The Journal of Cell Biology
The Journal of Cell Biology, Volume 166, Number 6, September 13, 2004 787–800
The Rockefeller University Press, 0021-9525/2004/09/787/14 $8.00
Quantitative kinetic analysis of nucleolar breakdown
and reassembly during mitosis in live human cells
Anthony Kar Lun Leung,
and Angus I. Lamond
Yun Wah Lam,
Division of Gene Regulation and Expression, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee,
Dundee DD1 5EH, Scotland, UK
European Molecular Biology Laboratory (EMBL), D6901 Heidelberg, Germany
ne of the great mysteries of the nucleolus surrounds
its disappearance during mitosis and subsequent
reassembly at late mitosis. Here, the relative dy-
namics of nucleolar disassembly and reformation were
dissected using quantitative 4D microscopy with fluorescent
protein-tagged proteins in human stable cell lines. The data
provide a novel insight into the fates of the three distinct
nucleolar subcompartments and their associated protein
machineries in a single dividing cell. Before the onset of
nuclear envelope (NE) breakdown, nucleolar disassembly
started with the loss of RNA polymerase I subunits from the
fibrillar centers. Dissociation of proteins from the other
subcompartments occurred with faster kinetics but com-
menced later, coincident with the process of NE breakdown.
The reformation pathway also follows a reproducible and
defined temporal sequence but the order of reassembly
is shown not to be dictated by the order in which individ-
ual nucleolar components reaccumulate within the nucleus
The nucleolus is a dynamic nuclear structure that assembles
and disassembles during each mitotic cell division (Carmo-
Fonseca et al., 2000; Olson et al., 2000; Hernandez-Verdun
et al., 2002; Gerbi et al., 2003; Leung and Lamond, 2003).
Nucleoli form around clusters of tandemly repeated ribo-
somal DNA (rDNA) genes, the so-called nucleolar organizing
regions (NORs), where RNA polymerase I transcribes the
rDNA repeats and generates the 45S rRNA precursor. The
precursor is then cleaved and modified within nucleoli to
generate mature 18S, 5.8S, and 28S rRNAs that are sub-
sequently assembled with ribosomal proteins to form ribo-
some subunits. These processes occur at distinct nucleolar
subcompartments, namely the fibrillar centres (FCs), dense
fibrillar components (DFCs), and granular components
(GCs). The rDNA clusters are located either within, or at
the periphery of, FCs and rRNA transcripts appear at the
boundary between the FC and DFC (Huang, 2002). The
maturing rRNA transcripts progress through the DFC and
then GC regions, before export of the ribosome subunits to
the cytoplasm. The structural integrity of nucleoli may also
play a general role in coordinating cellular stress responses.
For example, disintegration of the nucleolus has been pro-
posed as a common feature in cellular responses that activate
the p53 pathway (Rubbi and Milner, 2003).
During mitosis, mammalian nucleoli disassemble and their
components disperse. When cells exit mitosis nucleolar com-
ponents reassemble around the respective NORs, which can
later coalesce to form either one or multiple functional nucle-
oli. Partially processed rRNA transcripts, together with as-
sociated processing factors, form structures during mitosis
termed “prenucleolar bodies” (PNBs; Jimenez-Garcia et al.,
The online version of this article contains supplemental material.
Address correspondence to Angus I. Lamond, Division of Gene Regula-
tion and Expression, School of Life Sciences, Wellcome Trust Biocentre,
University of Dundee. Dundee DD1 5EH, Scotland, UK. Tel.: 44-
1382-345473. Fax: 44-1382-345695.
A.K.L. Leung’s present address is Center for Cancer Research, Dept. of
Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
G. Miller’s present address is Fred Hutchinson Cancer Research Center,
MS A1-162, 1100 Fairview Ave. N., Seattle, WA 9810.
C. Lyon’s present address is Cyclacel, James Lindsay Place, Dundee
Technopole, Dundee DD1 5JJ, UK.
Key words: nucleolus; nucleus; mitosis; fluorescent protein; 4D imaging
Abbreviations used in this paper: B23, nucleophosmin/nucleolar phos-
phoprotein B23/numatrin; DFC, dense fibrillar component; FC, fibrillar
centre; FIB, fibrillarin; FP, fluorescent protein; GC, granular compo-
nent; IBB, importin-
binding; LB1, lamin B1; LBR, lamin B receptor;
NE, nuclear envelope; NOR, nucleolar organizing region; PNB, prenu-
cleolar body; rDNA, ribosomal DNA; RL27, ribosomal protein L27;
RPA39, RNA polymerase I subunit RPA39; RRN3, RNA polymerase I
transcription factor RRN3; UBF, upstream binding transcription factor.
788 The Journal of Cell Biology
Volume 166, Number 6, 2004
1994; Dundr et al., 2000; Savino et al., 2001). Compo-
nents are subsequently transferred from the PNBs into the
reforming nucleoli at NORs (Dundr et al., 2000; Savino et
al., 2001). In contrast, the process of nucleolar disassembly
when cells enter mitosis is not well characterized. Both the
timing and mechanism of breakdown is unclear although it
has been reported that a subset of nucleolar factors, includ-
ing RNA polymerase I (Scheer and Rose, 1984; Gilbert et
al., 1995) and the RNA Pol I upstream binding transcrip-
tion factor (UBF) and transcription factor SL1 (Roussel
et al., 1993; Jordan et al., 1996), remain associated with
chromosomes at NORs, although no nascent rRNA tran-
scripts are synthesized during mitosis (Weisenberger and
Fluorescent protein (FP)-tagged nuclear factors provide
markers in live cells for the location of specific nuclear bod-
ies, such as nucleoli, or even subcompartments within nucle-
oli (Gerlich and Ellenberg, 2003a; Janicki and Spector,
2003). Proteins tagged with different spectral variants of ei-
ther GFP, or RFP, can be coexpressed to detect two or more
subnuclear structures in a single cell. The 3D localization of
these markers can be recorded and quantitated over time,
i.e., “4D” imaging, to provide detailed information about
their dynamic movement and kinetic behavior (Gerlich et
al., 2001; Dundr et al., 2002b; Gerlich and Ellenberg,
2003a). Here, we have applied quantitative 4D imaging us-
ing cell lines expressing different combinations of two or
three FP-tagged markers to analyze the processes of nucle-
olar breakdown and reassembly during mitosis in live cells
and to correlate this with parallel events affecting other nu-
In vivo analysis of RNA polymerase I localization
A HeLa cell line termed HeLa
expressing YFP-tagged RNA polymerase I subunit RPA39
(RPA39), which is a subunit of RNA polymerase I and also
RNA polymerase III (see Materials and methods). The YFP-
RPA39 fusion protein concentrated in bright foci within nu-
cleoli corresponding to the FC and also in a diffuse nucleo-
plasmic pool (Fig. 1 A). Both YFP-RPA39 and a transiently
expressed CFP fusion of the transcription factor RNA poly-
merase I transcription factor RRN3 (RRN3) that is essential
for initiation of transcription by RNA polymerase I (Miller
et al., 2001) colocalize in the nucleolar foci (Fig. 1 A, arrow-
heads). These data indicate that YFP-RPA39 colocalizes in-
side nucleoli with RRN3 at sites including active RNA poly-
merase I. Analysis of HeLa
that the expression of YFP-RPA39 does not delay cell cycle
progression (unpublished data).
We next addressed whether the YFP-RPA39 subunit is in-
corporated into functional RNA polymerase I complexes. In
vitro RNA polymerase I transcription assays were performed
using nuclear extracts prepared from both HeLa
parental HeLa cell lines (Fig. 1 B). Extracts from both cell
lines show equivalent levels of RNA polymerase I activity
(Fig. 1 B, lanes 8 and 9). Active RNA polymerase I transcrip-
tion complexes can be immunoprecipitated using anti-GFP
antibodies specifically from the HeLa
(Fig. 1 B, compare lane 2 with lanes 3–7). The in vitro activ-
ity is stimulated by addition of purified UBF and transcrip-
was established stably
cells by FACS showed
RPA39 cell lines. (A) HeLaYFP-RPA39 cells
were fixed 10 h after transient transfection
with CFP-RRN3. Arrowheads indicate
the colocalization of punctate structures
labeled by YFP-RPA39 and CFP-RRN3
within nucleoli. The nuclear boundaries
are denoted by dotted ovals. Bar, 5 ?m.
(B) In vitro transcription assay. Nuclear
extracts prepared from HeLaYFP-RPA39 and
parental HeLa cells were tested for spe-
cific transcription initiation activity on
the rDNA promoter, which is shown by
the presence of nascent rDNA transcript
(lanes 8 and 9). Lanes 1–7 assay tran-
scription from RNA Polymerase I
complexes immunoprecipitated with
complexes from HeLaYFP-RPA39 nuclear
extracts were transcriptionally active
upon the addition of rRNA gene pro-
moter template DNA and ribonucleoside
triphosphates (lane 3). Addition of SL1
alone (lanes 4 and 6) or SL1 and UBF
(lanes 5 and 7) further stimulated tran-
scription. In the absence of anti-GFP
antibody no transcription was observed
(lane 1) and in the presence of anti-GFP-protein G beads but absence of a GFP tag (parental HeLa nuclear extract) no transcription was
observed (lane 2). (C) Pulse-labeled rRNA transcripts transiently colocalize within the DFC after a 6-min chase. The left panel shows the
localization of DFC labeled by YFP-FIB in stable HeLaYFP-FIB cell lines and the middle panel illustrates the localization of accumulated BrUTP
at 6 min and the pattern of incorporation within nucleoli are shown in detail in the inserts. Bar, 5 ?m.
Characterization of YFP-
Nuclear dynamics in mitosis
Leung et al. 789
tion factor SL1 to the assays (Fig. 1 B, compare lane 3 with
lanes 4–7). We analyzed further the relationship between
YFP-RPA39 and transcription sites in vivo by performing
pulse-chase incorporation of BrUTP in both HeLa
and HeLa cells, the latter being a cell line stably express-
ing YFP-fibrillarin (FIB) as a marker for the DFC (Fig. 1
C; see Online supplemental material). The localization of
BrUTP-labeled RNA within the nucleolus was analyzed by
immunofluorescence, after a 6-min chase after removing
BrUTP from the culture medium (see Materials and meth-
ods). These data show an accumulation of labeled RNA at
the periphery of the foci containing YFP-RPA39 (i.e., FC),
within the boundary region between the FC and DFC, as de-
tected by YFP-FIB (Fig. 1 C). At longer chase times, after re-
moval of BrUTP, we observe the labeled RNA colocalizing
with the GC (within
20 min) and then appearing in the cy-
60 min; unpublished data). The appear-
ance of labeled RNA is inhibited by Actinomycin D at low
levels that inhibit selectively RNA polymerase I activity (un-
published data). These data are consistent with previous stud-
ies on the sites of rRNA transcription in nucleoli (Huang,
2002). In summary, we conclude that YFP-RPA39 is incor-
porated into active RNA polymerase I transcription com-
plexes and serves in vivo as a valid marker for detecting sites
within nucleoli containing active RNA polymerase I.
We next used the HeLa
ization of FP-RPA39 throughout an entire mitosis, using
both confocal and deconvolution fluorescence microscopy.
Time-lapse microscopy showed that RPA39 remained con-
centrated in chromatin-associated foci for most of mitosis,
consistent with previous data (Scheer and Rose, 1984; Gil-
bert et al., 1995). However, detailed time-lapse analysis
of single live cells consistently showed a window during
30 min, when RPA39 is no longer de-
tected in chromatin foci (Fig. 2, Metaphase, arrowheads;
Video 1, available at http://www.jcb.org/cgi/content/full/
jcb.200405013/DC1). Loss of RNA polymerase I subunits
from chromatin, specifically during metaphase, was also ob-
served by immunolocalization, using monoclonal antibodies
specific for the RPA20 subunit (see Online supplemental
material). Although RPA20 has been reported as a core sub-
unit of RNA polymerases I, II, and III, during interphase
the immunofluorescence signal of RPA20 detected using
this antibody predominantly colocalizes in bright foci within
nucleoli that contain RRN3 (Jones et al., 2000; see Online
supplemental material). An independent study has also
found that RNA polymerase I subunits RPA194, RPA39,
and RPA16, but not RPA43, are transiently lost from chro-
matin-associated foci during metaphase (Dundr, N., and T.
Misteli, personal communication). We suggest that RNA
cells to monitor the local-
with an anti-UBF antibody to denote the NORs and stained with DAPI to show the condensed chromosomes at different stages of mitosis.
Arrows indicate colocalization of YFP-RPA39 and UBF, whereas arrowheads indicate the presence of UBF but absence of YFP-RPA39. The
cells were also immunolabeled with an anti-RPA20 antibody, showing the exact same pattern as YFP-RPA39 (Fig. S1 D and not depicted,
available at http://www.jcb.org/cgi/content/full/jcb.200405013/DC1). Bars, 5 ?m.
The localization of UBF and RPA39 in different stages of mitosis. Unsynchronized HeLaYFP-RPA39 cells were fixed and immunolabeled
790 The Journal of Cell Biology
Volume 166, Number 6, 2004
diHcRed and followed through mitosis. The inserts indicate the change in punctate structure labeled by CFP-FIB from the middle nucleolus.
The top panels show the xy projections and the bottom panels show the xz projections of the fluorescence corresponding to FIB and RPA39
to indicate the loss of DFC and FC, respectively. (B) HeLa
mitosis. Arrowheads indicate the change in structure labeled by DsRed2-B23. (C) HeLa
diHcRed and followed through mitosis. For A–C, the time shown on the top of each panel indicates the time for the cell progressing through
mitosis and 00:00 indicates the time at which the nuclear intensity of IBB domain dropped to 50% of its initial value (midpoint). Bars, 5
(D) The mean fluorescence intensities of nucleolar components RPA39, FIB, B23, and RL27 within nucleoli were averaged, normalized and
plotted against time, where time equals 00:00 is the midpoint for IBB and
Disassembly of subnucleolar components when cells enter mitosis.
were transiently transfected with H2B-
were transiently transfected with DsRed2-B23 and followed through
were transiently transfected with H2B-
number of cells examined. The distribution of the original data
Nuclear dynamics in mitosis
Leung et al. 791
polymerase I either transiently leaves chromosomes during
metaphase, or else that multiple subunits dissociate tran-
siently from the polymerase ternary complex. However, im-
munolabeling with antibodies specific for the RNA poly-
merase I UBF, which binds to the rRNA gene repeats,
showed that UBF associated with chromatin foci through-
out mitosis (Fig. 2, UBF). Thus, although UBF colocalizes
in foci with RPA39 during prophase, prometaphase, ana-
phase, and telophase, it remains in similar foci throughout
metaphase when the RNA polymerase I subunits are no
longer detected (Fig. 2). The loss of RNA polymerase I sub-
units from chromatin foci is therefore not a detection prob-
lem at this stage of metaphase. In summary, the data indi-
cate that the previous view that RNA polymerase I remains
associated with chromatin throughout the entire period of
mitosis may need to be revised.
Disassembly of nucleolar subcompartments
To study nucleolar disassembly, we performed 4D imaging
on cell lines expressing combinations of three separate
marker proteins labeled with either CFP, YFP, HcRed or
DsRed (see Materials and methods). Cells coexpressing YFP-
RPA39 (FC), CFP-FIB (DFC), and HcRed histone H2B,
showed that RNA polymerase I was consistently lost from
FCs before the dissociation of FIB from the DFC in all cells
examined (Fig. 3 A). This is most clear in the xz side projec-
tions for the YFP-RPA39 and CFP-FIB expressing cells (Fig.
3 A, bottom). Interestingly, the punctate distribution of FIB
within the DFC changed to a more diffuse pattern, though
still confined to the DFC, coincident with the loss of RNA
polymerase I from nucleoli (Fig. 3 A, top, insets).
A similar 4D analysis was performed using cells coexpress-
ing YFP-RPA39 (FC), DsRed-nucleophosmin (B23;GC)
and CFP-H2B (Fig. 3 B). DsRed-B23 in the GC persists for
4–5 min after loss of RNA polymerase I from nucleoli. A
subtle change in the pattern of B23 distribution in the GC is
visible coincident with the loss of RNA polymerase I from
nucleoli (Fig. 3 B, arrowheads, compare
G2). Similar kinetics were observed using FP-tagged riboso-
mal protein L27 (RL27) as an alternative marker for the GC
(Fig. 3 D; not depicted). To compare the timing of loss of
markers from the DFC and GC subcompartments, a 4D
analysis was performed on cells coexpressing CFP-B23 (GC),
YFP-FIB (DFC), and HcRed-H2B (Fig. 3 C and Video 2,
available at http://www.jcb.org/cgi/content/full/jcb.200405013/
DC1). Both CFP-B23 and YFP-FIB leave the nucleolus at
approximately the same time. We conclude that nucleolar
disassembly is initiated at the FC, whereas concurrent loss of
both DFC and GC components occurs later.
01:54 with late
is reported in Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200405013/DC1). (E) Pairwise midpoint-midpoint comparison between
markers expressed within the same cell. For example, a negative value of
FIB reaches its midpoint 4.20 min later than RPA39, averaging from five experiments. A possible time line to summarize the relationship
between all the nucleolar markers examined and IBB is represented in the bottom panel. If the midpoint for IBB is defined at time equals 00:
00, then RPA, FIB, RL27, and B23 would be
02:04, 00:56, 01:20, and 01:20, respectively. Bar, 30 s. Note that although IBB is not present
in this experiment, the time line correlation is made according to a set of multiple experiments.
1.35 on the column FIB and the row RPA39 indicates that
HeLaYFP-LB1 cells were transiently transfected with DsRed2-B23 for 16 h before imaging through mitosis. The location of nucleoli was denoted
by DsRed2-B23 (green and in the bottom panel for xz projection) and condensing chromosomes by Hoechst 33234. The time shown on the
top of each panel indicates the time for the cell progressing through mitosis and 00:00 indicates the time at which the nuclear intensity of IBB
dropped to 50% of its initial value (midpoint). Bars, 5 ?m.
Comparison of nucleolar disassembly and the structural disintegration of NE upon entering into mitosis. (A) HeLaYFP-LBR and (B)
792 The Journal of Cell Biology
Volume 166, Number 6, 2004
Nuclear dynamics in mitosis
Leung et al. 793
To quantitate the pathway of nucleolar disassembly, we
measured the levels of FP-fluorescence in defined cellular
structures during mitotic progression (Fig. 3 D; see Online
supplemental material). Data obtained from 5 to 25 separate
experiments for each fluorescent marker show that the rate
of loss of RPA39 from nucleoli is slower than either RL27 or
B23, though comparable to FIB (Fig. 3 D). However, the
loss of RPA39 is initiated earlier than any of the other mark-
ers tested. A comparison of the times at which the levels of
fluorescence for each marker dropped to 50% of their initial
values in nucleoli indicates that loss of RPA39 precedes the
other markers by
4 min (Fig. 3, D and E). The loss of
RNA polymerase I subunits is therefore the earliest event we
have detected at the onset of nucleolar disassembly.
Comparison of nucleolar and nuclear envelope (NE)
We next compared the relative timing of nucleolar and NE
disassembly using stable cell lines expressing either YFP-
Lamin B receptor (HeLa
), or YFP-lamin B1 (HeLa
These are the first and last components, respectively, to disas-
semble from NE during mitosis (Beaudouin et al., 2002).
We performed 4D analyses using both these cell lines with
DsRed-B23 expressed transiently (Fig. 4). Nucleoli contain-
ing B23 are still detected when lamin B receptor (LBR) starts
to dissociate from NE (Fig. 4 A). This is most clear on the xz
projection (Fig. 4 A, bottom). Loss of B23 from nucleoli oc-
1.5 min after the decrease in LBR signal from NE
(Fig. 5 D). In contrast, analysis of HeLa
that the loss of B23 from nucleoli occurs
dissociation of lamin B1 (LB1) from NE (Fig. 4 B; Fig. 5 D).
Therefore, nucleolar disassembly occurs predominantly within
the window during which NE components dissociate.
Next, we compared the loss of NE function, as judged by
leakage of HcRed-importin
the cytoplasm, with the timing of loss of RPA39 (Fig. 5 A)
and FIB from nucleoli (Fig. 5 B). Cytoplasmic IBB is ac-
tively recruited to the nucleus by importin
of a GTP/GDP gradient maintained by an intact NE and
therefore is a good reporter of NE integrity. These experi-
ments were performed using HeLa cells stably expressing
both CFP-H2B and either YFP-RPA39 (FC), or YFP-FIB
(DFC), with HcRed-IBB transiently expressed in both cell
lines (Fig. 5, A and B). Loss of RPA39 from nucleoli pre-
cedes the loss of IBB from the nucleoplasm (Fig. 5 A, nu-
clear boundary shown by dotted yellow line). In contrast,
loss of IBB from the nucleus precedes the loss of FIB within
nucleoli (Fig. 5 B). Loss of NE function, as judged by
2 min before
binding (IBB) domain into
in the presence
leakage of IBB into cytoplasm, precedes NE breakdown
(Fig. S4, available at http://www.jcb.org/cgi/content/full/
jcb.200405013/DC1). These data indicate that loss of
RPA39 is one of the earliest nuclear events when cells enter
A quantitative analysis of the rates of loss of both nucle-
olar and NE markers showed that initiation of RPA39 disas-
sembly occurs before the loss of all other proteins tested
from either the nucleolus or NE (Fig. 5, C and D; see On-
line supplemental material). However, dissociation of the
GC and DFC nucleolar markers was only observed after the
functional integrity of the NE is lost, as judged by loss of
IBB from the nucleus. Note that the rate of loss of IBB is
faster than other markers. For example, although IBB begins
to decrease at least 5 min after loss of RPA39 is detected, it
has fallen to 50% of its initial value within 1–2 min and is
3 min before RPA39. IBB was
not assembled into a subnuclear body and may leave the nu-
cleus faster than proteins that must undergo disassembly
processes. This quantitative analysis demonstrates that com-
plete nucleolar breakdown occurs while a substantial frac-
tion of the nuclear lamina remains intact, but after the NE
no longer prevents loss of soluble proteins from the nucleus.
Reassembly of NORs
We next analyzed nuclear and nucleolar reassembly after mi-
tosis in cells stably expressing YFP-FIB, and CFP-H2B and
transiently expressing HcRed-IBB (Fig. 6). FIB is associated
with chromatin in the daughter nuclei at an early stage, be-
fore the re-import of IBB (Fig. 6 A). This chromatin associa-
tion is at least 5 min before the detection of any foci corre-
sponding to the reformation of nucleoli (Fig. 6 A and Fig. 7
A). RPA39 also associates early with daughter nuclei, before
the nuclear accumulation of IBB (Fig. 6 B). However,
RPA39 is immediately detected in chromatin-associated foci,
which only accumulate FIB
rowheads). Interestingly, although FIB is already present in
the nuclei, it only accumulates at the RPA39 foci when IBB
accumulates in the daughter nuclei (Fig. 6 B and C). At the
same time we observe a clear increase in the number of foci
containing both FIB and RPA39 (Fig. 6 B, 05:26–12:59).
This suggests that reestablishment of nuclear protein import
is important for stepwise reformation of nucleoli.
Quantitative measurements of these nuclear protein con-
centrations confirm that FIB associates with nuclei before ei-
ther RPA39 or IBB (Fig. 6 D; see Online supplemental
material). However, IBB accumulation in the nucleus is de-
tected 1–2 min earlier than RPA39 by this quantitative
5 min later (Fig. 6 C, foci, ar-
to the medium 30 min before the experiment to label the condensing chromosomes in live cells. The time shown on the top of each panel
indicates the time for the cell progressing through mitosis and 00:00 indicates the time at which the nuclear intensity of IBB dropped to 50%
of its initial value (midpoint). Bar, 5
m. (C) The mean fluorescence intensities of nucleolar components RPA39, FIB, and B23 within nucleoli
and that of NE components LBR and LB1 (left) and nuclear import substrate IBB (right) within nuclei were averaged, normalized, and plotted
against time, where time equals 00:00 is the midpoint for IBB and
reported in Fig. S2. (D) The time at which the nucleolar, NE and nuclear import markers dropped to 50% of their respective initial values
were compared and tabulated as in Fig. 3 E. A possible time line to summarize the relationship between nucleolar, nuclear import and NE
markers is proposed in the right panel. If the midpoint for IBB is defined at time equals 00:00, then LBR, B23, and LB1 would be at 00:23,
01:20, and 03:09, respectively. Bar, 30 s.
The timing of nucleolar subcompartment disassembly and the loss of functionality of NE.
cells were transiently transfected with diHcRed-IBB for 16 h before imaging through mitosis and Hoescht 33234 was added
number of cells examined. The distribution of the original data is
794 The Journal of Cell Biology
Volume 166, Number 6, 2004
Nuclear dynamics in mitosis
Leung et al. 795
analysis, even though by visual inspection of the correspond-
ing micrographs the concentrated RPA39 in nuclear foci is
more striking than the diffuse nuclear accumulation of IBB.
For comparison, we analyzed the rate of fluorescence in-
crease over time within reforming nucleoli for both RPA39
and FIB, as a fraction of total nuclear fluorescence (Fig. 6 E;
see Online supplemental material). This shows that FIB as-
sembles into nucleoli later than RPA39 (
50% accumulation), although it had accumulated earlier
within the total nuclear volume (Fig. 6, compare D with E).
Moreover, FIB is only incorporated into foci containing
RPA39 (i.e., the reforming NORs) when the increase in nu-
clear RPA39 level is maximal. This suggests that the recruit-
ment of FIB into the reforming nucleoli may require RNA
Interestingly, there is a close connection between the tim-
ing of FIB accumulation in the DFC and the time at which
a functional NE is reestablished. Similarly, during nucleolar
disassembly, loss of FIB from the DFC closely correlates
with the time at which the functional integrity of NE is
compromised (compare Fig. 5 with Fig. 6). This raises the
interesting possibility that one or more factors normally ex-
cluded from the nucleus by an intact NE can contribute to
signaling the dissociation of FIB from the nucleolus.
4 min later for
Reassembly of functional nucleoli
We define the reformation of functional nucleoli as requir-
ing the presence of all the major interphase markers for the
FC, DFC and GC subcompartments. To monitor this in
live cells we analyzed cell lines stably expressing both CFP-
B23 and YFP-FIB and transiently expressing DsRed-RL27
(Fig. 7). Together with the data from Fig. 6 showing the
stepwise assembly of FC and DFC, these data showed for
both the B23 and RL27 markers that the GC forms later
than either the FC or DFC, consistent with previous immu-
nofluorescence data (Dundr et al., 2000; Savino et al.,
2001). Quantitative analysis showed that the GC forms
18 min after the DFC and
pearance of the GC after mitosis correlates closely with the
time at which BrUTP-labeled RNA appears in the GC in in-
terphase pulse-chase experiments (unpublished data). Next,
we addressed quantitatively the concentration kinetics of nu-
cleolar factors in cells exiting mitosis (Fig. 7 B). These exper-
iments indicate that FIB accumulates before both GC mark-
ers B23 and RL27, which reassemble with similar initial
kinetics. In summary, these data support the view that the
reassembly of nucleoli is normally coupled to the activation
of ribosome subunit synthesis. The relationship between dif-
27 min after the FC. The ap-
ferent nucleolar subcompartment markers in terms of their
timing in nucleolar reassembly is summarized in Fig. 7 C.
To investigate spatial aspects of nucleolar formation, we
quantitated the number of separate DFC clusters over time
as cells exit mitosis (Fig. 7 D; see Online supplemental ma-
terial). We observed an increase in the mean number of
DFC clusters, up to
7 per nucleus, during the first 20 min
after mitosis (zero time defined as the time of chromosome
separation during anaphase; Fig. 7 D). However, briefly af-
ter the onset of GC formation, the mean number of DFC
clusters decreased on average to
between neighboring clusters (Fig. 7 D; Fig. 7 A, green ar-
rows). In the time-lapse micrographs it is apparent that the
GC markers (e.g., B23) fuse before the fusion of the DFC
marker FIB (Fig. 7 A, compare inset 31:20 with inset 38:
00). Therefore, the timing of the nucleolar fusion events
may be driven, at least in part, by the formation of the GC
around the DFCs. Intriguingly, we observed a statistically
significant variation in the number of FC foci and nucleoli
formed in the two daughter nuclei, although they are more
similar to each other than to unrelated nuclei exiting mitosis
(Fig. S3, available at http://www.jcb.org/cgi/content/full/
jcb.200405013/DC1). This raises the possibility that sto-
chastic events can influence nucleolar reassembly.
4.5, due to fusion events
Here, we used a quantitative 4D imaging approach to ana-
lyze the processes of nucleolar breakdown and reassembly
during mitosis in single live cells. HeLa cell lines were con-
structed that stably express either one or two FP-tagged
markers for either different nucleolar subcompartments, nu-
clear lamina components, nuclear transport reporters and/
or chromatin. By establishing double-transformed stable
cell lines and parallel transient transfection, we could per-
form multi-wavelength 3D microscopy over time to correlate
changes in the relative distributions and concentrations of
multiple nuclear marker proteins in the same live cell during
mitosis. The data for nucleolar disassembly and reassembly
during mitosis (Fig. 8) show that nucleolar breakdown be-
gins with the loss of RNA polymerase I subunits from FCs,
before the onset of NE breakdown. The subsequent disas-
sembly of the DFC and GC subcompartments coincides
with NE disassembly.
The RPA39 dissociates from the NORs during a brief pe-
riod within metaphase, although the rRNA promoter bind-
ing factor, UBF, remains bound (Fig. 2). The pathway of
nucleolar reformation in live cells after mitosis showed a
substrate marker IBB-diHcRed for 16 h before imaging. The inserts in B represent the distribution of RPA39 signal within reforming NORs.
Arrowheads indicate the development of the same FC foci over time. (C) HeLa
indicate the location of a reforming NOR over time. For A–C, the time shown on the top of each panel indicates the time for the cell progressing
through mitosis and 00:00 indicates the beginning of chromosome migration toward its respective pole. Bars, 5
intensities of nuclear import substrate IBB and nucleolar subcompartment marker RPA39 and FIB within the reforming nuclei were averaged,
normalized and plotted against time, where time equals 00:00 when the chromosomes start to migrate toward respective poles and
number of cells examined. (E) The total fluorescence intensities of nucleolar components RPA39 and FIB entering the reforming nucleoli as a
fraction of the total nuclear fluorescence were averaged, normalized, and plotted against time in the same manner as of panel D and the curve
for nuclear accumulation of IBB was used as a reference. The distribution of the original data is reported in Fig. S2.
The reassembly of NOR.
and (B) HeLa
cells were transiently transfected with nuclear import
cells were followed through mitosis. Arrowheads
m. (D) The mean fluorescence
796 The Journal of Cell Biology
Volume 166, Number 6, 2004
imaging. The time shown on the top of the panel indicates the time for the cell progressing through mitosis and 00:00 indicates the beginning
of chromosome migration toward its respective pole. Bar, 5
m. Pairs of arrows were used to follow two reforming NORs to form a single
nucleolus and the color changes from red to green to represent the fusion events. (B) The mean fluorescence intensities of the nucleolar com-
ponents FIB, B23 and RL27 within nucleoli were averaged, normalized and plotted over 5 (left) and 15 h (right) in the same manner as in Fig.
6 D. The distribution of the original data is reported in Fig. S2. (C) Collating data from Figs. 6 and 7, a possible time line to summarize the
relationship between different nucleolar subcompartments and nuclear import markers, is proposed. The top side indicates the time for different
The reassembly of functional nucleoli.
cells were transiently transfected with DsRed2-RL27 for 16 h before
Nuclear dynamics in mitosis
Leung et al. 797
strictly defined and reproducible temporal sequence of in-
corporation of markers in the order of FC, DFC, and finally
GC. However, this did not correlate with the order in which
the factors were imported into nuclei, as the DFC marker
FIB accumulated early in nuclei but only later was incorpo-
rated into nucleolar foci, coincident with the maximal nu-
clear import of RNA polymerase I.
The advantages of live cell imaging have recently been ap-
plied to study several dynamic nuclear processes (Clute and
Pines, 1999; Gerlich et al., 2001; Beaudouin et al., 2002;
Gerlich and Ellenberg, 2003a; Prasanth et al., 2003). A fea-
ture of the live cell approach used here is that we can conduct
quantitative studies on nucleolar dynamics during mitosis at
the single cell level. Moreover, we have directly correlated
quantitatively the temporal changes in each of the separate
nucleolar, NE and chromatin components within the nu-
cleus. This has allowed us to detect small differences in the
timing of events that would not be apparent by conventional
immunofluorescence approaches, where temporal informa-
tion is not available. Similarly, biochemical methods sample
the mean properties of cell populations, rather than the be-
havior or sequence of events in individual cells. For example,
we could reproducibly detect a timing difference as small as
4 min in the loss of RPA39 from the FC, before loss of
DFC or GC markers during nucleolar breakdown. Quantita-
tive analyses of these time-lapse data obtained from multiple
cells in each experiment also provided information regarding
the kinetic behavior of nuclear proteins across the cell popu-
lation and showed the degree of variation between nuclei.
Recent studies on the dynamics of nucleoli during mitosis
have focused mainly on the process of postmitotic nucleolar
reformation (Dundr et al., 2000; Savino et al., 2001). Two
previous studies have included the use of live cell imaging to
analyze events during the formation of nucleoli (Dundr et al.,
2000; Savino et al., 2001). Both studies used a combination
of immunofluorescence and cell lines expressing a single
GFP-tagged marker to analyze the temporal order of forma-
tion of PNBs and nucleoli. In particular, both the composi-
tion of PNBs, which accumulate partially processed rRNA
precursors and associated components, and how these com-
ponents are subsequently transferred into NORs for nucle-
olar assembly have been studied (Jimenez-Garcia et al., 1994;
Dundr et al., 2000; Savino et al., 2001). Here, we have fo-
cused on quantitating the temporal pathway of nucleologene-
sis as well as studying the spatial organization of reforming
NORs. In each of the live cells analyzed, multiple FP-tagged
markers used for different nucleolar subcompartments allows
us to distinguish reforming NORs and nucleoli from PNBs.
For example, PNBs do not contain RNA polymerase I, which
reassociates with the NOR early during nucleologenesis.
Thus, we could perform a correlative as well as quantitative
analysis of components from all three nucleolar subcompart-
ments in parallel. More importantly, we have extended these
analyses by comparing the processes of nucleolar breakdown
as well as reassembly in the same cells. A novel conclusion
from this work is that RNA polymerase I subunits RPA39
and RPA20 transiently leave the NORs during metaphase,
whereas UBF remains associated with NORs throughout mi-
tosis. Our findings are supported by the data from a recent
independent study showing that other RNA polymerase I
subunits, specifically, RPA194, RPA39, and RPA16, but not
RPA43, also leave the NORs during metaphase (Dundr, M.,
and T. Misteli, personal communication). These results differ
from the current view that RNA polymerase I, as well as
UBF, remains associated with NORs throughout mitosis,
based on immunofluorescence data using fixed cells, where it
is difficult to detect a transient absence of RNA polymerase I.
Our data in contrast indicate that multiple RNA polymerase
I subunits either leave the chromosomes transiently, or else
decrease in concentration below our detection limit, for a
brief period at metaphase. We note that this can explain the
previous observation based on high resolution in situ hybrid-
ization studies that metaphase chromosomes do not contain
nascent rRNA (Weisenberger and Scheer, 1995). Our data
are also consistent with the findings from run-on assays that
rRNA genes in metaphase chromosomes, whereas still in the
same open configuration as interphase chromosomes, are less
transcriptionally active (Conconi et al., 1989). Our present
data therefore indicate that the mitotic behavior of RNA
polymerase I may be more similar to RNA polymerase II
than was previously apparent.
We observe distinct kinetic behavior of individual proteins
during nucleolar disassembly (Fig. 3 D and Fig. 5 C). For ex-
ample, although dissociation of B23 and RL27 from nucleoli
initiates later than RPA39 or FIB, their dissociation rate is
higher. Both GC components dissociate from nucleoli at sim-
ilar rates, with comparable kinetics to cytoplasmic dispersal of
IBB upon NE breakdown, which is most likely a diffusion-
limited event. Although the molecular mechanism of nucle-
olar disassembly remains poorly understood, the present data
raise the possibility that distinct processes could operate se-
quentially and/or independently during the disassembly of
FC, DFC, and GC subcompartments. We observe that
RPA39 leaves the nucleolus before breakdown of nuclear
lamina components, whereas the DFC and GC markers are
lost during the period of nuclear lamina breakdown. It is
known from previous studies that lamina disassembly is trig-
gered via cyclin B-CDK1 mediated phosphorylation of mul-
tiple components, including LB1 (Pines and Rieder, 2001;
Burke and Ellenberg, 2002). Coincidently, the same cyclin
complex is involved in repression of mitotic ribosomal tran-
scription and nucleolar reformation. For example, phosphor-
nucleolar components to reassemble into nucleoli, whereas the bottom side indicates the time required for nucleolar markers RPA39 and FIB
as well as nuclear import markers IBB to enter nucleoli after mitosis. The time line begins at 00:00 when the chromosomes start to migrate
toward its respective pole. For nucleolar entry, RPA39, FIB, B23, and RL27 reach 50% of its saturated value at 14:42, 19:00, 53:00, and 60:00,
respectively. For nuclear import, FIB, IBB, and RPA39 reach 50% of its saturated value at 09:06, 11:18, and 13:48, respectively. Bar, 5 min.
(D) The number of clusters represented by the DFC marker FIB was counted and plotted against time, where time equals 00:00 when the
chromosomes start to migrate toward its respective pole and
the number of cells examined. An orange window was added in the same
time scale to denote the first appearance of GC within the reforming nucleoli. The distribution of the original data counting the number of
DFC clusters is reported in Fig. S3 A (available at http://www.jcb.org/cgi/content/full/jcb.200405013/DC1).
798 The Journal of Cell Biology
Volume 166, Number 6, 2004
ylation of UBF and transcription factor SL1 by CDK1 causes
shut-off of RNA polymerase I transcription (Heix et al.,
1998; Klein and Grummt, 1999). The CDK1 inhibitor ros-
covitine also causes reactivation of RNA polymerase I tran-
scription during mitosis but not the recruitment of rRNA
processing factors to the rRNA gene repeats (Sirri et al., 2000,
2002). Our data indicate that the loss of RNA polymerase I
and hence transcription of rRNA genes is likely to be the ini-
tial event during mitotic disassembly of nucleoli. However,
loss of rRNA gene transcription alone may not be sufficient
to cause subsequent disassembly of the entire nucleolus. For
example, although inhibition of ribosomal transcription dur-
ing interphase causes RNA polymerase I subunits to leave nu-
cleoli in vivo (unpublished data), the inhibition of ribosomal
transcription by Actinomycin D in isolated nucleoli does not
cause nucleoli to disintegrate in vitro (unpublished data).
Therefore, we propose that the mitotic disassembly of the
DFC and GC subcompartments is a result of an active mech-
anism rather than an indirect effect of the loss of transcrip-
tional activity. This is consistent with a recent study that
germ cell proteins FRGY2a and FRGY2b can reversibly disas-
semble somatic nucleoli in Xenopus egg cytoplasm indepen-
dent of rRNA transcription (Gonda et al., 2003), suggesting
that transcription activity and nucleolar integrity may not be
obligatorily coupled. It will thus be interesting in future to
test whether molecular mechanisms such as phosphorylation
During disassembly, (a and b) the RPA39 (blue) dissociates from the nucleoli ?2 min before the loss of functional integrity of the NE as
illustrated by the leakage of IBB (gray). (b and c) Within ?4 min, the DFC (yellow) and GC (magenta) subcompartments disintegrate, within
the period of NE breakdown and (c) subtle changes in the distribution of proteins localized to these two subcompartments are observed
(Fig. 3 C). (d) A brief period during metaphase follows, in which the RPA39 is not associated with the NORs, whereas the UBF remains
bound. (e) Before anaphase, the RNA polymerase I subunits reassociate with the UBF-containing NORs and at the same time FIB reassociates
with the chromosomes (shown by a change of color on the chromosome). At this stage PNBs (orange dots), which consists of partially pro-
cessed rRNA transcripts and associated processing factors, are formed in the nucleoplasm. (f) Within 5 min, FIB starts to reassociate with
the reforming NORs and the number of reforming NORs increases upon the activation of nuclear import. (g and h) Within 27 min after step e,
the GC reforms, coincident with the maximal number of DFC fusion events. (i) Variations in the number of nucleoli between daughter nuclei
imply that stochastic events can influence nucleolar reassembly.
The dynamics of nucleolus during mitosis. The nucleolar disassembly and reassembly pathway are shown in a–c and d–i, respectively.
Nuclear dynamics in mitosis | Leung et al. 799
by the cyclin B–CDK1 complex may play a role in either
RPA39 dissociation from FCs or in other steps in the nucle-
olar breakdown pathway.
In contrast with the precise temporal regulation in the or-
der of events during both disassembly and reformation of
nucleoli during mitosis, higher order aspects of nucleolo-
genesis appear to be less strictly controlled. A statistical
evaluation of the number of FC foci and functional nucle-
oli that appear after mitosis suggested that daughter nuclei
are more similar to each other than to unrelated nuclei exit-
ing mitosis elsewhere, consistent with the expected conser-
vation in global chromosome positioning (Gerlich and
Ellenberg, 2003b; Walter et al., 2003). Nonetheless, we ob-
serve statistically significant differences between the two
daughter nuclei. At least from the point at which RNA
polymerase I subunits reassociate with NORs, it appears
that local effects, which can differ between daughter nuclei,
influence the overall pathway of nucleologenesis. Effects
acting at the level of the local chromosome environment,
such as variations in chromosome orientation and decon-
densation (Thomson et al., 2004), variable activation of
rRNA genes within the repeat clusters and variation in pro-
tein concentration kinetics (Dundr et al., 2002a), may all
influence both the association of RNA polymerase I with
NORs and the probability of NOR fusion and hence the
number of active nucleoli formed. It is interesting to com-
pare this view with a recent FISH analysis on the process of
induced RNA polymerase II gene activation at the single
cell level (Levsky et al., 2002). Both this FISH analysis and
our work suggest that the pattern and level of gene activa-
tion varies at the single cell level, which had not been ap-
parent from previous biochemical studies from cell popula-
tions. Although it is increasingly appreciated that nuclear
structure, including the relative 3D distribution of chromo-
somes (Parada and Misteli, 2002; Bickmore and Chubb,
2003), can influence gene expression by RNA polymerase
II, we infer from this work that nuclear structure may also
have an important effect on events connected with RNA
polymerase I transcription.
Materials and methods
Cell culture, transfection, and establishment of stable cell line
HeLa cell lines were grown in DME supplemented with 10% FCS and 100
U/ml penicillin and streptomycin (Invitrogen). Single and double stably-
transformed cell lines were established using selection with G418 and/or
blasticidin, after transfection with ?2 ?g of each respective plasmid
construct (Table SI, available at http://www.jcb.org/cgi/content/full/jcb.
200405013/DC1) per 100-mm dish. 24 independent clones were isolated
for each cell line and three selected for analysis after further characteriza-
tion. A description of each cell line is provided in Table SII. Plasmid trans-
fection was performed using Effectene reagent (QIAGEN) according to the
To prepare the fixation buffer, 2? PHEM buffer (18.14 g Pipes, 6.5 g
Hepes, 0.99 g MgSO4, 3.8 g EGTA in 500 ml H2O, pH 7.0) were added to
freshly prepared 37% PFA in 1:5 ratio. The fixation buffer was then
warmed to 37?C and added directly to the cells slowly after pouring the
media out of the dish. The cells were fixed for 10 min before being washed
thrice with 1? PBS very gently. The cells were then permeabilized, stained
and mounted as previously published (Leung and Lamond, 2002). Primary
antibodies used were anti-UBF (1:5; Santa Cruz Biotechnology, Inc.) and
anti-RPA20 (1:1; B6-2; Jones et al., 2000).
Immunoprecipitations and in vitro transcription assays
50 ?g of HeLaYFP-RPA39 or HeLa nuclear extract were precleared for 30 min
with 5 ?l of protein G–Sepharose beads and used in immunoprecipitations
with 4 ?g of anti-GFP antibodies bound to 7.5 ?l of protein G–Sepharose
beads in 0.25 M KCl/TM10 (50 mM Tris–HCl, pH 7.9, 12.5 mM MgCl2, 1
mM EDTA, 10% glycerol, 1 mM sodium metabisulfite and 1 mM DTT)
buffer. The beads were incubated with nuclear extract for 1 h, with shak-
ing, at 4?C. After immunoprecipitation the beads were washed in TM10/
0.25 M KCl buffer, then equilibrated in TM10/0.05 M KCl buffer and used
in in vitro transcription assays. In vitro transcription reactions were per-
formed as described previously (Miller et al., 2001) at a final salt concen-
tration of 50–70 mM KCl. Supercoiled prHu3 plasmid DNA, which con-
tains the human rRNA gene promoter from ?515 to ?1548, were used as
templates in the transcription reaction. The resulting transcripts were ana-
lyzed in an S1 nuclease protection assay after annealing the RNA to a 5?-
end–labeled oligonucleotide, which was identical to the region between
?20 and ?40 of the promoter template strand.
Coverslips seeded with HeLa cells were rinsed with hypotonic KH buffer
(30 mM KCl, 10 mM Hepes, pH 7.4) briefly and incubated with 50 ?l KH
buffer containing 10 mM BrUTP (Sigma-Aldrich) for 5 min in a 5% CO2 in-
cubator at 37?C. The cells on coverslips were “chased” with DME contain-
ing 20% FCS and 200 ?g/ml (final concentration) G418 for a defined time
in the incubator to chase the transcripts before fixation. Before methanol
fixation for 20 min at ?20?C, the coverslips were rinsed with PBS. The
cells were then permeabilized with acetone for 30 s and air-dried for 10
min, followed by rehydration with PBS for 5 min and immunostaining us-
ing the anti-BrdU (1:5) antibody.
Mitotic studies of living cells
At each time point a 3D image stack spanning the entire nuclear volume
was recorded for each of the three wavelengths. Low light imaging condi-
tions were chosen to ensure that cells were able to progress through the en-
tire mitosis. For these experiments a series of “double stable” cell lines
were established that express combinations of two FP-tagged markers (Ta-
ble SII). The third marker in each experiment was expressed by transient
transfection (Table SI). Cells were seeded in a Labtek II chambered cover-
glasses on the previous night before imaging and an imaging medium con-
taining 20% FCS and 0.5 mg/ml L-ascorbic acid replaced the growth
medium for 3–4 h before imaging. 4D imaging was performed on a cus-
tomized confocal laser scanning fluorescence microscope (model LSM510;
Carl Zeiss MicroImaging, Inc.) kept at 37?C and equipped with a z-scan-
ning stage (HRZ 200) for fast 4D acquisition using a Plan Apochromat 63?
DIC oil immersion objective. Triple-color imaging of CFP, YFP and DsRed/
HcRed was achieved by alternating the 413 nm Kr, 514 nm Ar, and 543
nm HeNe laser for selective excitation. Bidirectional scanning and detec-
tion were performed as published previously (Gerlich et al., 2001).
Quantitation and statistical analysis
For object identification, a reference channel was chosen that represented
the structure of interest. For example, FP-H2B and FP-RPA39 are used to
define the nuclear volume and reforming NORs, respectively. The images
in this channel were processed by an anisotropic diffusion filtering and
subsequent thresholding (Gerlich et al., 2001) to obtain a binary mask rep-
resenting areas of interest. Mean fluorescence intensities were then mea-
sured in all channels of the original unfiltered images within these areas.
Automated analysis of 4D data was achieved by implementation of a com-
puter macro, which was executed in the image processing toolbox Heu-
risko 4.05 (Aeon). Importantly, a single threshold was selected for the anal-
ysis of entire 4D images. Although the specific threshold chosen by the
user affects absolute values, the kinetics of protein concentrations mea-
sured during mitotic progression were largely unaffected (Gerlich et al.,
2001). The data collected were then normalized and analyzed using
GraphPad Prism 4.0 software.
Online supplemental material
Fig. S1 includes (A) representative Western blots showing expression levels
of FP-fusion proteins in cell lines used, (B) FP-RPA39 localization at EM
level, the localization of (C) pulsed labeled rRNA transcripts and (D)
RPA20 in HeLaYFP-RPA39 cells. Distributions of quantitative measurements in
Figs. 3, 5–7 are shown in Fig. S2. The statistical analysis for the differential
behavior of daughter cells after mitosis is shown in Fig. S3. Fig. S4 shows
that the loss of functionality of the NE precedes its structural disintegration.
The plasmid and stable cell lines used in this work are tabulated in Table
800 The Journal of Cell Biology | Volume 166, Number 6, 2004
S1. The localization of RPA39, FIB and B23 are shown in Videos 1 and 2.
Online supplemental material is available at http://www.jcb.org/cgi/
We thank Drs. H. Kimura and P.R. Cook (University of Oxford, Oxford,
UK) for the gift of mAbs B6-2, Mr. G. Rabut (EMBL) for providing macros to
ensure fast acquisition after multi-point visit and multi-wavelength time se-
ries scanning as well as autofocussing during imaging of multi-point time
series, and Ms. E. Zanin (EMBL) for making the IBB-FP fusion construct.
A.K.L. Leung was supported by a Croucher Foundation Scholarship, an
EMBL Advanced Light Microscopy Fellowship and a Company of Biologists
Ltd. travelling Fellowship. D. Gerlich is supported by an EMBO long-term
fellowship. G. Miller was a Wellcome Trust Prize Student. A.I. Lamond is a
Wellcome Trust Principal Research Fellow. J. Zomerdijk is a Wellcome
Trust Senior Research Fellow. The Human Frontier Science Program is ac-
knowledged for a research grant entitled “Functional organization of the
cell nucleus investigated through proteomics and molecular dynamics”.
Submitted: 4 May 2004
Accepted: 23 July 2004
Beaudouin, J., D. Gerlich, N. Daigle, R. Eils, and J. Ellenberg. 2002. Nuclear en-
velope breakdown proceeds by microtubule-induced tearing of the lamina.
Bickmore, W.A., and J.R. Chubb. 2003. Dispatch. Chromosome position: now,
where was I? Curr. Biol. 13:R357–R359.
Burke, B., and J. Ellenberg. 2002. Remodelling the walls of the nucleus. Nat. Rev.
Mol. Cell Biol. 3:487–497.
Carmo-Fonseca, M., L. Mendes-Soares, and I. Campos. 2000. To be or not to be
in the nucleolus. Nat. Cell Biol. 2:E107–E112.
Clute, P., and J. Pines. 1999. Temporal and spatial control of cyclin B1 destruction
in metaphase. Nat. Cell Biol. 1:82–87.
Conconi, A., R.M. Widmer, T. Koller, and J.M. Sogo. 1989. Two different chro-
matin structures coexist in ribosomal RNA genes throughout the cell cycle.
Dundr, M., T. Misteli, and M.O. Olson. 2000. The dynamics of postmitotic reas-
sembly of the nucleolus. J. Cell Biol. 150:433–446.
Dundr, M., U. Hoffmann-Rohrer, Q. Hu, I. Grummt, L.I. Rothblum, R.D. Phair,
and T. Misteli. 2002a. A kinetic framework for a mammalian RNA poly-
merase in vivo. Science. 298:1623–1626.
Dundr, M., J.G. McNally, J. Cohen, and T. Misteli. 2002b. Quantitation of GFP-
fusion proteins in single living cells. J. Struct. Biol. 140:92–99.
Gerbi, S.A., A.V. Borovjagin, and T.S. Lange. 2003. The nucleolus: a site of ribo-
nucleoprotein maturation. Curr. Opin. Cell Biol. 15:318–325.
Gerlich, D., and J. Ellenberg. 2003a. 4D imaging to assay complex dynamics in
live specimens. Nat. Cell Biol. 5:S14–S19.
Gerlich, D., and J. Ellenberg. 2003b. Dynamics of chromosome positioning dur-
ing the cell cycle. Curr. Opin. Cell Biol. 15:664–671.
Gerlich, D., J. Beaudouin, M. Gebhard, J. Ellenberg, and R. Eils. 2001. Four-
dimensional imaging and quantitative reconstruction to analyse complex
spatiotemporal processes in live cells. Nat. Cell Biol. 3:852–855.
Gilbert, N., L. Lucas, C. Klein, M. Menager, N. Bonnet, and D. Ploton. 1995.
Three-dimensional co-location of RNA polymerase I and DNA during in-
terphase and mitosis by confocal microscopy. J. Cell Sci. 108:115–125.
Gonda, K., J. Fowler, N. Katoku-Kikyo, J. Haroldson, J. Wudel, and N. Kikyo.
2003. Reversible disassembly of somatic nucleoli by the germ cell proteins
FRGY2a and FRGY2b. Nat. Cell Biol. 5:205–210.
Heix, J., A. Vente, R. Voit, A. Budde, T.M. Michaelidis, and I. Grummt. 1998.
Mitotic silencing of human rRNA synthesis: inactivation of the promoter se-
lectivity factor SL1 by cdc2/cyclin B-mediated phosphorylation. EMBO J.
Hernandez-Verdun, D., P. Roussel, and J. Gebrane-Younes. 2002. Emerging con-
cepts of nucleolar assembly. J. Cell Sci. 115:2265–2270.
Huang, S. 2002. Building an efficient factory: where is pre-rRNA synthesized in
the nucleolus? J. Cell Biol. 157:739–741.
Janicki, S.M., and D.L. Spector. 2003. Nuclear choreography: interpretations from
living cells. Curr. Opin. Cell Biol. 15:149–157.
Jimenez-Garcia, L.F., M.L. Segura-Valdez, R.L. Ochs, L.I. Rothblum, R. Hannan,
and D.L. Spector. 1994. Nucleologenesis: U3 snRNA-containing prenucle-
olar bodies move to sites of active pre-rRNA transcription after mitosis. Mol.
Biol. Cell. 5:955–966.
Jones, E., H. Kimura, M. Vigneron, Z. Wang, R.G. Roeder, and P.R. Cook. 2000.
Isolation and characterization of monoclonal antibodies directed against sub-
units of human RNA polymerases I, II and III. Exp. Cell Res. 254:163–172.
Jordan, P., M. Mannervik, L. Tora, and M. Carmo-Fonseca. 1996. In vivo evi-
dence that TATA-binding protein/SL1 colocalizes with UBF and RNA
polymerase I when rRNA synthesis is either active or inactive. J. Cell Biol.
Klein, J., and I. Grummt. 1999. Cell cycle-dependent regulation of RNA polymer-
ase I transcription: the nucleolar transcription factor UBF is inactive in mi-
tosis and early G1. Proc. Natl. Acad. Sci. USA. 96:6096–6101.
Leung, A.K., and A.I. Lamond. 2002. In vivo analysis of NHPX reveals a novel nu-
cleolar localization pathway involving a transient accumulation in splicing
speckles. J. Cell Biol. 157:615–629.
Leung, A.K., and A.I. Lamond. 2003. The dynamics of the nucleolus. Crit. Rev.
Eukaryot. Gene Expr. 13:39–54.
Levsky, J.M., S.M. Shenoy, R.C. Pezo, and R.H. Singer. 2002. Single-cell gene ex-
pression profiling. Science. 297:836–840.
Miller, G., K.I. Panov, J.K. Friedrich, L. Trinkle-Mulcahy, A.I. Lamond, and J.C.
Zomerdijk. 2001. hRRN3 is essential in the SL1-mediated recruitment of
RNA Polymerase I to rRNA gene promoters. EMBO J. 20:1373–1382.
Olson, M.O., M. Dundr, and A. Szebeni. 2000. The nucleolus: an old factory with
unexpected capabilities. Trends Cell Biol. 10:189–196.
Parada, L., and T. Misteli. 2002. Chromosome positioning in the interphase nu-
cleus. Trends Cell Biol. 12:425–432.
Pines, J., and C.L. Rieder. 2001. Re-staging mitosis: a contemporary view of mi-
totic progression. Nat. Cell Biol. 3:E3–E6.
Prasanth, K.V., P.A. Sacco-Bubulya, S.G. Prasanth, and D.L. Spector. 2003. Se-
quential entry of components of the gene expression machinery into daugh-
ter nuclei. Mol. Biol. Cell. 14:1043–1057.
Roussel, P., C. Andre, C. Masson, G. Geraud, and D. Hernandez-Verdun. 1993.
Localization of the RNA polymerase I transcription factor hUBF during the
cell cycle. J. Cell Sci. 104:327–337.
Rubbi, C.P., and J. Milner. 2003. Disruption of the nucleolus mediates stabiliza-
tion of p53 in response to DNA damage and other stresses. EMBO J. 22:
Savino, T.M., J. Gebrane-Younes, J. De Mey, J.B. Sibarita, and D. Hernandez-
Verdun. 2001. Nucleolar assembly of the rRNA processing machinery in liv-
ing cells. J. Cell Biol. 153:1097–1110.
Scheer, U., and K.M. Rose. 1984. Localization of RNA polymerase I in interphase
cells and mitotic chromosomes by light and electron microscopic immuno-
cytochemistry. Proc. Natl. Acad. Sci. USA. 81:1431–1435.
Sirri, V., P. Roussel, and D. Hernandez-Verdun. 2000. In vivo release of mitotic si-
lencing of ribosomal gene transcription does not give rise to precursor ribo-
somal RNA processing. J. Cell Biol. 148:259–270.
Sirri, V., D. Hernandez-Verdun, and P. Roussel. 2002. Cyclin-dependent ki-
nases govern formation and maintenance of the nucleolus. J. Cell Biol.
Thomson, I., S. Gilchrist, W.A. Bickmore, and J.R. Chubb. 2004. The radial posi-
tioning of chromatin is not inherited through mitosis but is established de
novo in early G1. Curr. Biol. 14:166–172.
Walter, J., L. Schermelleh, M. Cremer, S. Tashiro, and T. Cremer. 2003. Chromo-
some order in HeLa cells changes during mitosis and early G1, but is stably
maintained during subsequent interphase stages. J. Cell Biol. 160:685–697.
Weisenberger, D., and U. Scheer. 1995. A possible mechanism for the inhibition of
ribosomal RNA gene transcription during mitosis. J. Cell Biol. 129:561–575.