First published online 11 December 2002
doi: 10.1242/jcs.00250
A plant cyclin B2 is degraded early in mitosis and its ectopic expression shortens G2-phase and alleviates the DNA-damage checkpoint
Magdalena Weingartner1,4,
Helvia R. Pelayo2,
Pavla Binarova3,
Karin Zwerger1,
Balázs Melikant1,
Consuelo de la Torre2,
Erwin Heberle-Bors1 and
László Bögre4,*
1 Institute of Microbiology and Genetics, University of Vienna, Vienna
Biocenter, Dr Bohrgasse 9, A-1030 Vienna, Austria
2 Centro de Investigaciones Biológicas CSIC, Velázquez 144,
E-28006-Madrid, Spain
3 Institute of Microbiology, Academy of Sciences of the Czech Republic,
Víde
ská 1083, 142 20 Prague 4, Czech Republic
4 School of Biological Sciences, Royal Holloway, University of London, Egham
TW20 0EX, UK
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Fig. 1. Cl-tetracycline-inducible expression of cyc-B2-HA mRNA and
Cyc-B2-HA-associated Cdk activity in tobacco plants and in cultured cells. (A)
Northern blot of mRNA samples from leaves from lines 1, 2, 4, 5 and 6
incubated with (+) or without (-) 0.1 mg/l Cl-tetracycline for 1 day. (B) Time
course of cycB2-HA mRNA induction by 0.1 mg/l Cl-tetracycline in cultured
cells generated from line 2. In both A and B the upper panel represents
CycB2-HA mRNA and the lower panel represents control (cnt6) mRNA. (C) Western
blot of total protein samples from cultured cells generated from line
CycB2-HA-2, which was induced for 12 hours by indicated concentrations of
Cl-tetracycline. CycB2-HA and Cdc2 protein were detected using HA-specific and
PSTAIRE-specific antibodies, respectively. (D) Histone H1 kinase activity of
immunopurified CycB2-HA-associated kinase and p13suc1-bound Cdks
from the extracts shown in C.
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Fig. 2. Entry into mitosis is advanced by Cl-tetracycline-induced CycB2-HA
expression. (A) Mitotic indices after release from aphidicolin-induced S phase
block in the presence (+) or absence (-) of 0.1 mg/l Cl-tetracycline. (B)
Northern blot analysis of tobacco cyclin B1, 1, histone H4, cyclin B2-HA and
cnt6 mRNA levels in synchronized cells as in A. (C) Proportion of mitotic
microtubule structures [pre-prophase band (PPB), spindle and phragmoplast] in
synchronized cells in the same experiment.
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Fig. 3. The tetracycline-induced expression of cyclin B2 allows caffeine and
okadaic acid to override the HU-induced replication checkpoints. (A) Mitotic
indices of the asynchronously growing transgenic cell line without (lanes 1-6)
or with (lanes 7-12) tetracycline-induced cyclin B2 expression. Cells were
either treated with no drug (1 and 7), with caffeine (3 and 8) or okadaic acid
(4 and 9) for 1 hour, or first with hydroxyurea for 16 hours (4 and 10), and
then incubated for 1 hour with caffeine (5 and 12) or okadaic acid (6 and 12).
(B) Cyclin B2-associated histone H1 kinase activities after immunopurification
using the HA-epitope tag (H1 CycB2-HA), immunodetection of CycB2-HA protein
using an anti HA-antibody, and CDKs using a PSTAIRE antibody, from cells
treated in the same way as in A.
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Fig. 4. Cyclin B2 is located in the nucleus and degrades in prometaphase. (A)
Triple-labeling of CycB2-HA-expressing cells with antibodies against the
HA-epitope (red) and  -tubulin (green); chromatin is stained with DAPI
(blue). A representative cell is shown in interphase (Int), preprophase (PPB),
pro-metaphase (PM), metaphase (Met), anaphase (Ana) and telophase (Tel). (B)
CycB2-GFP fluorescence and the corresponding DIC images in cultured cells.
Arrows in the top images indicate metaphases, and in the bottom images a cell
after cytokinesis. (C) CycB2-HA is absent in stationary-phase cells and
becomes detectable before S phase. Stationary phase cells from line CycB2-HA-2
were diluted with fresh medium and immediately incubated with BrdU labeling
reagent. Aliquots were taken at 3 hour intervals and the initiation of S phase
was determined by immunostaining for BrdU incorporation using an antibody
against BrdU. The percentage of cells with CycB2-HA protein was determined by
immunostaining using an antibody against the HA-epitope. Scale bars: 10
µm.
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Fig. 5. CycB2-HA localization in cells treated with roscovitine, APM, taxol and
MG-132. (A) Cells were treated in G2 phase with 100 µM roscovitine and
samples were taken after five hours incubation; arrows indicate a
prophase-arrested cell. Immunostaining of CycB2-HA expressing cells with the
anti HA-antibody is shown in the left-hand panels and DAPI-staining of the
chromatin in the right-hand panels. (B) Cells were treated in G2 phase with 10
µM amiprophos methyl (APM) (upper panels) or 50 µM taxol (lower panels)
and samples were taken after 5 hours incubation; arrows indicate
metaphase-arrested cells. (C) Cells were treated in G2-phase with 100 µM MG
132 and samples were taken after 5 hours. The arrows indicate a cell arrested
in mitosis. Scale bars: 10 µm.
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Fig. 6. Cyclin B1-GFP but not cyclin B2-GFP is stabilized in living cells arrested
in mitosis by activation of the spindle checkpoint or by inhibition of the
proteosome. (A) Visualization of GFP fluorescence in living cells expressing
cyclin B1-GFP (left panels) or cyclin B2-GFP (right panels) after treatment
with propyzamide. Arrows indicate cells arrested in mitosis. (B) Visualization
of GFP fluorescence of Cyclin B1-GFP (left panels) or cyclin B2-GFP (right
panels) in living cells after treatment with epoxomicin. Arrows indicate cells
arrested in mitosis. Upper panels, Fluorescent images; lower panels, DIC
images. Scale bars: 10 µm.
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Fig. 7. Protein levels of ectopically expressed CycB2-HA and endogenous cyclin B1
in synchronized CycB2-HA-2 cells and after treatment with propyzamide, MG 132,
epoxomicin or lactacystin. (A) Immunodetection of CycB2-HA protein with an
anti HA-antibody in protein samples prepared from non induced control cells or
in samples prepared at 2 hour intervals from 7 to 15 hours after aphidicolin
release and at 15 hours in cells that were treated in late G2-phase with
propyzamide (P), epoxomicin (E), lactacystin (L) or MG132 (Mg). (B)
Immunodetection of endogenous CycB1 protein with an anti N-terminal CycB1;1
peptide antibody in the same synchronized samples as described in A, and in
samples treated with propyzamide (P) epoxomicin (E) and lactacystin (L).
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Fig. 8. Root regeneration is inhibited by ectopic expression of CycB2-HA, which
correlates with shortening of the G2 phase in leaf cells. (A) Leaf disks from
line CycB2-HA-2 were incubated on root-inducing medium containing 0.5 mg/l
auxin (NAA) and 0.1 mg/l cytokinin (BAP) in the presence (+) or absence (-) of
0.1 mg/l Cl-tetracycline and pictures of regenerates were taken after 3 weeks.
(B) Flow cytometry measurements of G1 and G2 DNA contents in isolated nuclei
from leaves treated as in A and incubated for 3 days.
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