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J. Biol. Chem., Vol. 277, Issue 18, 15661-15665, May 3, 2002
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From the
Received for publication, January 31, 2002, and in revised form, March 11, 2002
DNA topoisomerase I is a nucleolar protein, which
relocates to the nucleoplasm in response to drugs stabilizing
topoisomerase I·DNA intermediates (e.g. camptothecin).
Here we demonstrate that this phenomenon is solely caused by the
drug's impact on the interplay between mobility and localization of
topoisomerase I in a living cell nucleus. We show by photobleaching of
cells expressing biofluorescent topoisomerase I-chimera that the enzyme
moves continuously between nucleoli and nucleoplasm. Complex kinetics
of fluorescence recovery after photobleaching indicates that two enzyme
fractions with different mobility coexist in nucleoli and nucleoplasm.
However, the whole complement of topoisomerase I is in continuous
flux between these compartments and nucleolar accumulation can
plausibly explained by the enzyme's 2-fold lesser overall mobility in
nucleoli versus nucleoplasm. Upon addition of camptothecin,
topoisomerase I relocates within 30 s from the nucleoli to radial
nucleoplasmic structures. At these sites, the enzyme becomes retarded
in a dose-dependent manner. Inside nucleoli the mobility of
topoisomerase I is much less affected by camptothecin. Thus, the
enzyme's distribution equilibrium is shifted toward the nucleoplasm,
which causes nucleolar delocalization. In general, topoisomerase I is
an entirely mobile nuclear component, unlikely to require specific
signaling for movements between nuclear compartments.
DNA topoisomerase I changes the pitch of DNA helices by cutting
one DNA strand and allowing passage of the complementary strand through
the transient nick (1). One important role for this mechanism is the
release of torsional stress created by DNA transcription. Thus,
topoisomerase I activity is in principle required in the nucleoplasm
for mRNA synthesis (2) and in the nucleolus for rRNA synthesis (3).
However, the latter is considered the major working place of the
enzyme, which has been recognized as a predominantly nucleolar protein
(3, 4). Camptothecin and other agents stabilizing covalent
topoisomerase I·DNA intermediates cause relocation of topoisomerase I
from the nucleoli to the nucleoplasm (5). This was also observed with
inhibitors of RNA synthesis prompting the conclusion that the
phenomenon might be related to reduced activity of rRNA biosynthesis in
the nucleolus (6). Alternatively, it has been proposed that
camptothecin-induced conjugation of topoisomerase I with small
ubiquitin-like modifiers might serve as a signal triggering
translocation of topoisomerase I from the nucleoli to the nucleoplasm
(7).
Until now the dynamic properties of topoisomerase I in the nucleus or
between nuclear compartments have not been assessed directly. Thus, it
is not clear whether the enzyme is mobile or not, whether it resides in
the nucleolus in a static manner and moves into the nucleoplasm in
response to specific signals (as suggested in ref. 7) or whether it
shuttles continuously between nucleolus and nucleoplasm. Consequently,
it is also unclear as to what is the molecular event that leads to
nucleolar delocalization of topoisomerase I upon exposure to
camptothecins. We addressed these questions by determining the mobility
of topoisomerase I in substructures of the living cell nucleus using
high resolution confocal fluorescent microscopy and photobleaching techniques.
GFP1-topoisomerase I
was stably expressed in the human embryonal kidney cell line 293 (German Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany) using a bicistronic expression vector (8). The
first cistron was hybrid genes of GFP and either human topoisomerase I
or the active site mutant topoisomerase IPhe-723. The
second cistron contained the selection marker
puromycin-N-acetyl transferase. Cells were grown in
Dulbecco's modified Eagle's medium with Glutamax-I
(Invitrogen, Karlsruhe, Germany) and transfected using
LipofectAMINE (Invitrogen). Stably transfected cell lines were selected
and maintained with 0.35 µg ml Constitutive Expression of Active GFP-topoisomerase I in 293 Cells--
This investigation is based on the constitutive expression
of biofluorescent chimera of human topoisomerase I at physiological levels. To avoid overexpression, we used a vector conferring a balanced
coexpression of a hybrid gene of topoisomerase I fused at its N
terminus to GFP and the selection marker
puromycin-N-acetyltransferase from a single bicistronic
transcript (8). Significantly less clones emerged from transfection of
human 293 cells with GFP-topoisomerase I than with GFP alone, attesting
to a narrow tolerance margin for stable transgenic expression of human
topoisomerase I. However, growth rate and morphology of cell lines
supporting constitutive expression of GFP-topoisomerase I were similar
to untransfected cells or cells expressing GFP alone. This suggested a
quasi physiological expression of GFP-topoisomerase I,
provided that the chimeric enzyme was active, not overexpressed, and
colocalized with endogenous topoisomerase I. Control experiments
addressing these requirements are summarized in Fig.
1.
When Western blots were probed with GFP antibodies (Fig. 1A,
top), GFP-topoisomerase I was readily detected in
transfected cells as a single protein band (Fig. 1A,
lane 3) not apparent in untransfected cells (Fig.
1A, lane 1) or cells expressing GFP alone (not
shown). Thus, rearrangements of the chimeric gene could be excluded,
and green fluorescence of the cells could be unambiguously assigned to
full-length GFP-topoisomerase I. When Western blots were probed with
topoisomerase I antibodies (Fig. 1A, bottom), GFP-topoisomerase I appeared as an additional band of slower migration and similar intensity as compared with endogenous topoisomerase I. In
untransfected 293 cells the additional band was absent. It is
interesting to note that expression of GFP-topoisomerase I was
accommodated by a slight reduction in expression of endogenous topoisomerase I (Fig. 1A, bottom, lane
3). Thus, the GFP-tagged species was not overexpressed, and the
overall expression of topoisomerase I was the same as in untransfected cells.
The activity of GFP-topoisomerase I was tested by immuno-band depletion
(11). Before immunoblotting, cells were treated with camptothecin,
which stabilizes covalent topoisomerase I·DNA intermediates
inherent in the enzyme's catalytic cycle. Since these intermediates
are too large to enter the gel, the active fraction of the enzyme
becomes depleted from the blots. Apparently, endogenous as well as
GFP-linked topoisomerase I became efficiently depleted within the same
cell sample (Fig. 1A, lane 4). Moreover, the dose
response to camptothecin was similar for GFP-tagged and endogenous
topoisomerase I (Fig. 1B). In contrast, GFP-chimera of the
active site mutant topoisomerase IPhe-723 was not
depleted at all by camptothecin (Fig. 1A, lanes 5 and 6), attesting to the specificity of the assay.
Considering finally that fusion to GFP might disrupt the cellular
targeting of topoisomerase I, we compared within individual cells the
fluorescent patterns of GFP-topoisomerase I (Fig. 1C, top)
and topoisomerase I labeled with antibodies (Fig. 1C,
middle). Untransfected cells (Fig. 1C, left) gave
rise to antibody-derived signals only, whereas cells expressing
GFP-topoisomerase I (Fig. 1C, right) also emitted
GFP fluorescence. The patterns of GFP fluorescence and
immunofluorescence were virtually the same, confirming colocalization
of the GFP signal with endogenous topoisomerase I. Moreover,
immunostaining patterns of untransfected and transfected cells were
similar (Fig. 1C, compare left and
right), excluding that transgenic expression of
GFP-topoisomerase I disrupted localization of the endogenous enzyme.
Thus, the transgenic cell lines had fully integrated the GFP-chimera
into their cellular pool of topoisomerase I and could be used to study
the behavior of the enzyme.
Localization and Mobility of GFP-topoisomerase I in Living
Cells--
Fig. 2A shows time
lapsed epifluorescence microscopy of a cell expressing
GFP-topoisomerase I. Apparently, the cell lived under the microscope
because it divided while observed. Monitoring begins in late
G2 phase/early prophase (0 min), where GFP-topoisomerase I
was concentrated in the nucleoli. As the cell moved into prophase (4 min), nucleoli disappeared, and a granular fluorescent pattern emerged
indicating association of GFP-topoisomerase I with condensing chromatin
from early on. The enzyme stayed chromosome-bound until telophase
(12-48 min). Finally, during the G1 phase (77 min to 3 h) it accumulated again in the reforming nucleoli and was
otherwise distributed in the nucleoplasm in a uniform manner.
Next we applied photobleaching to probe the dynamic properties of
GFP-topoisomerase I in interphase nuclei of living cells. To determine
FRAP kinetics (12), cells expressing GFP-topoisomerase I were cultured
under a confocal laser scanning microscope and GFP fluorescence was
irreversibly bleached by high-powered laser pulses in circular areas of
the nucleoplasm or the nucleoli, respectively. Subsequently,
fluorescence recovery in the bleached spots as a consequence of other
GFP-topoisomerase I molecules moving in from unbleached areas was
recorded over time by sequential imaging scans (Fig. 2B). As
a control for a freely diffusible protein, we used cells expressing GFP
alone, which exhibited FRAP kinetics too fast for recording with our
experimental settings (Fig. 2B, inset). As a
control for immobile proteins we used cells expressing GFP-histone H3
(Fig. 2B, inset) known to be firmly
chromatin-bound (13). FRAP of GFP-topoisomerase I was complete after
30 s in nucleoplasm and nucleoli, indicating that the enzyme is
entirely mobile in both compartments (as opposed to histone H3).
However, GFP-topoisomerase I was clearly much slower than GFP alone,
suggesting that the enzyme is not freely diffusible. Moreover,
fluorescence recovery of GFP-topoisomerase I in the nucleoli was slower
than in the nucleoplasm, demonstrating that the enzyme's mobility at the two locations was restrained to a different extent. Nonlinear regression of these data (hatched lines in Fig.
2B) indicated with significance (p < 0.0001) that in both cases two different mobility states of the
fluorescent enzyme contributed to the apparent FRAP kinetics. A major
portion appeared to be moving fast (t1/2 = 1.1 ± 0.1 s and 1.9 ± 0.2 s for nucleoplasm and nucleoli,
respectively), whereas a minor portion was moving much slower
(t1/2 = 14.3 ± 2.3 s and 12.5 ± 1.4 s for nucleoplasm and nucleoli, respectively). The slow
portion amounted to 28 ± 3% in nucleoli as opposed to only
16 ± 2% in nucleoplasm. This difference explains why overall
mobility of GFP-topoisomerase I in the nucleoli was about 2-fold less
than in the nucleoplasm.
Rapid fluorescence recovery in nucleoli and nucleoplasm, as observed
here with GFP-topoisomerase I, suggests free and unrestricted exchange
of the protein between nuclear compartments. We corroborated this
notion by the FLIP approach (12). These experiments are summarized in
Fig. 2C. When one nucleolus was repeatedly bleached, all
topoisomerase I-linked GFP fluorescence was eventually lost from other
nucleoli of the same nucleus and also from the surrounding nucleoplasm.
Similar results were obtained when repeated bleaching was applied to
the nucleoplasm (not shown). These data imply that fluorescent
topoisomerase I molecules originally localized in both compartments
were eventually hit by bleach pulses aimed to only one of them, thus
demonstrating a rapid, continuous, and unrestricted traffic of all
enzyme molecules between individual nucleoli and between nucleoplasm
and nucleoli.
The Impact of Camptothecin on Topoisomerase I Mobility--
In the
light of these findings, we were curious how mobility and distribution
of topoisomerase I would be influenced by camptothecin, which
stabilizes covalent enzyme·DNA complexes. For this purpose, we
cultured cells expressing GFP-topoisomerase I under a confocal laser
scanning microscope and added camptothecin to the culture medium, while
taking serial confocal scans every 3.1 s (Fig.
3A and supplemental QuickTime
movie). The first image (0 s) recorded immediately before adding
camptothecin shows the normal nucleolar accumulation of topoisomerase
I. Subsequent images demonstrate a very rapid redistribution of
topoisomerase I to the nucleoplasm. After 20-30 s nucleoli were
largely reduced in fluorescence, and most of the enzyme was localized
in radial substructures within the nucleoplasm.
Fig. 3B shows FRAP kinetics determined in the nucleoplasm of
cells treated for 20 min with various concentrations of camptothecin. Nonlinear regression (Fig. 3B, hatched lines)
again indicated with significance (p < 0.0001) the
contribution of two different mobility states of the fluorescent enzyme
to each of these curves. Upon plotting of the half-times (Fig.
3C) and the relative proportions (Fig. 3D) of
slow (
A comparison of FRAP kinetics determined in nucleoli and nucleoplasm of
camptothecin-treated cells (Fig. 3E) showed that in the
nucleoli topoisomerase I was retarded to a much lesser extent than in
the nucleoplasm, suggesting that camptothecin acted preferentially on
topoisomerase I in the nucleoplasm. As a consequence, the enzyme was
moving much faster in the nucleolus than in the nucleoplasm in a
camptothecin-treated cell, whereas in an untreated cell it was the
other way around (Fig. 3C, compare open and
closed symbols). Thus, nucleolar accumulation and
camptothecin-induced nucleolar delocalization of topoisomerase I seem
to be driven by differences in mobility. Support of this notion was
gained from the behavior of the catalytically inactive mutant
topoisomerase IPhe-723. FRAP kinetics of GFP-topoisomerase
IPhe-723 were virtually the same in nucleoli and
nucleoplasm (Fig. 3F, compare circles and
triangles) and with and without camptothecin (Fig. 3F,
compare open and closed triangles), attesting to
the fact that mobility of the inactive enzyme was the same in both compartments and not affected by camptothecin. Consequently,
topoisomerase IPhe-723 did not accumulate in nucleoli and
did not delocalize from nucleoli upon exposure to camptothecin (Fig.
3F, inset). Interestingly, FRAP kinetics of
topoisomerase IPhe-723 were in general slower
(t1/2 = 2.5 ± 0.2 s and 22.4 ± 2.2 s for fast and slow subpopulations in both compartments,
respectively) than those of the active enzyme, suggesting that DNA
catalysis somehow facilitates detachment of the enzyme from its binding sites.
We present here for the first time data on the mobility and the
dynamics of human topoisomerase I in living cells. We show that the
enzyme is fully mobile and in continuous flux between nucleoli and the
nucleoplasm. These findings conform to the general concept that nuclear
compartments are generated by a binding equilibrium of entirely mobile
proteins (14). In keeping, we show here that topoisomerase I is not
restricted to the nucleoli but imposes as a nucleolar protein, because
it is moving more slowly in the nucleoli than in the nucleoplasm,
whereas the inactive mutant topoisomerase IPhe-723 has the
same mobility in both compartments and is therefore more evenly
distributed between them.
A comparison of FRAP kinetics of GFP-topoisomerase I and untagged GFP
shows that, overall, topoisomerase I moves slower than to be expected
from free diffusion, which is a usual feature of proteins with a
nuclear function (14). We show in addition that in each nuclear
compartment topoisomerase I is divided into a slow and a fast fraction.
We do not know whether these fractions are interconvertible, but the
most plausible interpretation is that the enzyme switches between an
off state, where it is more or less freely diffusible
(t1/2 = 1-2 s), and an on state, where
it's mobility is about 10-fold reduced (t1/2 = 12-14 s). Since topoisomerase I is continuously scanning the entire
nuclear space, interactions of the enzyme with processes and places
where its activity is required must implicitly be transient. It stands
to reason that such interactions will involve less mobile nuclear
components, such as chromatin, nuclear matrix, multiprotein complexes,
etc. and, therefore, slow down the enzyme (14, 15). Thus, the slow fraction (the on state) most likely represents topoisomerase
I engaged in processes, whereas the fast fraction (the off
state) represents topoisomerase I moving between processes. This
interpretation of our data is in good agreement with a
"stop-and-go" model recently suggested for the interaction of
histone H1 with chromatin (16, 17).
How does camptothecin fit into such a revised perception of
topoisomerase I? The drug is known to bind and stabilize the covalent topoisomerase I·DNA intermediate (18). We show here that camptothecin further attenuates the slow fraction of nucleoplasmic topoisomerase I,
which supports the above notion of this fraction being the one engaged
in DNA turnover. At least in living cells, the drug does not immobilize
topoisomerase I completely, which fits some previous biochemical data
(19) showing that topoisomerase I·DNA intermediates stabilized by
camptothecin have a comparatively short half-life (<1 min). Why does
topoisomerase I disappear so rapidly from its favored nucleolar
residence when camptothecin is present? It has been proposed that
nucleolar delocalization is triggered by conjugation of the enzyme with
small ubiquitin-like modifiers (7). However, this process is operating
on a minute scale (20), whereas we show here that nucleolar
delocalization occurs within seconds. Moreover, it is unlikely that an
entirely mobile protein like topoisomerase I should require specific
signals to move from one place in the nucleus to another. As elaborated in the first paragraph, topoisomerase I is prone to accumulate in the
nucleoli, because here it is moving more slowly than in the
nucleoplasm. In the presence of camptothecin, however, the situation is
rapidly reversed. Now, the enzyme is moving more slowly in the
nucleoplasm than in the nucleoli. Accordingly, it accumulates in the
nucleoplasm and delocalizes from nucleoli, although it is still freely
exchanging between the two compartments. Thus, nucleolar delocalization
of topoisomerase I upon exposure to camptothecin seems to be a
plausible epiphenomenon of the enzyme's nuclear traffic and not a
reflection of some specific cellular response to the drug. In keeping,
topoisomerase IPhe-723, which cannot be stabilized in
covalent DNA complexes by camptothecin, does not delocalize from
nucleoli upon exposure to the drug, as should be the case, if nucleolar
delocalization was due to some kind of a coordinated stress reaction
and not a direct consequence of the altered mobility of the enzyme.
We are grateful to Jörg Hacker
and Hilde Merkert for generously providing access to a confocal
laser scanning microscope.
*
This work was supported by the Deutsche
Forschungsgemeinschaft (Bo 910/3-1, Bo 910/4-1, GRK 639, and HA
1434/13-1) and the Danish Cancer Society (97-100-32).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
(49-931)201-70080; Fax: 49(931)201-70980; E-mail:
christian.mielke@mail. uni-wuerzburg.de.
Published, JBC Papers in Press, March 20, 2002, DOI 10.1074/jbc.C200066200
The abbreviations used are:
GFP, green
fluorescent protein;
FLIP, fluorescence loss in photobleaching;
FRAP, fluorescence recovery after photobleaching.
Changes in Mobility Account for Camptothecin-induced Subnuclear
Relocation of Topoisomerase I*,
,,,, and¶
Department of Clinical Chemistry,
Medizinische Poliklinik, University of Würzburg, Klinikstrasse
6-8, D-97070 Würzburg, Germany and the § Department of
Molecular and Structural Biology, University of Aarhus, DK-8200
Aarhus-C, Denmark
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 puromycin.
Immunoblotting and immunhistochemistry followed published protocols
(9). Epifluorescent images were acquired with a Zeiss Axiovert 100 inverted microscope equipped with a heated live-cell chamber system
(Bioptechs Inc., Butler, PA) for inspection of living cell specimen.
For confocal imaging, FRAP, and FLIP, we used a Zeiss LSM 510 inverted
confocal laser scanning microscope equipped with a
CO2-controlled on-stage heating chamber and a heated
63×/1.4 NA oil-immersion objective. Culturing of cells at 37 °C
under the microscope was crucial for obtaining consistent data of
localization and mobility of topoisomerase I, whereas erratic results
were obtained when native cell specimen were analyzed at ambient
temperature. For FRAP measurements, fluorescent images of a single
optical section were taken at 1.6-s time intervals before
(n = 5) and after bleaching of a circular area at 20 milliwatt nominal laser power with three iterations. Imaging scans were acquired with the laser power attenuated to 0.1-1% of the bleaching intensity. The same laser settings were used for FLIP experiments, where the cells were repeatedly bleached and imaged at intervals of
15 s. For quantitative analysis of FRAP, fluorescence intensities of the bleached region and the entire cell nucleus were measured at
each time point. Data were corrected for extracellular background intensity and for the overall loss in total intensity as a result of
the bleach pulse itself and of the imaging scans. FLIP measurements were corrected only for the loss of fluorescence intensity caused by
the imaging scans, which was determined in neighboring cell nuclei not
subjected to bleaching. The relative intensity of the bleached area
Irel was calculated according to Ref. 10, and the computer software Prism (GraphPad Software Inc., San Diegeo, CA)
was used for nonlinear regression analysis and plotting of the data.
Kinetic models assuming the coexistence of one, two, or three
individual enzyme fractions with different mobility were tested. In all
cases, best fits (according to R2-value and F-test
significance) were obtained assuming the coexistence of two enzyme
fractions with different mobility. Values of maximal recovery derived
from nonlinear regression were used to calculate the proportion of the
two enzyme fractions with different mobility, whereas a third, immobile
enzyme fraction was calculated by adding up the individual fractions to
100%.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (78K):
[in a new window]
Fig. 1.
Characterization of HEK 293 cells expressing
GFP-topoisomerase I. A, immunoblotting of 293 cells not
transfected (lanes 1 and 2), expressing
GFP-topoisomerase I (lanes 3 and 4), or
expressing GFP-topoisomerase IPhe-723 (lanes 5 and 6). Blots were probed with antibodies against GFP
(top) or human topoisomerase I (bottom). The
positions of GFP-linked and endogenous topoisomerase I are indicated on
the right margin. Cells in lanes 2, 4,
and 6 were cultured with camptothecin (20 µM,
20 min) prior to analysis. B, cells expressing
GFP-topoisomerase I were cultured for 20 min with various
concentrations of camptothecin and subjected to immunoblotting with
antibodies against topoisomerase I. C, colocalization of
GFP-topoisomerase I with endogenous topoisomerase I. Untransfected 293 cells (left double column) and GFP-topoisomerase I
(right double column) were grown on microscopic slides,
para-formaldehyde-fixed, permeabilized, and double
stained with topoisomerase I antibodies (middle) and
4',6-diamidino-2-phenyindole (bottom). The top
row shows corresponding images of GFP fluorescence
(GFP). Each double column shows representative cells in
interphase (left) and pro/metaphase
(right).
View larger version (60K):
[in a new window]
Fig. 2.
Localization and mobility of
GFP-topoisomerase I. A, time-lapsed imaging of a cell
proceeding from late G2 to early G1 phase.
Cells expressing GFP-topoisomerase I were cultured at 37 °C under an
inverted epifluorescence microscope. A cell in late G2
phase was selected and imaged at the indicated time points by phase
contrast (top) and green fluorescence (bottom)
until it reached G1 phase. B, FRAP analysis of
living cells expressing GFP-topoisomerase I. Circular areas (Ø = 2 µm) of the nucleoplasm (top) or a nucleolus
(bottom) of interphase nuclei were bleached. Consecutive
images at 1.6-s time intervals were taken before and at the indicated
time points after the bleach pulse. Areas to be bleached are indicated
by a circle in the pre-bleach panels. Corresponding
quantitative data of fluorescence recovery kinetics are plotted below.
Fluorescence intensities in the bleached nucleoplasmic ( 
) or
nucleolar (
) region were measured and expressed as the relative
recovery over time after the bleach pulse (at 0 s). Mean values
from at least six individual cells and three independent experiments
are shown. Standard deviations were in each case less than 5% of the
mean values (not displayed). Hatched lines represent the
results of nonlinear regression analyses of the data. The
inset shows FRAP kinetics obtained with cells expressing
unfused GFP (
) or GFP-histone H3 (
). C, FLIP analysis of cells expressing GFP-topoisomerase
I. A circular area (Ø = 3 µm; white circle) of a
nucleolus was repeatedly bleached. Cells were imaged before each new
bleach pulse (selected images are shown at the top).
Fluorescence intensities of neighboring nucleoli and nucleoplasm
(black and white circles, respectively) were
determined and plotted below (, nucleoplasm; , nucleolus).
View larger version (37K):
[in a new window]
Fig. 3.
The impact of camptothecin on localization
and mobility of GFP-topoisomerase I. A, time-lapsed
confocal images of a cell exposed to camptothecin. Cells expressing
GFP-topoisomerase I were cultured at 37 °C under the microscope, and
confocal midsection scans of green fluorescence were taken every
3.1 s. Representative images of an individual cell are shown
before (0 s) and at the indicated time points after addition of 20 µM camptothecin. The complete sequence can be viewed in
the supplemental QuickTime movie. B, FRAP analysis (compare
with Fig. 2B) of GFP-topoisomerase I in the nucleoplasm of
cells exposed to increasing concentrations of camptothecin ( ,
control; 
, 1 µM; 
, 4 µM; 
, 20 µM; 
, 200 µM). For clarity, data points
are displayed by a connecting line, and only selected time points are
marked by a symbol. Hatched lines represent the results of
nonlinear regression analyses of the data. C, half-times of
slow and fast fractions of GFP-topoisomerase I derived from
nonlinear regression of the data in B are plotted over the
log molar concentration of camptothecin. D, percentages of
slow , fast , and immobile () fractions of GFP-topoisomerase I
derived from nonlinear regression of the data in B are
plotted over the log molar concentration of camptothecin. E,
comparative FRAP analysis of GFP-topoisomerase I in nucleoli ( and
) and nucleoplasm ( and ) of untreated cells (open
symbols) and cells treated with camptothecin (20 µM,
20 min; closed symbols). Data acquisition and plotting were
done as in B. F, FRAP analysis of
GFP-topoisomerase IPhe-723 in nucleoli () and in the
nucleoplasm () of untreated cells and in the nucleoplasm of cells
treated with campto- thecin (; 20 µM, 20 min). The inset
shows representative images of an individual cell expressing
GFP-topoisomerase IPhe-723 before (0 min) and 20 min after
addition of 20 µM camptothecin.
) and fast () enzyme fractions against the log molar
concentration of camptothecin, it becomes apparent that the drug acted
preferentially on the slow fraction of topoisomerase I. This fraction
was further retarded by camptothecin in a dose-dependent manner, whereas half-times of the fast fraction were not significantly altered (Fig. 3C). Coincidentally, an increasing proportion
of the enzyme was recruited to the slow state, whereas only an
insignificant portion became actually immobile (Fig. 3D,
compare and ).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
The on-line version of this article (available at
http://www.jbc.org) contains a QuickTime movie.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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