Molecular Biology of the Cell
Vol. 16, 2395–2413, May 2005
Dynamic Sorting of Nuclear Components into Distinct
Nucleolar Caps during Transcriptional Inhibition
Yaron Shav-Tal,*†Janna Blechman,* Xavier Darzacq,†Cristina Montagna,‡
Billy T. Dye,§?James G. Patton,§Robert H. Singer,†and Dov Zipori*
*Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100 Israel;
Departments of†Anatomy and Structural Biology and Cell Biology, and‡Molecular Genetics, Albert Einstein
College of Medicine, Bronx, NY 10461; and§Department of Molecular Biology, Vanderbilt University,
Nashville, TN 37235
Submitted November 16, 2004; Revised February 16, 2005; Accepted February 21, 2005
Monitoring Editor: Joseph Gall
Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be
mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and
the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been
proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of
nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during
transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to
nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either
nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping
of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at
least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as
determined using photobleaching and require energy for their formation. These findings demonstrate that the process of
nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and empha-
size the dynamic characteristics of nuclear domain formation in response to cellular stress.
The nucleus is a dynamic organelle consisting of interacting
chromosomal and protein compartments. One of the major
pathways of nuclear translocation is the movement of pre-
ribosomal particles from the nucleolus into the cytoplasm
for the assembly of functional ribosomes. The main nucleo-
lar functions involve RNA polymerase (pol) I transcription,
posttranscriptional maturation of pre-rRNA transcripts and
their subsequent assembly with ribosomal proteins into pre-
ribosomal particles. Other functions have been attributed to
the nucleolus (for reviews, see Carmo-Fonseca et al., 2000;
Olson, 2004b) and include the processing of RNA pol III
transcripts, RNA editing, sequestration of cell cycle compo-
nents in yeast, and Mdm2 protein in mammalian cells. The
localization of telomere proteins and telomerase RNA in
nucleoli suggests a role for the nucleolus in aging.
Nucleolar components are found in all cells and tissues
although the size, shape, and number of nucleoli may
change depending on the species, cell type, and functional
state. Transmission electron microscopy (TEM) has revealed
three major structures within nucleoli: fibrillar centers (FC),
dense fibrillar components (DFC), and the granular compo-
nent (GC; for reviews, see Busch and Smetana, 1970; Goes-
sens, 1984; Shaw and Jordan, 1995; Scheer and Hock, 1999).
rDNA transcription units are found in the FC and consist of
tandem repeats of these genes. rRNAs are harbored within
the DFC and are processed there. It is therefore thought that
rRNA transcription occurs at the interface between the FC
and the DFC. Later stages of rRNA processing take place in
the GC. Thus, the processing of rRNA is spatially arranged
in accordance to the ultrastructure of these compartments.
Great variability is found between nucleoli of cells ob-
served at different stages of cellular metabolic activity. In
quiescent cells or cells subjected to transcriptional arrest a
phenotype of nucleolar segregation is observed, in which the
fibrillar and granular zones disengage to form separate but
juxtaposed structures (Smetana and Busch, 1974; Vera et al.,
1993; Malatesta et al., 2000). In some cases, for example in
developing Xenopus oocytes (Van Gansen and Schram,
1972), these structures resemble cap-like formations situated
on the outer part of the segregated nucleolus. Although the
processes of nucleolar segregation and nucleolar capping are
physiological occurrences assumed to reflect the inhibition
of RNA synthesis, they have not been pursued and have
only been structurally characterized, mostly by TEM, using
agents that induce transcriptional inhibition (for reviews,
see Bernhard and Granboulan, 1968; Busch and Smetana,
1970; Simard et al., 1974; Smetana and Busch, 1974). Based on
differences in phase contrast light microscopy, the formation
of two types of “nucleolar caps” was observed during tran-
scriptional arrest by inhibitors such as actinomycin D (ActD;
This article was published online ahead of print in MBC in Press
on March 9, 2005.
?Present address: Institute for Molecular Biology, University of
Wisconsin-Madison, Madison, WI 53706.
Address correspondence to: Yaron Shav-Tal (firstname.lastname@example.org)
(starting August 2005: email@example.com).
Abbreviations used: ActD, actinomycin D.
© 2005 by The American Society for Cell Biology2395
Journey and Goldstein, 1961; Reynolds et al., 1963, 1964).
Multiple “dark nucleolar caps” (DNCs) had a concave base
and appeared to be pressed onto the surface of the nucleolar
body, thus forming an interface between the two. The less
frequent “light nucleolar caps” (LNCs) had a convex appear-
ance without a clear margin between them and the nucleolar
body, therefore seeming closely attached or protruding
slightly into the nucleolar body. Time-lapse microscopy
showed that this cap originated from the center of the nu-
cleolus. Independently, Schoefl observed similar structures:
RNP granules embedded in a protein matrix and a fibrillar
RNP component (Schoefl, 1964). Another study called the
granular structures the P2fraction, forming on the surface of
the nucleolar body termed P1and separate from other
smaller caps he termed the “fibrillar substance” (Recher et
These studies have led to the general assumption that
nucleolar caps consist of nucleolar proteins originating from
the disintegrating nucleolus. However, the static view of the
nucleolus in 1960s and 70s has since been replaced by our
knowledge that the nucleolus is a dynamic structure that has
the ability to disassemble and reassemble (for review see
Hernandez-Verdun, 2004). We have previously shown how
a nucleoplasmic protein, normally excluded from the nucle-
olus, is highly enriched in the nucleolar region (Shav-Tal et
al., 2001b). Another study, has shown by use of proteomics
that a number of proteins are enriched in the nucleolar
fraction during transcriptional arrest induced by ActD
(Andersen et al., 2002). The purpose of the present study was
to determine which nucleoplasmic proteins can be directed
to the nucleolar caps and whether they belong to a specific
family of proteins. The analysis of more than 70 endogenous
nuclear proteins allows us to assemble a comprehensive
picture of nuclear compartments during transcriptional ar-
rest. By characterizing the different types of nucleolar caps
and identifying their protein and RNA composition, we
show that nucleolar segregation is a concerted process in-
volving specific spatial reshuffling of nucleoplasmic and
nucleolar components in response to transcriptional arrest.
We find that nucleolar caps are dynamic structures con-
stantly exchanging with the nucleoplasm and that their for-
mation is an energy-dependent process.
MATERIALS AND METHODS
For indirect immunofluorescence and in situ hybridization, HeLa cells were
grown on glass coverslips in DMEM supplemented with 10% fetal calf serum
(FCS). Human U2OS osteosarcoma cells were cultured in low-glucose DMEM
(Invitrogen, Carlsbad, CA) with 10% FCS and for live cell experiments were
maintained in phenol-red free Leibovitz’s L15 medium at 37°C using a tem-
perature-controlled Delta T4 culture dish system with a heated lid and an
objective heater (Bioptechs, Butler, PA). For transcriptional inhibition of RNA
polymerase I and II 5 ?g/ml ActD (Sigma, St. Louis, MO) were added to cells
for 2 h (Ochs, 1998). ActD at 0.01 ?g/ml for 3–6 h was used for inhibition of
RNA pol I only and 5,6-dicholoro-?-d-ribofuranosylbenzimidazole (DRB) at
25 ?g/ml for 2 h or ?-amanitin at 30 ?g/ml for 6 h was used inhibition of pol
II only. 2,3-butanedione monoxime (BDM, 20 mM) was used for myosin I
inhibition. For full metabolic depletion, 6 mM 2-deoxyglucose (Sigma) and 10
mM Na-azide were added to cell cultures in either Hanks’ balanced salt
solution (HBSS) or DMEM. Glucose, 25 mM, or 2 mM pyruvate were added
to HBSS as indicated.
Protein Extraction and Western Blotting
HeLa cells were lysed on ice in 50 mM Tris-HCl, pH 8, containing 1% Nonidet
P-40 (NP-40), 150 mM NaCl, 5 mM EDTA, 1 mM phenyl methylsulfonyl
fluoride, 3 ?g/ml aprotinin, 20 ?g/ml leupeptin, 10 mM iodoactetate, 1 mM
sodium orthovanadate, 10 mM sodium fluoride. Protein extracts were boiled
in SDS-sample buffer (5% glycerol, 2% SDS, 62.5 mM Tris-HCl, pH 6.8, 2%
2-mercaptoethanol, 0.01% bromophenol blue) and analyzed by SDS-PAGE.
Immunoblot analysis of cell lysates (40 ?g/lane) was performed using the
anti-PSF B92 monoclonal antibody (mAb). Specific binding was detected with
goat anti-mouse horseradish peroxidase (HRP)-coupled antibody (Sigma) and
enhanced chemiluminescence (ECL) reagents.
Primary Antibodies to Nuclear Proteins
The primary antibodies to nuclear proteins are shown in Tables 1 and 2.
Fluorophore-labeled secondary antibody purchased from Jackson Immu-
noResearch (West Grove, PA) were as follows: for double labelings with
primary mouse and rabbit antibodies: fluorescein (FITC)-conjugated donkey
anti-rabbit F(ab?)2, Cy3-conjugated goat anti-mouse IgG; for triple labeling:
Cy5-conjugated goat anti-rabbit F(ab?)2; other antibodies used for verification
of stainings: R-phycoerythrin (PE)-conjugated donkey anti-rabbit F(ab?)2,
FITC-conjugated donkey anti-mouse IgG, Alexa 488 donkey anti-goat IgG
(Molecular Probes, Eugene, OR). For other double labelings: FITC-conjugated
mouse anti-rat IgG, Texas Red–conjugated goat anti-human IgG and Cy3-
conjugated anti-mouse IgM (Jackson ImmunoResearch), FITC-conjugated an-
ti-guinea pig IgG (B. Geiger, Weizmann Institute), FITC-conjugated anti-
mouse IgM (Serotec, Oxford, United Kingdom).
Immunofluorescence and In Situ Hybridization
HeLa cells were fixed for 2 min in 4% paraformaldehyde with 0.5% Triton
X-100 and for an additional 20 min in 4% paraformaldehyde. After washing
and blocking in 5% bovine serum albumin (BSA), cells were stained with the
indicated antibodies for 45 min, washed twice and then incubated with the
appropriate secondary antibodies for 45 min, and counterstained with
Hoechst for DNA labeling. Before all confocal double-labeling experiments
each antibody was first checked on its own by immunofluorescent micros-
copy. Immunofluorescence was viewed and analyzed using a Bio-Rad con-
focal microscope (Bio-Rad, Richmond, CA) or a Zeiss Axioplan microscope
equipped with SPOT-II (Diagnostic Instruments, Sterling Heights, MI) cooled
For in situ hybridization the following probes were synthesized, labeled
with either Cy3 or Cy5, and hybridized as previously described (Chartrand et
al., 2000): U3: CGCTACCTCTCTTCCTCGTGGTTTTCGGTG-CTCTACACGT-
TCATCAATGGCTGAC (two probes); U2: AGTGGACGGAGCAAGCTC-
CTATTCCATCTCCCTGCTCCAAAAATCCATTT; U6: CGTGTCATCCTT-
GCGCAGGGGCCATGCTAATCTTCTCTGTATCGTTCCAA; U93: CATGC-
GCACGAAC; RNAseP: ATTGAACTCACTTCGCTGGCCGTGAGTCTG-
TTCCAAGCTCCGGCAAAGGA; 5? ETS: CTCTCAGATCGCTAGAGAAG-
CTCATTTGGATGTGTCTGGAGTCTTGGAAGCTTGACT; and E3: CTG-
For poly(A) mRNA detection an oligo(dT) probe was used. After hybrid-
ization, immunofluorescence was performed from the blocking step as above.
For chromosomal DNA FISH, U2OS cells were seeded at low density in
two-well chamber slides (Nunc, Napierville, IL) and grown over night. One
well was treated with ActD for 2 h. Cells were fixed in ice cold methanol for
10 min. Chromosome painting probes were generated by degenerate oligo-
nucleotide primed PCR amplification (DOP-PCR) of flow-sorted chromo-
somes (University of Cambridge, United Kingdom) with direct incorporation
of Spectrum Orange-dUTP (Vysis, Abbot Laboratories, Abbott Park, IL).
Detailed protocols for probes generation and FISH are available at (http://
www.riedlab.nci.nih.gov). Slides were denatured in formaldehyde/SSC for
1.5 min and hybridized overnight at 37°C in a moist chamber.
Images were acquired with an Olympus BX61 epifluorescence microscope
(Olympus America, Melville, NY) and a Roper Scientific CoolSNAP HQ
camera (Roper Scientific, Tucson, AZ).
For studies in fixed cells, HeLa cells were transfected with GFP-PSF constructs
(Dye and Patton, 2001) or GFP-p54nrb(Peng et al., 2002) or GFP-PML (A.
Ben-Ze’ev, Weizmann Institute; Shtutman et al., 2002) or YFP-ASF/SF2 (Bubu-
lya et al., 2004) using either the calcium phosphate transfection method or
Fugene. Cells were fixed and processed for immunofluorescence after over-
night transfection. For live cell imaging, U2OS cells were electroporated with
the following constructs: GFP-fibrillarin, GFP-Nopp140 (T. Meier, AECOM;
Dundr et al., 2004), GFP-p14(ARF) (G. Peters, Cancer Research UK, London;
Llanos et al., 2001). For GFP-TLS, the TLS sequence was amplified by PCR
from pBS-TLS and subcloned into the SacI-BamHI sites of pEGFP-C3 (Clon-
Y. Shav-Tal et al.
Molecular Biology of the Cell 2396
tech, Palo Alto, CA). Stable GFP-PSF U2OS cells were generated by electro-
poration and selected under G418.
Fluorescent Recovery after Photobleaching
Transfected U2OS cells, with or without ActD treatment, were imaged at 37°C
with a Leica TCS SP2 AOBS laser scanning confocal microscope equipped
with a 63?, 1.4 NA objective (Leica Microsystems, Exton, PA) and scanned
using a 488-nm laser for the detection of GFP at low laser powers (?1.5%) to
avoid bleaching and cytotoxicity. For photobleaching, one scan at 100% of the
laser was required using the Leica fluorescence recovery after photobleaching
(FRAP) module. Specific scanning times and intervals between images were
used for each GFP-tagged protein depending on the measured recovery times.
Measurements of intensity were performed using the Leica software. For each
time point, the background taken from a ROI outside of the cell was sub-
tracted from all other measurements. T(t) and I(t) were measured for each
time point as the average intensity of the nucleus and the average intensity in
the bleached ROI, respectively. One image was collected before bleaching and
these initial conditions are referred to as Ti? nuclear intensity and Ii?
intensity in ROI before bleaching. Ic(t) is the corrected intensity of the
bleached ROI at time t (Phair and Misteli, 2000):
Transmission Electron Microscopy
HeLa cells were fixed with 4% paraformaldehyde, 0.1% glutaraldehyde in 0.1
M sodium cacodylate buffer, dehydrated through a graded series of ethanol
using a progressively lowering of temperature technique, embedded in Lowi-
cryl HM20 resin, and polymerized with UV light at ?20°C. Ultrathin sections
were cut on a Reichert Ultracut UCT and viewed on a JEOL 1200EX trans-
mission electron microscope at 80 kV. For immunogold labeling, thin sections
mounted on grids were incubated with blocking solution with the addition of
5% BSA and 1% cold water fish gelatin, followed by primary antibody and
secondary antibody conjugated to 10 nm gold (Aurion, Wageningen, The
PSF and GFP-PSF Colocalize within ActD-induced
We have previously found that a nucleoplasmic protein, PSF
(PTB-associated splicing factor; Shav-Tal and Zipori, 2002)
formed peri-nucleolar structures in HL-60 cells treated with
transcriptional inhibitors ActD and DRB (Shav-Tal et al.,
2001b). This phenomenon occurred also in GFP-PSF trans-
fected HeLa cells (Dye and Patton, 2001). This finding is of
interest because nucleolar segregation is thought to involve
separation of nucleolar components rather than addition of
nucleoplasmic components. We were therefore interested in
defining the conditions that caused PSF to relocalize to the
nucleolus and the structures to which it was targeted. PSF in
the nucleoplasm exhibited both diffuse and punctate stain-
ing that was excluded from the nucleolus (Figure 1A). In
addition, several brighter foci were observed (Shav-Tal et al.,
2000). During ActD treatment of HeLa cells, both monoclo-
nal (B92) and polyclonal (1121) anti-PSF antibodies identi-
fied the PSF protein in 2–3 concave cap-like structures sur-
rounding the nucleolus (Figure 1A). There was a consequent
reduction in nucleoplasmic PSF and the bright foci. In addi-
tion, the nucleolar caps were surrounded by a dense chro-
matin ring. Concentration of PSF in nucleolar caps was
observed during inhibition of RNA polymerases I and II
using concentrations of ActD reported to result in fully
segregated nucleoli (Ochs, 1998). Similarly, nucleolar relo-
calization of PSF was seen when the RNA pol II specific
inhibitors DRB or ?-amanitin were used, but not when only
Table 1. Antibodies to components of nucleolar caps
Antigen in nucleolar capsAntibodyClone Antibody provided by
P4 E. Canaani, Weizmann Institute, Israel
D. Ginsberg, Weizmann Institute, Israel
A. Lamond, University of Dundee, UK
Y. Takagaki, University of Virginia School of Medicine
O. Delattre, Curie Institute, France
I. Raska, Academy of Sciences of the Czech Republic, Czech Republic
W. Filipowicz, Friedrich Miescher Institute for Biomedical Research,
D. Black, University of California
K. Bomsztyk, University of Washington
S. Baserga, Yale University School of Medicine
T. Meier, Albert Einstein College of Medicine
C. Adamson, University of Arizona
J. Harper, Baylor College of Medicine
P. Tucker, University of Texas at Austin
F. Fuller-Pace, University of Dundee, UK
R. Van Driel, University of Amsterdam, The Netherlands
A. Ben-Ze’ev, Weizmann Institute, Israel
NeoMarkers, Fremont, CA
Lee et al. (1996)
Shav-Tal et al. (2000)
A. Lamond, University of Dundee, UK
H. Will, Heinrich-Pette Institute for Experimental Virology and
Immunology at the University of Hamburg, Germany
Transduction Labs, Lexington, KY
Santa Cruz Biotechnology, Santa Cruz, CA
D. Ron, NYU School of Medicine
R. Goodman, Vollum Institute, Portland
R. Lührmann and E. Makarov, Max-Planck Institute for Biophysical
A. Rosen, John Hopkins School of Medicine
E. Chan, Scripps Research Institute
hnRNP F and H
Nucleolar Structure after ActD
Vol. 16, May 20052397
RNA pol I activity was inhibited by low concentrations of
ActD (unpublished data). We therefore focused on condi-
tions sufficient to inhibit both RNA pol I and II.
GFP-PSF entered the same nucleolar caps identified by the
anti-PSF antibodies (Figure 1B). This required the C-terminal
portion of PSF (amino acids 338–707) containing two RNA
binding domains (RRMs) and nuclear localization sequences
(NLSs; Figure 1C). The N-terminal part of PSF did not enter
the nucleus. GFP-PSF localized to nucleolar caps after dele-
tion of either or both RRMs (amino acids 490–707), required
for normal localization in nucleoplasmic foci (Dye and Pat-
ton, 2001). This targeting was reduced in constructs contain-
ing only the very C-terminal portion containing the last NLS
(604–707 and 699–707; Figure 1D).
We proceeded to clarify which type of nucleolar cap con-
tained PSF after ActD treatment. Using phase-contrast mi-
Table 2. Antibodies to proteins that are not components of nucleolar caps
Antigen not in nucleolar capsSpecies CloneAntibody provided by
FBP11 and FBP21
HMGN1 and 2
hnRNP R & Q
nuclear myosin 1?
10b I. Correas, Universidad Autonoma de Madrid, Spain
J. Pines, Wellcome/CRC Institute, UK
M. Bedford, University of Texas, MD Andersen Cancer Center
G. Dreyfuss, University of Pennsylvania School of Medicine
Upstate Biotechnology, Lake Placid, NY
R. Hock, University of Würzburg, Germany
G. Dreyfuss, University of Pennsylvania School of Medicine
E. Canaani, Weizmann Institute, Israel
G. Dreyfuss, University of Pennsylvania School of Medicine
M. Sendtner and W. Rossoll, University of Würzburg, Germany
C. Query, Albert Einstein College of Medicine
StressGen, Victoria, British Columbia, Canada
BD Biosciences, San Diego, CA
D. Black, University of California
V. Parnaik, Center for Cellular and Molecular Biology, India
E. Canaani, Weizmann Institute, Israel
D. Schümperli, University of Bern, Germany
O. Bensaude, ENS-CNRS Paris, France
V. Rotter, Weizmann Institute, Israel
C. Astell, University of British Columbia, Canada
P. de Lanerolle, University of Illinois at Chicago
S. Piñol-Roma, Mount Sinai School of Medicine
M. Olson, University of Mississippi Medical Center
D. Compton, Dartmouth Medical School
R. Bastos, Hospital Clinic Universitari, Spain
K. Wiman and M. Lindström, Karolinska Hospital, Sweden
B4 & 3E
p62 of TFIIH
J. Egly and B. Sandrock, Louis Pasteur University, France
E. Wahle and U. Kühn, Martin-Luther-Universität Halle-Wittenberg,
V. Rotter, Weizmann Institute, Israel
D. Helfman, Cold Spring Harbor Laboratory
R. Burgess, University of Wisconsin-Madison
Y. Shav-Tal, Albert Einstein College of Medicine
N. Hernandez, Cold Spring Harbor Laboratory
RNA pol II
RNA pol II
RNA pol III RPC155 and
RNA pol III RPC62
R. Roeder, Rockefeller University
F. Fackelmayer, Heinrich-Pette Institute for Experimental Virology and
Immunology at the University of Hamburg, Germany
E. Canaani, Weizmann Institute, Israel
TFIIF? RAP74 and RAP30
U5 snRNP 116K
2B1 G. Dreyfuss, University of Pennsylvania School of Medicine
S. Kitajima, Tokyo Medical and Dental University, Japan
M. Carmo-Fonseca, University of Lisbon, Portugal
R. Lührmann, Max-Planck Institute for Biophysical Chemistry, Germany
S. Stamm, Friedrich-Alexander-University Erlangen, Germany
Y. Shav-Tal et al.
Molecular Biology of the Cell2398
croscopy, PSF was detected in large and dark nucleolar caps
(DNCs; Figure 1E). Immunogold labeling showed that PSF
was enriched in the large nucleolar caps (Figure 1F, stars),
previously described as having a concave base and appear-
ing pressed onto the surface of the central body that was
originally the GC (Reynolds et al., 1964). For the sake of
clarity, we will refer to nucleolar caps using the original
terminology (Reynolds et al., 1964). The above nucleolar caps
will be referred to as concave caps or DNCs. This is in
contrast to the smaller and convex shaped nucleolar caps
termed “light nucleolar caps” (LNCs), closely attached to the
central body (CBY) and occasionally protruding into it (Fig-
ure 1F, top arrow and see Figure 4D for phase). It is known
that in the segregated nucleolus, the FC is removed from the
with the B92 mAb and the 1121 polyclonal antibody in untreated and in ActD-treated cells. (B) GFP-PSF–transfected cells were labeled with
the B92 antibody to PSF. Hoechst DNA counterstain is shown in blue. The forth column on the right is a computer-generated overlap of the
PSF and DNA stain showing the dense chromatin ring surrounding the nucleolar caps. Bar, 10 ?m. (C) GFP-PSF 338–707 forms nucleolar
caps. (D) Other GFP-PSF constructs were tested for nucleolar cap formation: ?, form caps; ?, do not form caps. P/Q, proline- and
glutamine-rich domain; RRM, RNA recognition motif; NLS, nuclear localization sequences. (E) PSF (left panel: immunofluorescence) is found
in phase dark nucleolar caps (DNCs; right panel: phase contrast). (F) Immunogold labeling with anti-PSF showed localization in large
concave caps (stars) situated on the central body (CBY). The smaller caps (arrows) represent the segregated DFC and FC.
PSF and GFP-PSF localize in the same nucleolar caps. (A) Immunofluorescence images of double staining for nucleoplasmic PSF
Nucleolar Structure after ActD
Vol. 16, May 20052399
DFC and forms nucleolar caps termed fibrillar caps that
cannot be seen by light microscopy but that are in close
association with LNCs (Figure 1F, bottom arrow and Figure
4D for phase). The remaining granular component will be
called the central body (CBY; Figure 1F).
Composition of Dark Nucleolar Caps
We identified components of either dark or light nucleolar
caps using antibodies to more than 70 endogenous nucleo-
plasmic or nucleolar proteins. PSF was used as a reference
for dark nucleolar caps. TLS/FUS is a nucleoplasmic protein
that colocalized with PSF in dark nucleolar caps (DNCs;
Figure 2A). It has previously been shown to enter nucleolar
structures through its oncogenic N-terminus (Zinszner et al.,
1997). This was also true for GFP-TLS and colocalization
with PSF was also corroborated in cells cotransfected with
GFP-PSF and HA-TLS (unpublished data). We also identi-
fied EWS, a homologue of TLS, and p54nrb/NonO (and
GFP-p54nrb), a heterodimer of PSF, as components of DNCs
(Table 3). The latter corroborates the localization of PSF to
nucleolar caps, because p54nrbshares homology with the
C-terminus of PSF.
Other known cap-localizing proteins detected in concave
nucleolar caps were the U1–70K component of U1 snRNP
(Carmo-Fonseca et al., 1991), cdk2 (Liu et al., 2000; Figure 2B),
and hnRNP K (Kamath et al., 2001; Table 3). Paraspeckle
proteins PSP1, PSP2/CoAA, and p54nrbtogether with the
helicases p68 and p72 are also present (Fox et al., 2002).
Indeed, we confirmed that PSF, p68 (Figure 2C) and p54nrb
colocalized to the same concave DNC and thus conclude
that PSP1, PSP2, and p72 are also components of DNCs.
We identified previously unknown components of DNCs
such as p220NPAT, which interacts with cdk2-cyclin E com-
plex; the TFIID subunit TAFII70; CstF-64, which is involved
in the polyadenylation process; the hnRNP F and H proteins
(Figure 2D); and the SR splicing factor ASF/SF2 (Table 3).
On the other hand, PTB (polypyrimidine tract-binding pro-
tein) known to be in complex with PSF under normal con-
ditions (Patton et al., 1993; Meissner et al., 2000) remained
dispersed in the nucleoplasm and did not relocalize with
PSF to nucleolar caps (Figure 2E and Table 4). Interestingly,
the peri-nucleolar compartment (PNC; Matera et al., 1995;
Huang et al., 1997) normally containing PTB (Figure 2E)
disappeared under these conditions. Sin3A, another nuclear
factor directly associated with PSF (Mathur et al., 2001) was
not found in DNCs either (Table 4).
Because many of the identified DNC proteins are nor-
mally associated with mRNAs, we were interested to deter-
mine whether other components of the transcriptional ma-
chinery can be found in DNCs. A recent study has identified
proteins of the transcription and splicing machineries in
functional complexes (termed X1 and X2) formed at the
mRNA 5? splice site (Kameoka et al., 2004). Some of the
RNA-binding proteins identified were PSF, p54nrb, TLS, and
U1–70K, all components of DNCs. We therefore tested the
presence of other X1/X2 proteins in the segregated nucleo-
lus. X1/X2 complexes contain the active forms of RNA pol II
together with transcription, splicing, and elongation factors.
However, we did not detect the spliceosome-associated pro-
teins hPrp5, U5–166K, FBP11, and FBP21, or the hypo- or
hyperphosphorylated forms of RNA pol II CTD, or the basal
transcription factors TFIIF and TFIIH, or the elongation fac-
tor P-TEFb in DNCs (Table 4). These data indicate that even
though an influx of a unique group of nucleoplasmic pro-
teins into the region of the segregating nucleolus is ob-
served, and many of them are RNA binding proteins, this
relocation is not a general trait of all the RNA transcribing
and processing machinery. Moreover, even though nucleo-
lar caps are commonly regarded as part of the nucleolus, we
DNCs. Confocal images of untreated and ActD-treated cells showing
colocalization of (A) PSF and TLS, (B) PSF and cdk2, (C) PSF and p68,
(D) PSF and hnRNP F. (E) PSF and PTB do not colocalize in concave
nucleolar caps. The strong foci seen in PTB labeling of untreated cells
are the perinucleolar compartments (PNC). Right-hand column in this
and the following figures shows the merged output. Bar, 10 ?m
Nucleoplamic proteins that colocalize with PSF in concave
Y. Shav-Tal et al.
Molecular Biology of the Cell2400
could not detect nucleolar proteins in the large and concave
Composition of LNCs and Fibrillar Caps
p80 coilin, a nucleoplasmic protein, is normally found in
Cajal bodies, which redistribute to the nucleolar periphery
during inhibition of transcription (Raska et al., 1990). It was
shown to form nonconcave caps distinct from U1–70K caps
(Carmo-Fonseca et al., 1992). We found that PSF and p80
coilin each formed two distinct nucleolar caps, observed
tandemly around the nucleolar body (Figure 3A). p80 coilin
positive areas around the nucleolus were a peripheral subset
(Raska et al., 1990) distinct from nucleolar caps formed by
fibrillarin, a DFC marker (Ochs et al., 1985). The incomplete
overlap between fibrillarin and p80 coilin can be seen in
Figure 3B. Fibrillarin containing nucleolar caps are termed
“light nucleolar caps” (LNCs; see Figure 4D for phase).
Other nucleolar proteins that compartmentalize with
fibrillarin in LNCs upon transcriptional inhibition are
Nopp140 (Chen et al., 1999; Figure 3C), gar1 (Pellizzoni et al.,
2001), and MSP58 (Ren et al., 1998; Table 3). Interestingly, we
found that the Drosophila trithorax homologue, ALL-1, nor-
mally found in a nucleoplasmic complex consisting of RNA-
binding proteins (Yano et al., 1997; Nakamura et al., 2002)
also was found in LNCs (Figure 3D). Another nucleolar
protein, p110, which is associated with U3 snoRNP, was not
found in fibrillarin containing caps at low ActD concentra-
tions (Adamson et al., 2001), but did colocalize with high
levels of ActD (Table 3).
Proteins related to the RNA pol I transcriptional machin-
ery have been shown to colocalize in small nucleolar caps in
regions juxtaposed to fibrillarin (LNCs). These caps have
been termed on occasion fibrillar caps as they contain FC
proteins. These proteins include: RNA pol I, UBF, TBP,
TAFI63, and TAFI110 (Reimer et al., 1987; Zatsepina et al.,
1993; Jordan et al., 1996) and topoisomerase I (Christensen et
al., 2004; Table 3). It has been shown that some nucleolar
proteins can be found in both types of cap regions that
Table 3. Endogenous nuclear proteins and RNAs that we detected in nucleolar caps (using immunofluorescence and FISH)
DNCLNC FC Central bodyCajal bodyPML body
p14(ARF)p80 coilin PML
MRP RNAU93 scaRNA
Table 4. Endogenous nuclear proteins and RNAs that did not compartmentalize in nucleolar caps
RNA processing and spliceosome
Nuclear structure and
hnRNP R and Q
RNA pol II
RNA pol III
Nuclear myosin I
HMGN1 and 2
7SK RNA RNaseP RNA
Nucleolar Structure after ActD
Vol. 16, May 2005 2401
contain DFC or FC proteins, such as Nopp140 (Chen et al.,
1999) and MPP10. The latter was seen to partially colocalize
with fibrillarin caps during incubation with low levels of
ActD (Westendorf et al., 1998); however, it remained par-
tially in DNCs using high levels of ActD (Table 3). This is the
only nucleolar protein we identified in DNCs.
The presence of three distinct nucleolar caps was then
confirmed by costaining with antibodies to PSF, fibrillarin,
LNCs. Confocal images of untreated and
ActD-treated cells showing: (A) Untreated:
PSF nucleoplasmic staining and p80 coilin la-
beling of Cajal bodies. Treated: PSF and p80
coilin were found in two distinct nucleolar
caps. (B) Untreated: fibrillarin was found
mainly in nucleoli but also in Cajal bodies.
Treated: partial overlap between p80 coilin
caps and fibrillarin caps. (C) Fibrillarin and
Nopp140 were found in the same nucleolar
caps. (D) Fibrillarin and ALL-1 also colocal-
ized in these caps. Bar, 10 ?m.
Nuclear proteins that colocalize in
Y. Shav-Tal et al.
Molecular Biology of the Cell 2402
UBF and gar1 (Figure 4, A–D). Fibrillarin and UBF segregate
to distinct but adjacent nucleolar caps; fibrillarin is in phase-
dense LNCs, whereas caps containing UBF were not ob-
served (Figure 4D). To summarize, concave DNCs were
larger and wider and contained incoming nucleoplasmic
proteins. The smaller and rounder caps found in between
DNCs contained two regions with either nucleolar DFC or
FC proteins, whereas p80 coilin formed a separate cap. In
addition, although DNCs were always found on the periph-
ery of the nucleolar body, fibrillar caps were occasionally
situated on top of concave caps (Figure 4C).
p14(ARF) Remains in the Central Body after ActD
As it became apparent that the formation of nucleolar caps
involved nucleoplasmic proteins, we were interested in
identifying proteins in the central body. As known, nucleo-
lin (C23), which is found both in the DFC and GC, and
nucleophosmin (B23) which is a GC protein, dispersed in the
nucleoplasm after ActD treatment (Figure 5A, Table 4).
Staining of PSF, nucleolin and p80 coilin showed nucleolar
caps situated on the periphery of the central body (Figure
5A, inset). RNA helicase RH-II/Gu normally found in the
distinct compartments. Confocal images of
(A) untreated: PSF is nucleoplasmic, whereas
fibrillarin was in the nucleoli; treated: PSF
and fibrillarin relocalized in the two different
nucleolar caps. This was true also for (B) PSF
and UBF, (C) PSF and gar1. Bar, 10 ?m. (D)
Fibrillarin is found in phase light nucleolar
caps (LNCs), whereas UBF, representing the
transcriptional machinery, is found in the ad-
jacent fibrillar caps.
DNCs, LNCs, and fibrillar caps are
Nucleolar Structure after ActD
Vol. 16, May 20052403
GC has been described to disperse in a similar manner
(Valdez et al., 1998).
p14(ARF) was the only GC nucleolar protein that re-
mained in the central body during ActD treatment, although
some of it was dispersed in the nucleoplasm (Figure 5B,
Table 3). GFP-p14(ARF) behaved in the same manner (un-
published data). ARF is involved in the attenuation of the
Mdm2-mediated ubiquitination and degradation of p53.
During cellular stress ARF binds to Mdm2, resulting in an
upregulation of p53. It has been suggested that this interac-
tion occurs in the nucleolus (Weber et al., 1999; Llanos et al.,
2001). However, we found by antibody staining, that Mdm2
did not show any accumulation in the central body of the
segregated nucleolus (Table 4).
A large number of nucleoplasmic proteins related to dif-
ferent nuclear functions were then tested and most were
found to remain in the nucleoplasm without compartmen-
talization during transcriptional inhibition (Table 4). Nu-
transfected cells were labeled with antibodies to nucleolin and p80 coilin. Untreated: GFP-PSF was nucleoplasmic, nucleolin was in nucleoli
and p80 coilin was in Cajal bodies. No colocalization was seen in the merged image. Treated: GFP-PSF concave nucleolar caps were situated
on the central body and p80 coilin fibrillar caps were found inbetween the concave caps. A segregated nucleolus is seen in the enlarged
merged image. (B) Untreated: PSF was nucleoplasmic, whereas p14(ARF) was in the GC. Treated: p14(ARF) was both in the central body and
in the nucleoplasm. Concave PSF caps formed on this body. A segregated nucleolus is seen in the enlarged merged image. (C) Untreated:
PSF foci dispersed throughout the nucleoplasm in comparison to the large SC-35 speckles. Treated: PSF and SC-35 localized in distinct
compartments. Bar, 10 ?m.
Nuclear proteins that do not localize in nucleolar caps during transcriptional inhibition. Confocal images of (A) GFP-PSF–
Y. Shav-Tal et al.
Molecular Biology of the Cell2404
clear speckles, which normally contain proteins like SC-35,
4.1R, lamin A, hPrp5, FBP11, and FBP21, became rounded
and sometimes enlarged, but were distinct and spatially
distant from nucleolar caps (Figure 5C). The relative concen-
tration of SC-35 in speckles is only twice the intensity of the
nucleoplasmic pool (Fay et al., 1997). Measurements of the
average intensities of PSF staining in the nucleolplasm and
nucleolar caps using confocal microscope line scans showed
that in ActD-treated cells PSF was 3–5 times more concen-
trated in concave caps than in the nucleoplasm, whereas
SC-35 in speckles was 2–3 times more concentrated than the
corresponding nucleoplasmic signal (unpublished data).
Unique Nucleolar Cap for PML Body Components
Because PML bodies are normally associated with regions of
active transcription (Wang et al., 2004), we tested whether
proteins commonly found in PML bodies would move to
nucleolar caps. Indeed, a portion of the PML protein moved
to the nucleolar periphery in distinct nucleolar caps either
adjacent to PSF caps or on top of them (Figure 6A). This was
also seen with GFP-PML (unpublished data). Similarly, a
portion of Sp100, another component of PML bodies, was
also found in these caps (Table 3). Because p80 coilin from
Cajal bodies formed similar nucleolar caps, we checked
whether these proteins share the same nucleolar cap. How-
ever, this was not the case. PML protein formed a novel
nucleolar cap distinct from p80 coilin (Figure 6, B and C),
Nopp140 (LNCs; Figure 6D), and UBF (fibrillar caps; Figure
6E). Using an antibody that detects PML bodies in EM
preparations (Figure 6F), we could detect PML both in small
structures adjacent to large nucleolar caps (Figure 6G) or on
top of DNCs (Figure 6H).
Nucleolar Caps Are Surrounded by Heterochromatin
In ActD-treated cells, the segregated nucleolar regions be-
came surrounded by dense chromatin (Figure 1, A and B).
Antibodies specific for dimethylation of lysine 9 on histone
H3 (Figure 7A), a characteristic of heterochromatin, stained
the dense chromatin regions. These domains remained un-
stained with antibodies that detect either dimethylation of
lysine 4 (Figure 7A) or acetylation of lysine 9 on histone H3,
characteristic of euchromatin. Using DNA FISH against hu-
man chromosomes, we determined that chromosomes con-
taining nucleolar organizer regions (NORs; chr 13, 14, 15, 21,
22) retained their nucleolar proximity during transcriptional
inhibition (Figure 7B), except for chromosome 13 that was
not always positioned in contact with nucleoli. Other chro-
mosomes that normally showed peripheral positioning in
the nucleus did not tend to associate with nucleolar regions
during transcriptional arrest (Figure 7B).
Nucleolar RNAs Segregate to Different Subtypes of
Our finding that the DNCs contain nucleoplasmic RNA
binding proteins led us to examine the distribution of sev-
eral functional nuclear RNAs during the process of nucleolar
segregation. The ribosomal genes transcribe a large precur-
sor RNA that is cleaved at the A0 site, which removes the 5?
external transcribed sequence (5?ETS). This step occurs in
the nucleolus and the 5?ETS is then degraded by the exo-
some. Using a probe to the 5?ETS, which specifically recog-
nizes pre-rRNAs, we found that the precursor rRNAs seg-
regated to the DNCs enriched in nucleoplasmic proteins and
colocalized with PSF (Figure 8A). U1 snRNA also localized
to these nucleolar caps (Carmo-Fonseca et al., 1991), whereas
U2 snRNA remained in speckles as described (Carmo-Fon-
seca et al., 1992) and 7SK RNA was enriched in nuclear
speckles, after ActD treatment (Table 4). Nucleoplasmic mR-
NAs identified using an oligo(dT) probe or a probe to the
abundant ?-actin transcript were not detected in nucleolar
caps (Table 4).
Small nucleolar RNAs (snoRNAs), required for the pro-
cess of ribosome assembly, colocalized with fibrillarin in
LNCs: U14 snoRNA (Figure 8B), U3 snoRNA (Carmo-Fonseca
et al., 1991; Puvion-Dutilleul et al., 1997; Leary et al., 2004), and
E3 snoRNA (Figure 8D; Table 3). The nucleolus plays a role
also in the formation of RNA pol III transcripts. Three of these
transcripts were tested, and we found that MRP RNA re-
mained in the central body (Figure 8C), U6 snRNA colocal-
ized with fibrillarin (Table 3; Carmo-Fonseca et al., 1991),
and RNase P RNA did not enter nucleolar caps (Table 4).
Cajal body–specific RNAs (scaRNAs) have been found in
Cajal bodies (Darzacq et al., 2002) and so we were interested
to see whether they would follow a similar fate as described
above for Cajal body proteins. Indeed, U93 scaRNA, typi-
cally found in Cajal bodies (Kiss et al., 2002), as seen by
colocalization with GFP-fibrillarin in Cajal bodies but not
with GFP-fibrillarin and E3 snoRNA in the nucleolus (Figure
8D, top), was found to localize on top of E3 snoRNA con-
taining caps (that also contain fibrillarin; Figure 8D, bottom)
and not with GFP-PSF–containing caps (Figure 8D, bottom).
A similar distribution was described also for p80 coilin
Nucleolar Caps Are Dynamic Structures
Nuclei observed at different time points after addition of
ActD indicated that the process of nucleolar capping was
dynamic. One hour posttreatment with ActD, PSF staining
showed delicate bead-like structures around the nucleolus
(Figure 9A). These increased in volume and became cap-like
after 2–2.5 h. In parallel, condensation of chromatin was
observed. After longer exposure to the drug these compart-
ments took upon a more rounded structure. We have pre-
viously shown that GFP-PSF in apoptotic cells, induced by
prolonged exposure to ActD, formed large, rounded struc-
tures (Shav-Tal et al., 2001a). This correlated with hyper-
phosphorylation of PSF in apoptotic and mitotic cells. How-
ever, no change in the mobility of the PSF band was
observed during nucleolar capping upon short exposure to
ActD (Figure 9B). This meant that the hyper-phosphoryla-
tion of PSF was a later step in the molecular pathway in-
duced by ActD.
The dynamic characteristics of nucleolar caps were then
examined using FRAP. Several GFP-tagged proteins, which
were found to localize to segregated nucleoli as their endog-
enous counterparts, were photobleached, either in the nu-
cleoplasm/nucleolus of untreated cells or in segregated nu-
cleoli of ActD-treated cells, and the recovery of the
fluorescent signal was monitored over time (Figure 9C).
GFP-PSF demonstrated a half-time of recovery of ?2.7 s, in
contrast to the nucleolar proteins GFP-fibrillarin (t1/2?15 s;
Phair and Misteli, 2000), GFP-Nopp140 (t1/2? 17 s), and
GFP-p14(ARF) (t1/2? 43 s), which also exhibited an immo-
bile nondynamic nucleolar fraction (45, 10, and 35%, respec-
tively; Figure 9C, green curves). Surprisingly, under ActD
conditions, the rate of exchange of GFP-PSF in concave
DNCs was as rapid as the nucleoplasmic pool of GFP-PSF
under unperturbed conditions (t1/2? 2.4 s; Figure 9C, or-
ange curves). The recovery of GFP-fibrillarin (t1/2? 10 s)
and GFP-Nopp140 (t1/2? 14 s) in LNCs was similar, yet an
increased immobile fraction was seen for both proteins (55
and 30%, respectively). Notably, GFP-p14(ARF) in the cen-
tral nucleolar body of ActD-treated cells exhibited an ex-
tremely slow turnover (t1/2? 40 s) and the fixed fraction
Nucleolar Structure after ActD
Vol. 16, May 2005 2405
was greatly enhanced (from 50 to 80%). This analysis shows
that nucleolar caps contain two distinct protein pools, a
portion in constant exchange with the nucleoplasm and a
fixed immobile fraction. This demonstrates that the dynam-
ics of nuclear domains are at least partially retained also
under conditions of transcriptional inhibition.
The Formation of Nucleolar Caps Is an Energy-dependent
It has been shown that nuclear dynamics is sensitive to
metabolic stress (Muratani et al., 2002; Platani et al., 2002;
Shav-Tal et al., 2004). We therefore investigated nucleolar
found in PML bodies. Treated: PSF and PML were found in two different nucleolar caps. (B) Untreated: p80 coilin was in Cajal bodies and
PML was in PML bodies. Occasionally, there was close association between the two bodies. Treated: p80 coilin and PML were in two separate
caps. (C) GFP-PSF–transfected cells were labeled with antibodies to PML and p80 coilin. The three different cap structures were seen in the
treated cells. Bar, 10 ?m. (D) PML caps were distinct from Nopp140 LNCs and (E) UBF fibrillar caps. (F) Immunogold labeling with anti-PML
detected PML bodies in the nucleoplasm of untreated cells and (G) PML in small cap structures adjacent to DNCs or (H) on top of DNCs.
PML protein is found in distinct nucleolar caps. Confocal images of (A) Untreated: PSF was nucleoplasmic, whereas PML was
Y. Shav-Tal et al.
Molecular Biology of the Cell2406
cap formation under low cellular energy levels. When cells
were incubated at 4°C and concurrently treated with ActD,
no formation of nucleolar caps was observed (unpublished
data). Inhibition of metabolism by addition of Na-azide and
2-deoxyglucose to ActD-treated cells showed that nuclear
proteins remained in their normal distributions (compare
Figure 9D, a and b). The same phenomenon was observed
with other components of nucleolar caps (unpublished
data). This indicated that the redistribution of the proteins
required energy. In contrast, chromatin condensation seen
after ActD treatment was still observed (see DNA panels in
Figure 9D). It has been shown that the nucleus contains a
unique myosin 1? form (Pestic-Dragovich et al., 2000). Be-
cause this motor protein might be involved in the translo-
cation of proteins in the nucleus, cells were treated with
2,3-butanedione monoxime (BDM), an inhibitor of actin-
dependent myosins. No change in cap formation was seen
To identify the energy source required for cap formation,
treatment with ActD was performed in minimal medium
(HBSS) without metabolic supplements. In this case, most
nuclei did not contain nucleolar caps (Figure 9Ea), indicating
that reduction in metabolic levels due to lack in energy
sources did not allow the formation of caps. The addition of
either protein in the form of FCS (Figure 9Eb) or amino acids
(unpublished data) or glucose (Figure 9Ec) did not change
this condition. However, when pyruvate was added to the
minimal medium, all cells formed nucleolar caps. These
however, were not fully formed as those seen after 2.5 h of
treatment (Figure 9Ed). Finally, when ActD treatment was
performed in a carbohydrate-rich medium but without FCS,
nucleolar caps formed as usual (Figure 9Ee). In all the above
cases, condensation of chromatin was seen. We therefore
conclude that energy produced by the phosphorylative ox-
idation pathway is a prerequisite in the process of reshuf-
fling of nuclear proteins during transcriptional stress.
We have found that the process of nucleolar segregation
caused by the transcriptional arrest of RNA polymerases I
and II was accompanied by the sorting and rearranging of
nuclear proteins and RNAs into defined nuclear subdo-
mains. The course of events was a dynamic and specific
process, encompassing many major players that define in-
tranuclear structure. Several main themes of nuclear rear-
rangement were observed: 1) the segregation of the three
defining regions of the nucleolus (FC, DFC, GC) into three
distinct but juxtaposed domains that retain many of their
original protein and RNA components and that are termed
nucleolar caps and central body, 2) this segregation is ac-
companied by the release of several GC proteins into the
nucleoplasm, 3) the disassembly of nuclear bodies such as
Cajal bodies, SMN bodies, and PML bodies and the reloca-
tion of their protein and RNA components to discrete nu-
cleolar caps, 4) the formation of a large heterochromatin
domain surrounding the segregated nucleolus, 5) the influx
of a significant number of nucleoplasmic proteins, many of
which are RNA binding proteins, into large nucleolar caps,
whereas 6) most nucleoplasmic proteins and nuclear speckle
proteins retained their localization.
The nucleolus is sensitive to the transcriptional profile of
the cell, and the status of transcriptional activity is reflected
in nucleolar structure. Nucleolar segregation and capping is
a normal cellular process occurring under physiological cir-
cumstances that involve transcriptional shut down (Smetana
and Busch, 1974) and can be mimicked by drug-induced
transcriptional arrest (Bernhard and Granboulan, 1968; Zin-
szner et al., 1997; Dousset et al., 2000; Andersen et al., 2002;
Fox et al., 2002; Ospina and Matera, 2002). In the developing
Xenopus oocyte dramatic changes in nucleolar structure
were observed, which included a stage of nucleolar segre-
gation and cap formation reminiscent of ActD treatment
(Van Gansen and Schram, 1972). During ovulation of Xeno-
pus eggs, nucleoli disappear and transcription is shut off,
segregated nucleolus showed characteristics of
heterochromatin via staining with an antibody
against dimethylated lysine 9 of histone H3, but
not with the euochromatin marker: dimethyled
lysine 4 of histone H3. Bar, 5 ?m. (B) Chromo-
some labeling by DNA FISH showed that pe-
ripheral chromosomes (3, 4) retained this posi-
tioning during transcriptional arrest, whereas
NOR-containing chromosomes (15, 22) re-
mained associated with the nucleolar region. In
many cells, NOR containing chromosome 13
was not associated with nucleolar region after
transcriptional arrest. Chromosome X was not
associated with the nucleolus either. Bar, 10 ?m
(A) The chromatin surrounding the
Nucleolar Structure after ActD
Vol. 16, May 20052407
later to return in the embryo. In the procedure of nuclear
cloning, nuclei from somatic cells are injected into inter-
phase eggs and the somatic nucleoli are then found to seg-
regate and finally disassemble (Gonda et al., 2003). This
process is triggered by two germ cell proteins FRGY2a and
FRGY2b and is independent of rRNA transcription. Other
natural instances of segregation and capping occur in oo-
cytes (Mirre et al., 1980; Crozet et al., 1981), spermatocytes
(Stahl et al., 1991), developing embryos (Hyttel et al., 2000),
hepatocytes (Reddy and Svoboda, 1972), keratinocytes
(Karasek et al., 1972), mycoplasma infection (Jezequel et al.,
1967), and certain diseases (Karasek et al., 1970; Smetana et
al., 1972). Nucleolar capping of p80 coilin naturally occurs in
normal mouse tissues (Tucker et al., 2001) and in neuronal
cells (Raska et al., 1990; Carmo-Fonseca et al., 1993; Janevski
et al., 1997).
The study of electron micrographs of transcriptionally
arrested cells in the 1960s lead to the simple assumption that
the material found in nucleolar caps originated from the
segregation of granular and fibrillar components of the nu-
cleolus. Yet, one study noted that the granular material of P2
(concave) caps observed by TEM was not found in untreated
nucleoli (Recher et al., 1971). In other studies, nucleoplasmic
splicing factors were found associated with segregated nu-
cleoli of hibernating rodents (Malatesta et al., 2000) and SR
proteins were transiently associated with the nucleolus after
mitosis (Bubulya et al., 2004). Our study shows that DNCs
consisted mainly of nucleoplasmic proteins, whereas LNCs
and fibrillar caps evolved from nucleolar proteins. A few
RNA-binding nucleoplasmic proteins that were found in
nucleolar caps have been detected in the nucleolus by pro-
teomic analysis: PSF, p54nrb, hnRNP K, hnRNP H, and SF2/
ASF (Andersen et al., 2002; Scherl et al., 2002). However,
these proteins are normally found in the nucleoplasm and
are excluded from the nucleolus as observed either by im-
munofluorescent stainings or GFP tagging. Interestingly, a
different nucleolar caps. (A and B) U3 and
U14 snoRNAs were found to localize in fibril-
larin containing caps, whereas 5?ETS rRNA
was observed with GFP-PSF in concave caps.
(C) MRP RNA remained in the central body.
(D) U93 scaRNA was typically found in Cajal
bodies as was fibrillarin, but not E3 snoRNA.
After ActD treatment, U93 scaRNA was jux-
taposed to E3 snoRNA in nucleolar caps and
distinct from concave caps. Bar, 5 ?m.
Nucleolar RNAs are found in the
Y. Shav-Tal et al.
Molecular Biology of the Cell 2408
kinetic proteomic analysis has shown PSF to be one of the
highly enriched proteins in the nucleolus during ActD treat-
ment (Andersen et al., 2005). In the case of PSF, GFP-PSF
constructs showed that the C-terminal half of the protein is
important for the localization in caps. This property was
independent of the two RRMs in this region. From analyzing
concave nucleolar caps at different time points after addition of ActD. Hoechst DNA counterstain is seen in blue. (B) Western blot analysis
of PSF from protein extracts of cells at time points as in A. (C) Recovery curves after photobleaching of GFP-PSF, GFP-fibrillarin,
GFP-Nopp140, and GFP-p14(ARF) in the nucleoplasm or nucleolus before ActD treatment (green) and in nucleolar caps after ActD treatment
(orange). (D) PSF and nucleolin labeling of cells treated with: (a) ActD only, (b) ActD plus Na-azide and 2-deoxyglucose. (E) Formation of
PSF concave caps. ActD treatment was performed under different conditions: (a) HBSS medium, (b) HBSS medium plus 10% FCS, (c) HBSS
medium with glucose, (d) HBSS medium with pyruvate, (e) DMEM without FCS. Bar, 10 ?m.
Formation of nucleolar caps is a dynamic energy-requiring process. Immunofluorescence microscopy of (A) The formation of PSF
Nucleolar Structure after ActD
Vol. 16, May 2005 2409
constructs of the C-terminus we conclude that the middle
part of the C-terminal half of PSF is required for this local-
ization. The C-terminus is homologous to p54nrb, the het-
erodimer of PSF, which also translocated to DNCs. Align-
ment of amino acid sequences of DNC proteins did not
reveal any “cap localization signals,” although most of these
proteins contain RNA-binding domains such as RRMs or
RGG boxes, shown to be the most highly abundant motifs in
proteins identified in the nucleolar proteome (Leung et al.,
2003). Yet, these motifs are probably not sufficient for nucle-
olar cap targeting because other proteins tested, which also
contain RRM or RGG boxes, were not localized to nucleolar
caps (Table 4). The unexpected finding that nucleolar pre-
rRNA was preferentially detected in the DNCs that har-
bored mainly nucleoplasmic proteins still implies that the
localization of nucleoplasmic RNA-binding proteins to
DNCs was a consequence of the RNA-binding properties of
these proteins. FRAP experiments revealed that these struc-
tures were not static depots of proteins but were highly
dynamic structures in constant exchange with the nucleo-
plasm, complementary to FRAP experiments performed in
transcriptionally active cells (Janicki and Spector, 2003).
Our data strongly suggest that nucleolar segregation is
part of a general concerted process of nuclear rearrangement
taking place during transcriptional shut down and com-
prises both preexisting protein-RNA interactions and newly
established interactions. For example, we have detected a
trend in the association of nuclear body proteins with the
segregated nucleolus. Components of SMN bodies have
been shown to transiently pass through nucleolar caps
(Pellizzoni et al., 2001). p80 coilin from Cajal bodies was
found on the peripheral part of nucleolar caps that contain
fibrillarin, which is also, in part, a Cajal body component.
Nucleolar and Cajal body RNAs were also found to closely
segregate during ActD treatment. A Cajal body scaRNA was
found, as described above for p80 coilin, to be peripherally
situated on fibrillarin. Small nucleolar RNAs were found in
fibrillarin containing LNCs. We also detected a novel nucle-
olar cap to which a portion of PML and Sp100 proteins
localized that were distinct from DNCs, LNCs, or fibrillar
caps. PML caps were usually the smallest of the above and
formed on top or in between the other caps. Interestingly, a
different pathway of cellular stress involving the inhibition
of proteosome action caused PML and Sp100 to relocalize
inside the nucleolus (Mattsson et al., 2001). PML translocated
to the nucleolar periphery also in response to DNA damage
and colocalized there with Mdm2, but in an ARF-indepen-
dent manner (Bernardi et al., 2004). Another study has
shown that a variety of stress responses activating the p53
pathway affect nucleolar integrity (Rubbi and Milner, 2003).
The nucleolus therefore probably plays an important role in
sensing these stresses (Olson, 2004a) and can act as a dock-
ing site for many proteins released from disrupted struc-
tures and complexes.
The central body, on which nucleolar caps are formed, is
an intriguing structure. It was assumed to originate from the
GC region of the nucleolus. MRP RNA was detected in the
central body, whereas RNase P RNA was not. Although
different in sequence, these RNAs are structurally similar,
whereas MRP is involved in cleavage and maturation of the
precursor rRNA and RNase P acts in the processing of
pre-tRNA (van Eenennaam et al., 2000). However, under
these conditions MRP RNA and rRNAs were segregated in
distinct compartments. As for the protein composition of the
central body, we could only detect p14(ARF) in the central
body, whereas proteins found in the GC such as nucleophos-
min and nucleolin dispersed throughout the nucleoplasm.
FRAP experiments showed that p14(ARF) had the slowest
recovery rates in comparison to the other nucleolar proteins
tested, fibrillarin and Nopp140. These rates all differed from
the nuclear mobility of PSF in DNCs, which was comparable
to its mobility in the nucleoplasm of untreated cells and did
not show a fixed fraction. Moreover, most of p14(ARF) pro-
tein was not dynamic as observed by the increase in the
immobile fraction from 35 to 65%. This might indicate that
p14(ARF) has a function in retaining the structural integrity
Our study shows that the process of nucleolar segregation
and capping is an active process that requires active metab-
olism of the cell. However, it does not require active protein
synthesis (Goldblatt et al., 1970). The addition of metabolic
inhibitors to cells being treated with ActD hindered the
formation of nucleolar caps. The energy source required for
nucleolar cap formation was not in the form of amino acids,
proteins, or growth factors but was carbohydrate based. We
found that the addition of pyruvate (citric acid cycle) but not
glucose (glycolysis) to minimal medium could lead to the
formation of caps, although this was probably not the only
requisite. Because pyruvate is the end product of glycolysis,
it is possible that ActD is also inhibiting a certain step in this
process, as previously suggested (Laszlo et al., 1966). Pyru-
vate is also the input molecule for the citric acid cycle and
oxidative phosphorylation, the main producers of cellular
energy in the form of ATP and GTP. Azide is a metabolic
poison that blocks oxidative phosphorylation in the mito-
chondria, the organelle in which pyruvate is metabolized. It
was described for the TLS protein (DNC) that its entrance
into nucleolar caps is an active process requiring the integ-
rity of the Ran/TC4-RCC1 nuclear transport cycle (Zinszner
et al., 1997) and that the drug-induced translocation of nu-
cleophosmin from nucleoli to the nucleoplasm requires ATP
(Wu et al., 1995; Finch and Chan, 1996). These findings
indicate that the relocalization and nucleolar capping of
nuclear proteins is not just a byproduct of transcriptional
arrest but actually an active mechanism driving the reshap-
ing of nuclear compartments.
Although the physiological significance of nucleolar seg-
regation and capping is unclear, a correlation between re-
duction in RNA transcription and the formation of these
structures can be drawn, especially during differentiation
and development. In the normal situation, much of the
nuclear activity is devoted to transcription. In active cells,
transcriptional and posttranscriptional components are in
equilibrium; being recruited to DNA or to nascent mRNA
transcripts, respectively. Under ActD-induced transcrip-
tional arrest, the RNA polymerase II complexes are blocked
during the elongation process, thus titrating one fraction of
the transcription machinery to the DNA. Because mRNA
production has ceased, there exists an excess pool of free
posttranscriptional factors. In conjunction, in the nucleolus,
separation of the RNA polymerase I transcription machinery
from the rRNA occurs. The observed clustering of nucleo-
plasmic RNA-binding proteins, in part of the segregated
nucleolus that contains pre-rRNA, could be the result of the
high abundance of these proteins and the existence of newly
exposed RNA partners. A recent study showed that during
telophase, as RNA pol I activity in nucleolar NORs resumed,
nucleoplasmic SR-splicing factors become transiently asso-
ciated with the NORs (Bubulya et al., 2004). The interactions
we observe have specificity, as only a subset of nucleoplas-
mic RNA-binding proteins relocalize to these regions and
may reflect some of the protein complexes present in tran-
scriptionally active cells. Taken together, these data argue
for an additional pathway that RNA-binding proteins can
Y. Shav-Tal et al.
Molecular Biology of the Cell 2410
take when RNA pol II transcription is arrested. It has been
suggested that nuclear organization is driven by the state of
gene expression in the cell (Singer and Green, 1997) and our
data support this notion. Because nucleolar segregation can
occur under physiological states and can be followed by
nucleolar reassembly, it stands to reason that the nucleus
evolved a mechanism that uniquely redistributes specific
nuclear components during transcriptional arrest, while si-
multaneously up keeping certain basic interactions. Such a
mechanism would provide the flexibility required for re-
sponding to metabolic cues and would maintain a certain
degree of structure necessary for the efficient reassembly
once the transcriptional status of the cell changes.
We are immensely grateful to the researchers listed in Materials and Methods
who generously shared their antibodies and constructs with us. We thank
Tom Meier for critical comments on this manuscript. We thank Leslie Cum-
mings, Juan Jimenez, and Frank Macaluso of the Albert Einstein Imaging
Facility for assistance in the electron microscopy work and Jenetta Smith for
the DNA FISH. D.Z. is an incumbent of the Joe and Celia Weinstein profes-
sorial chair at the Weizmann Institute of Science. R.H.S. is supported by
National Institutes of Health grants EB2060 and DOE63056.
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