect spherical bodies to nuclear pore complexes and the inner nuclear membrane. One region in A is shown at higher magnification in C. (B) Quantification of the size distribution of intranuclear filaments (n=300 filaments).
|
|
View larger version (182K):
[in a new window]
|
Fig. 7. The pore-linked filament network is actin-dependent. (A,B) FeSEM images of Xenopus oocyte nuclei incubated with (A) DMSO alone, or (B) DMSO plus 2 µg/ml latrunculin A to depolymerize actin.
|
|
View larger version (153K):
[in a new window]
|
Fig. 8. Ultrastructural effects of jasplakinolide, which stabilizes actin filaments and can induce actin polymerization, on the pore-linked filament network in Xenopus oocyte nuclei. (A) Jasplakinolide does not disrupt the organization of the pore-linked filament network, visualized by feSEM. (B,C) Higher magnification feSEM images of pore-linked filaments after treatment with jasplakinolide; C is an enlargement of the region boxed in B. Arrows in C indicate three parallel rows of axially projecting short filaments.
|
|
View larger version (66K):
[in a new window]
|
Fig. 9. Immuno-gold localization of protein 4.1 epitopes on the PLF network. (A) Western blot of manually isolated Xenopus oocyte nuclei probed with antibodies against protein 4.1 shows a major isoform of 80 kDa, plus potential minor isoforms visible only upon longer exposure (data not shown). (B,C) FeSEM images of different PLFs showing protein 4.1 labeled with primary antibodies plus 10-nm-gold-labeled secondary antibodies. Gold particles were visualized directly in backscatter images (not shown), and are pseudo-colored yellow.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2004
