First published online 11 March 2003
doi: 10.1242/jcs.00311
Nucleolar association of pEg7 and XCAP-E, two members of Xenopus laevis condensin complex in interphase cells
Rustem Uzbekov1,2,
Elmira Timirbulatova2,
Erwan Watrin1,
Fabien Cubizolles1,
David Ogereau1,
Pavel Gulak3,
Vincent Legagneux1,
Vladimir Ju. Polyakov4,
Katherine Le Guellec1,* and
Igor Kireev2,5,
1 Groupe Structure Dynamique de la Chromatine, CNRS, UMR 6061, Faculte de
Medicine, 35043, Rennes, France
2 Cell Cycle Group, Division of Electron Microscopy, A. N. Belozersky Institute
of Physico-Chemical Biology, Moscow State University, 119899, Moscow,
Russia
3 Institute of Agricultural Biotechnology, 127550 Moscow, Russia
4 Division of Electron Microscopy, A. N. Belozersky Institute of
Physico-Chemical Biology, Moscow State University, 119899, Moscow,
Russia
5 Department of Cell and Structural Biology, University of Illinois at
Urbana-Champaign, Urbana 61801, USA
View larger version (27K):
[in a new window]
|
Fig. 1. Specificity of antibodies to Eg7G, XCAP-E1, TopoII and ß-tubulin in
XL2 cell extract. Proteins in X. laevis XL2 cell lysate
(2x105 cells) were separated onto 7% SDS-polyacrylamide gel,
transferred onto nitrocellulose membranes and immunodetected with mixture of
purified Eg7G and monoclonal antibodies against ß-tubulin (lane1) or
XCAP-E1 antibodies (lane 2) or monoclonal anti-topoII antibodies (lane 3).
|
|
View larger version (38K):
[in a new window]
|
Fig. 2. Western blot analysis of the quantity of pEg7, topoII and XCAP-E in the
synchronized cells of different stages of cell cycle. (A) After
electrophoresis and transfer onto nitrocellulose, the membrane was cut, the
upper part was incubated with antibodies to topoII, the second part with a
mixture of pEg7 and the third part with monoclonal antibodies against
ß-tubulin and the last part with monoclonal antibodies against pEg2
(clone 1C1). Immunocomplexes were revealed using an enhanced chemoluminescence
system. (B) Relative quantities of protein during the cell cycle
(protein:ß-tubulin ratio, with the ratio in G1 equal to 1). The level of
cycle-dependent protein kinase pEg2 increased from 1 at G1 to
15.44±3.21 at M phase; the quantity of topoII was maximal in G2 phase
(3.61±1.22) and practically the same in mitosis (3.52±1.04). The
quantity of pEg7 and XCAP-E increased from 1 at G1 to 1.48±0.09 and
1.48±0.04 times at M phase, respectively. Average data from four
measurements from two experiments are presented. Quantitative analysis was
performed using Image Quant computer program.
|
|
View larger version (170K):
[in a new window]
|
Fig. 4. Nucleolar localization of XCAPE and topoII in an interphase nucleus. XL-2
cells were grown and fixed as described in Materials and Methods. Cell were
then processed for double immunofluorescence staining (A-E) with anti-XCAP-E
affinity-purified polyclonal antibody (C) and anti-human topoII monoclonal
antibody (D). Double staining is shown in e. Cells were observed by phase
contrast microscopy (A) and stained with DAPI (B). For electron microscopy
(F-H), cells were fixed as described under Materials and Methods and then
labeled for double immunogold electron microscopy with an anti-XCAP-E
affinity-purified polyclonal antibody and an anti-human topoII monoclonal
antibody. Anti-XCAP-E and anti-topoII antibodies were revealed with a
secondary anti-rabbit antibody conjugated to 5 nm gold particle (for XCAP-E)
and an anti-mouse antibody conjugated to a 10 nm gold particles (for topoII,
arrows), respectively. Bar, 5 µm (A-E), 1 µm (F) and 0.2 µm
(G,H).
|
|
View larger version (148K):
[in a new window]
|
Fig. 3. Nucleolar localization of pEg7 and topoII in the interphase nucleus. XL-2
cells were grown and fixed as described under Materials and Methods. Cell were
then processed for double immunofluorescence staining (panels A-E) with an
anti-pEg7G affinity-purified polyclonal antibody (C) and an anti-human topoII
monoclonal antibody (D). The merged picture is shown in e. Cells were observed
by phase contrast microscopy (A) and stained with DAPI (B). For electron
microscopy (F-H) cell were fixed as described under Materials and Methods and
then labeled for double immunogold electron microscopy with anti-pEg7G
affinity-purified polyclonal antibody and anti-human topoII monoclonal
antibody. Anti-Eg7 and anti-topoII antibodies were revealed with a secondary
anti-rabbit antibody conjugated to a 5 nm gold particle (for pEg7) and an
anti-mouse antibody conjugated to 10 nm gold particle (for topoII, arrows),
respectively. Bar, 5 µm (A-E), 1 µm (F) and 0.2 µm (G,H).
|
|
View larger version (42K):
[in a new window]
|
Fig. 5. Immunolocalization of XCAP-E and UBF in control cells and after actinomycin
treatment. XL2 cells were incubated for 6 hours in the medium with 5 µg/ml
actinomycin D (E-H) or without the drug (A-D). After fixation, cells were
processed for immunofluorescence staining with polyclonal anti-XCAP-E1
antibodies (B,F) and human autoimmune serum to UBF (C,G). Cells were stained
with DAPI for DNA visualization (A,E). Triple DAPI/XCAP-E/UBF labeling is
shown (D,H). Bar, 5 µm.
|
|
View larger version (34K):
[in a new window]
|
Fig. 6. Immunolocalization of XCAP-E and fibrillarin in control cells and after
actinomycin treatment. XL2 cells were incubated for 6 hours in the medium with
5 µg/ml actinomycin D (E,H) or without the drug (A-D). After fixation,
cells were processed for immunofluorescence staining with polyclonal
anti-XCAP-E1 antibodies (B,F) and human autoimmune serum to fibrillarin (C,G).
Cells were stained for DNA visualization with DAPI (A,E). Triple
DAPI/XCAP-E/fibrillarin labeling is shown (D,H). Bar, 5 µm.
|
|
View larger version (26K):
[in a new window]
|
Fig. 7. Immunolocalization of XCAP-E and B23 in control cells and after actinomycin
treatment. XL2 cells were incubated for 6 hours in the medium with 5 µg/ml
actinomycin D (E-H) or without the drug (A-D). After fixation, cells were
processed for immunofluorescence staining with polyclonal anti-XCAP-E1 (B,F)
and monoclonal anti-B23 (C,G) antibodies. Cells were stained for DNA
visualization with DAPI (A,E). Triple DAPI/XCAP-E/B23 labeling is shown (D,H).
Bar, 5 µm.
|
|
View larger version (30K):
[in a new window]
|
Fig. 8. Immunolocalization of pEg7 and topoII on the nucleolus in control cells and
after actinomycin treatment. XL2 cells were incubated for 6 hours in the
medium with 5 µg/ml actinomycin D (E-H) or without the drug (A-D). After
fixation cells were processed for immunofluorescence staining with polyclonal
anti-pEg7 (C,G) and monoclonal anti-topoII (D,H) antibodies. Cells were
observed by phase contrast microscopy (A,E) and stained for DNA visualization
with DAPI (B,F). Bar, 5 µm.
|
|
View larger version (29K):
[in a new window]
|
Fig. 9. Immunolocalization of XCAP-E and topoII on the nucleolus in control cells
and after actinomycin treatment. XL2 cells were incubated for 6 hours in the
medium with 5 µg/ml actinomycin D (E-H) or without the drug (A-D). After
fixation, cells were processed for immunofluorescence staining with polyclonal
anti-XCAP-E1 (C,G) and monoclonal anti-topoII (D,H) antibodies. Cells were
observed by phase contrast microscopy (A,E) and stained for DNA visualization
with DAPI (B,F). Bar, 5 µm.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2003
