* Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724; and The National Center for Microscopy and Imaging
Research at San Diego, University of California, La Jolla, California 92093
The perinucleolar compartment (PNC) is a unique nuclear structure preferentially localized at the periphery of the nucleolus. Several small RNAs transcribed by RNA polymerase III (e.g., the Y RNAs, MRP RNA, and RNase P H1 RNA) and the polypyrimidine tract binding protein (PTB; hnRNP I) have thus far been identified in the PNC (Ghetti, A., S. PinolRoma, W.M. Michael, C. Morandi, and G. Dreyfuss. 1992. Nucleic Acids Res. 20:3671-3678; Matera, A.G., M.R. Frey, K. Margelot, and S.L. Wolin. 1995. J. Cell Biol. 129:1181-1193; Lee, B., A.G. Matera, D.C. Ward, and J. Craft. 1996. Proc. Natl. Acad. Sci. USA. 93: 11471-11476). In this report, we have further characterized this structure in both fixed and living cells. Detection of the PNC in a large number of human cancer and normal cells showed that PNCs are much more prevalent in cancer cells. Analysis through the cell cycle using immunolabeling with a monoclonal antibody, SH54, specifically recognizing PTB, demonstrated that the PNC dissociates at the beginning of mitosis and reforms at late telophase in the daughter nuclei. To visualize the PNC in living cells, a fusion protein between PTB and green fluorescent protein (GFP) was generated. Time lapse studies revealed that the size and shape of the PNC is dynamic over time. In addition, electron microscopic examination in optimally fixed cells revealed that the PNC is composed of multiple strands, each measuring ~80-180 nm diam. Some of the strands are in direct contact with the surface of the nucleolus. Furthermore, analysis of the sequence requirement for targeting PTB to the PNC using a series of deletion mutants of the GFP-PTB fusion protein showed that at least three RRMs at either the COOH or NH2 terminus are required for the fusion protein to be targeted to the PNC. This finding suggests that RNA binding may be necessary for PTB to be localized in the PNC.
Many nuclear functions including DNA replication, RNA transcription, processing, and transport have been extensively investigated at the
biochemical and molecular levels. However, much less is understood regarding the spatial organization of these
events within the three-dimensional context of the mammalian cell nucleus. Light and electron microscopic examination of cell nuclei has revealed many readily identifiable
nuclear structures including the nucleolus, electron dense
heterochromatin, and a variety of granular and fibrillar
structures including interchromatin granules, perichromatin granules, and perichromatin fibrils (for review see
Spector, 1993 In addition to the ubiquitous features of the nucleus, nuclear bodies have been described in specific cell types or
cells at different physiological states (Bouteille et al., 1967 Another intensively studied nuclear body is the promyelocyte (PML)1 oncogenic domain (POD), also named PML
or ND10, which consists of a dense fibrillar ring flanking a
central core (Ascoli and Maul, 1991 More recently, a unique structure localized at the periphery of the nucleolus, the perinucleolar compartment
(PNC), was identified (Ghetti et al., 1992 Cell Culture
Several human cell lines were grown to subconfluence on 22 × 22 mm
glass coverslips in 35-mm Petri dishes in the appropriate culture medium.
HeLa (epithelial carcinoma, cervix), T84 (colon carcinoma), SW620 (colon adenocarcinoma), WI-38 VA13 (WI-38 cells transformed with SV40
T), Wacar (normal skin fibroblasts), Homa (normal skin fibroblasts), and
MRC5 (normal lung fibroblasts) were grown in DME supplemented with
10% fetal bovine serum (GIBCO BRL, Gaithersburg, MD). The breast
cancer cell lines including MDA-MB-468 (adenocarcinoma) and Hs 578T
(ductal carcinoma) cells were grown in DME supplemented with 15%
FBS. Normal human mammary epithelium (NHME) cells were grown in
mammary epithelium basal medium (MEBM) supplemented with 5 mg/liter transferrin, 1 mg/liter hydrocortisone, 5 µM isoproterenol, 10 mg/liter
insulin, 5 µg/liter epithelium growth factor (EGF), and 0.2% bovine pituitary extract. MG63 (osteosarcoma), Detroit 551 (normal skin fibroblasts),
and WI-38 (normal lung fibroblasts) cells were grown in minimal essential medium (MEM) supplemented with 10% FBS. Cells were maintained at
37°C with 10% CO2.
Construction of GFP Fusion Proteins
Human PTB cDNA (Gil et al., 1991 Transfection
Expression constructs were transiently transfected into HeLa cells by
electroporation (Sambrook et al., 1989 Immunolabeling
SH54 monoclonal antibody was raised against HeLa nuclear extracts. The
antibody was selected by its specific labeling of the PNC in the nucleus.
SH54 recognizes a 57-kD protein on a Western blot (see Fig. 4 A) and was
confirmed to bind PTB specifically.
Subconfluent cells grown on the glass coverslips were fixed in freshly
made 2% formaldehyde in PBS for 15 min, and cells were washed 3 × 10 min each in PBS and incubated with anti-PTB primary antibody SH54 at a
dilution of 1:300, anti-Sm antibody (Lerner and Steitz, 1979 Photooxidation
Immunoelectron microscopic localization was performed according to the
preembedding photooxidation procedure (Deerinck et al., 1994 Cells were examined using a 35-mm microscope (Axiovert; Zeiss Inc.)
equipped with an MRC-1024 laser scanning confocal system (BioRad) using the 488 nm excitation line from an argon/krypton laser. During imaging the cells were kept at 10°C using a cold stage and were in 0.1 M sodium
cacodylate that had been deoxygenated by bubbling with argon to retard
photobleaching.
Once an area had been selected using a 40× 1.3 NA objective (Planapo; Zeiss Inc.), a cold oxygenated solution of 1 mg/ml diaminobenzidine tetrahydrochloride (DAB; Sigma Chemical Co.) in 0.1 M sodium cacodylate, pH 7.2, was added to the cells. Photooxidation of the DAB by eosin was accomplished by illuminating the region of interest with 515 nm light
from 100 W mercury lamp. The progress of the reaction was monitored by
transmitted light, and illumination was halted when a brownish reaction
product became visible (10-20 min). Both experimental and control preparations were reacted under identical conditions.
After photooxidation, the cells were rinsed five times for 2 min in 0.1 M
sodium cacodylate and then postfixed in 1% osmium tetroxide for 1 h.
Cells were then rinsed in double distilled water, dehydrated in an ethanol
series, and infiltrated with Durcupan ACM resin (Electron Microscopy
Sciences, Ft. Washington, PA). After polymerization of the resin for 24 h
at 60°C, the bottom glass coverslip was removed from the dish, and the region of interest was cut out and mounted for ultramicrotomy with an ultramicrotome (Ultracut E; Leica Inc., Deerfield, IL) using a diamond knife (Diatome U.S., Ft. Washington, PA). Electron micrographs were recorded from sections 80 nm in thickness at 60-80 kV with a transmission electron microscope (100CX; Jeol Ltd., Tokyo, Japan) and sections 1 µm
thick at 300 kV with an intermediate voltage transmission electron microscope (4000EX; Jeol Ltd.). Stereo-pair images were recorded by tilting the
specimen stage to ± 8°. Sections of photooxidized cells were not poststained.
Structural Characterization by Correlative
Electron Microscopy
To examine the structure of the PNC in optimally fixed cells at high resolution, transiently expressed GFP-PTB was used as a marker to indicate
the localization of the PNC. The corresponding nuclear region identified
by fluorescence of the GFP-PTB fusion protein was examined by electron
microscopy. Specifically, transfected cells were seeded on gridded coverslips. 12 h after transfection, cells were fixed in 4% paraformaldehyde
with 0.05% glutaraldehyde in PBS, and cells that showed the PNC were
quickly examined and photographed using an epifluorescence microscope
(FXA; Nikon Inc.) equipped with a SenSys cooled CCD camera (Photometrics Inc., Tucson, AZ) using Oncor Image software. Subsequently, cells
were fixed in 2% glutaraldehyde for 20 min and washed in PBS containing
0.3 M glycine and 0.1 M cacodylate buffer. Cells were then postfixed in
1% osmium tetroxide for 1 h and dehydrated by incubation in a series of
ascending concentrations of ethanol and embedded in epon/araldite at
60°C for 48 h. 80-nm sections were poststained with uranyl acetate/lead citrate and examined at 75 kV with a transmission electron microscope
(H-7000; Hitachi Scientific Instrs., Mountain View, CA). The same cell
photographed at the fluorescence microscopic level was located, and the
nuclear region that corresponded to the PNC was identified and photographed.
Observation of the PNC in Living Cells
Living cell studies were performed using an FCS2 live cell chamber
(Bioptechs, Inc., Butler, PA). Transfected cells were grown on the customized coverslips that were assembled into the FCS2 chamber, where
temperature was maintained at 37°C, and cells were supplemented with a
controlled flow of fresh medium. The chamber was directly mounted onto
the specimen stage of an inverted epifluorescence microscope (Axiovert
405M; Zeiss, Inc.) equipped with a cooled CCD (NU200; Photometrics
Inc.) camera. Images were captured using Oncor Image software.
The Presence of the PNC Correlates with the
Transformed Phenotype
Since PTB is the only protein component of the PNC thus
far identified, we used a monoclonal antibody, SH54, that
specifically recognizes PTB (see Materials and Methods
and Fig. 4 a) to characterize the PNC in cell nuclei. A large
number of human cancer cell lines and normal human diploid cell lines were examined for the presence of the PNC.
The results showed that the PNC is predominantly present in cancer cells and is rarely observed in normal primary
cells (Figs. 1 and 2). PNC prevalence, defined as the percentage of cells that contains one or more PNCs, showed a
large diversity among the cancer cell lines examined. Some
of the cell lines such as HeLa (endothelial carcinoma, cervix) and T84 (colon carcinoma) showed a PNC prevalence
of >80%. In contrast, the PNC prevalence of other cell
lines such as SW620 and MG63 ranged from 25 to 50%. The large variability of PNC prevalence among cancer,
transformed, and primary diploid cells suggests that PNC
prevalence may be correlated with the degree of malignancy. To examine this possibility, we compared PNC
prevalence in two human breast cell lines. We found that
PNC prevalence appeared to be fivefold higher in MDAMB-468 cells (American Type Culture Collection, Rockville, MD) isolated from a metastatic mammary adenocarcinoma that induces tumors in nude mice, than in Hs 578T
cells (American Type Culture Collection) isolated from a
mammary ductal carcinoma that does not induce tumors
in nude mice (Fig. 2). In addition, when primary human diploid fibroblasts (WI-38 cells) were transformed by
SV40 large T antigen (WI-38 VA13), the PNC prevalence
rose to >60% compared to 2% in the nontransformed parental cells (Fig. 2). It is also noticeable that the size of
PNCs varies from cell to cell (Fig. 1). In some cancer cells,
the structure tended to be larger (Fig. 1, HeLa and WI-38
VA13 cells) and occasionally extended into the nucleolus,
whereas, in other cancer cells, the structure was mostly observed as small dots (Fig. 1, MG63 cells). However, in the
very small percentage of normal cells that contains the
PNC, the PNC invariably appeared to be a small dot (data
not shown).
Characterization of the PNC through the Cell Cycle
We have further characterized the PNC in HeLa cell nuclei throughout the cell cycle by immunolabeling using
SH54. The PNC is clearly associated with nucleoli in interphase cells. The shape and size of the PNC varies from cell
to cell within the same cell line. As cells progress through
mitosis, the PNC dissociates in prophase cells (Fig. 3 D).
The dissociation appears to be a gradual event. When double labeled with the anti-fibrillarin antibody, a more concentrated PTB labeling is still visibly associated with the
partially disassembled nucleolus at early prophase (Fig.
3, D and E, arrows). Both PTB and fibrillarin are diffusely
localized in metaphase cells (Fig. 3, G and H). At late telophase, the PNC begins to form in the new daughter cell
nuclei before the re-entry of the entire nucleoplasmic population of PTB (Fig. 3 J, arrows). Double labeling experiments using the anti-fibrillarin antibody showed that the initial detectable PNCs are spatially associated with nucleolar
regions (Fig. 3, J and K, arrows). Double labeling experiments using the anti-Sm antibody showed that the re-
establishment of splicing factors in the new daughter cell
nuclei takes place before the formation of the PNC (data not
shown). In addition, the formation of the PNC did not appear to be affected by the presence of cycloheximide (data
not shown), suggesting that the newly formed PNC is composed of cellular components from the previous cell cycle.
Characterization of the PNC in Living Cells
To directly visualize the PNC in living cells, a mammalian
expression vector was constructed that expresses a GFP-
PTB fusion protein. Human PTB cDNA (Gil et al., 1991 Using the GFP-PTB fusion protein as a probe, we observed the PNC in living cells. Cells were transiently transfected with the GFP-PTB construct. 12 h after transfection, cells grown on glass coverslips were moved to a live
cell chamber (see Materials and Methods). The chamber
was mounted onto the stage of a fluorescence microscope,
and images were captured at various time intervals. The
exposure of cells to the excitation light was kept minimal. Time lapse observations demonstrated that the PNC is a
dynamic structure that makes small movements at the
periphery of and occasionally into the nucleolus over time
(Fig. 5). Closer examination revealed that the PNC appears to contain substructures whose organization also
changes through time (Fig. 5, insets). These substructures appeared to be strand like in some cases (Fig. 5, inset, 2 h). In many cells, the PNC changed shape and position within
2-3 h. GFP-PTB did not appear to have an immediate toxicity to transfected cells, as cells expressing GFP-PTB progressed through multiple cell cycles and newly divided
cells expressed the fusion protein (data not shown).
High Resolution Electron Microscopic Examination of
the PNC
To further analyze the structural characteristics of the
PNC at higher resolution, we examined the PNC in HeLa
cells by electron microscopy. Photooxidation pre-embedding immunolabeling was used to achieve a high degree of
sensitivity and good morphological preservation (Deerinck
et al., 1994
To visualize the basic structural features of the PNC in
optimally fixed cells at high resolution, we examined the
PNC in the same cell by fluorescence and electron microscopy. To mark the PNC without immunocytochemical
manipulations, the expression of GFP-PTB was used as a
marker to indicate the localization of the PNC in transiently transfected cells. Transfected cells were photographed,
optimally fixed, and processed for the electron microscopic examination (see Materials and Methods). The fluorescence and electron microscopic images of the same
cell are shown in Fig. 8. The nuclear region corresponding
to the PNC appeared to be an electron-dense structure,
which is composed of thick, short strands measuring ~80-
180 nm diam. Each strand appears to be surrounded by
lesser electron-dense areas. The strand-like structure may
correspond to the heterogeneous labeling of the PNC observed in the immunoelectron microscopic examination
using SH54 (Fig. 6). Some of the strands are directly
linked to the surface of the nucleolus (Fig. 8 C, arrows).
Similar structures were observed in multiple sections. In
some sections, the strands were as long as 1 µm. It is possible that in three dimensions the strands are connected,
forming a continuous structure.
Sequence Requirement for Targeting PTB to the PNC
To determine the sequence requirement for targeting
GFP-PTB to the PNC, we generated a series of deletion
mutants of the GFP-PTB fusion protein and analyzed the
subcellular localization of these mutant fusion proteins.
The mutant proteins were designed according to the structural characteristics of PTB, which has been previously shown to contain four RNA recognition motifs (RRMs;
Patton et al., 1991
Table I.
Characteristics of the GFP-PTB Deletion Mutant
Fusion Proteins
). The use of increasingly sophisticated molecular techniques and the availability of a large number of
antibodies and also nucleic acid probes has advanced our
understanding of the temporal and spatial organization of
nuclear functions, as well as revealed the complex nature
of the mammalian cell nucleus.
).
One of the more extensively studied examples is the coiled
body. Coiled bodies were first described by Ramon and
Cajal (1903) as nucleolar accessory bodies. These generally round structures, 0.5-1.0 µm diam, consist of coiled
fibrillar strands (Monneron and Bernhard, 1969
). In addition to small nuclear RNPs, several nucleolar components and a coiled body-specific protein, coilin, have been found
in these structures (for reviews see Brasch and Ochs, 1992
;
Lamond and Carmo-Fonseca, 1993
; Gall et al., 1995
).
However, [3H]uridine incorporation studies showed little
to no labeling of these bodies after a short pulse (Moreno
Diaz de la Espina et al., 1980), suggesting that coiled bodies are unlikely to be the sites of active RNA synthesis. In
addition, the absence of essential pre-mRNA splicing factors such as SC35 and SF2/ASF (Raska et al., 1991
; Huang
and Spector, 1992
; Lamond and Carmo-Fonseca, 1993
; Krainer, A., and D. Spector, unpublished observations) in
these structures suggests that they are probably not the
sites of active splicing. The number of coiled bodies per
nucleus and the percentage of cells that contain coiled
bodies increase dramatically in immortalized cells or cancer cells as compared to primary cells (Spector et al., 1992
).
In addition, coiled bodies have been found to be within the
nucleoli in some cell lines derived from breast cancer
tissues (Ochs et al., 1994
). However, the function of the
coiled body remains elusive. More recently, a novel nuclear
structure, gems, was identified in close proximity to coiled
bodies (Liu and Dreyfuss, 1996
). Gems are similar to
coiled bodies in number and size, response to metabolic
conditions, and their dynamics through the cell cycle. However, gems and coiled bodies contain different macromolecular components. Survival of motor neuron protein,
involved in the genetic disease spinal muscular atrophy,
is localized in gems. Components that are localized in
coiled bodies such as snRNPs, coilin, and fibrillarin are not
present in gems (Liu and Dreyfuss, 1996
). The function of
gems is currently unknown.
; Dyck et al., 1994
; Koken et al., 1994
, 1995; Weis et al., 1994
; Terris et al., 1995
).
The POD was defined by immunolabeling with an antibody specifically recognizing the PML protein in hematopoietic cells. Several other autoimmune antibodies also react with components in the POD (Ascoli and Maul, 1991
).
The POD becomes fragmented into a large number of microparticulates in acute promyelocytic leukemia (Dyck et
al., 1994
; Koken et al., 1994
; Weis et al., 1994
). The break
up of the POD into microparticulates is related to the formation of the PML-retinoic acid receptor
fusion protein resulting from a t(15;17) translocation (Dyck et al., 1994
;
Koken et al., 1994
; Weis et al., 1994
). When these cells are
treated with retinoic acid, the fragmented PML particulates fuse and reassemble into PODs along with the initiation of cellular differentiation and the loss of the PML-retinoic acid receptor
fusion protein. This observation
provided evidence that alterations of nuclear structure
may play a role in this form of carcinogenesis (Dyck et al.,
1994
; Koken et al., 1994
; Weis et al., 1994
). In addition to
being present in hematopoietic cells, the POD has recently been detected in a variety of tissues including hepatocytes,
endothelium, epithelium, and connective stroma (Koken
et al., 1995
; Terris et al., 1995
). The expression of PML and
its immunolabeling pattern, in some of the samples examined, appeared to change depending upon the proliferating or cancer state. However, the function of the POD is
presently unknown.
; Matera et al.,
1995
). The PNC has been shown to contain several small
RNAs transcribed by RNA polymerase III, including
RNase P, MRP RNAs, and multiple Y RNAs (Matera et
al., 1995
; Lee et al., 1996
), as well as the polypyrimidine
tract binding protein (PTB; Ghetti et al., 1992
; Matera et
al., 1995
). However, the structural and functional characteristics of the PNC have not yet been extensively studied.
In this paper, we examined the PNC in human cancer and
normal cell lines. We also characterized the PNC in fixed
cells using light and electron microscopy and in living cells
through time lapse observations. In addition, we analyzed
the sequence requirements to target a green fluorescent protein (GFP)-PTB fusion protein to the PNC.
Materials and Methods
) was amplified by PCR using Vent
DNA polymerase (New England Biolabs Inc., Beverly, MA). The amplified fragment was inserted in frame into a GFP expression construct,
pEGFP-C1 (Clontech Laboratories, Inc., Palo Alto, CA), at the HindIII
and BamH1 sites. The fusion protein contained GFP at the NH2 terminus
of PTB. Subsequently, deletion fragments of PTB were generated by PCR
using specific primers and were inserted into pEGFP-C1, which gave rise
to a series of mutant fusion proteins.
). Briefly, subconfluent cells in a
100-mm culture dish were collected by trypsinization and mixed with 20 µg
of DNA including 7 µg target DNA and 13 µg sheared salmon sperm
DNA. A 280 µl mixture of cells in DME with 10% FCS and DNA was
electroporated in a BioRad (Richmond, CA) electroporator at 270 V and
960 µFaraday. Cells were subsequently seeded onto glass coverslips in 35mm petri dishes and were grown for either 7 or 24 h.
Fig. 4.
(A) Both the GFP-
PTB fusion protein and endogenous PTB are expressed in transfected cells
and are detected at the expected size by Western blot
using antibody SH54 (lane
a). Only endogenous PTB is
detected in cells transfected
with the GFP construct alone (lane b). (B) The subcellular
localization of GFP-PTB is
indistinguishable from endogenous PTB. The endogenous PTB (a, red) and the
GFP-PTB fusion protein (b,
green) are colocalized to
both the PNC (arrows) and
the nucleoplasm. The PNC
observed in transfected cells
is not the result of the overexpression of the protein, as
some cells do not contain a PNC in spite of expressing large amounts of the fusion protein (c and d, left cell). c shows both the immunolabeling of endogenous PTB and transiently expressed GFP-PTB; d shows GFP-PTB. Bar, 10 µm.
[View Larger Versions of these Images (17 + 25K GIF file)]
) at 1:1,000,
anti-SC35 (Fu and Maniatis, 1990
) at 1:1,000, or anti-fibrillarin (Sigma
Chemical Co., St. Louis, MO) at 1:5 for 1 h at room temperature. Cells
were rinsed in PBS and then incubated with Texas red-conjugated goat
anti-human or FITC-conjugated goat anti-mouse antibody at a dilution of
1:40 for 1 h at room temperature followed by three washes in PBS. The coverslips were mounted onto glass slides with mounting medium containing 90% glycerol in PBS with 1 mg/ml paraphenylenediamine as an antifade agent. The mounting medium was adjusted to pH 8.0 with 0.2 M bicarbonate buffer. Cells were examined with a microscope (FXA; Nikon
Inc., Melville, NY) equipped with epifluorescence and differential interference contrast optics. Images were captured by a SenSys cooled CCD
camera (Photometrics, Tucson, AZ) using Oncor Image software.
). After incubation of the primary antibody (as described in the immunolabeling
procedure), cells were washed twice for 2 min in 0.1 M PBS and incubated
in 0.1 M PBS with 1% normal goat serum (NGS), 1% cold water fish gelatin, and 1% BSA (fraction V) for 20 min to block nonspecific staining.
Cells were then incubated with goat anti-mouse eosin-5-isothiocyanate
(conjugated as previously described, Deerinck et al., 1994
) diluted in 0.1 M
PBS with 1% BSA and 1% NGS for 1 h. Unbound conjugate was removed by washing six times for 5 min in 0.1 M PBS with 1% BSA and 1%
NGS followed by two times in 5 min washes with 0.1 M sodium cacodylate, pH 7.4. All washes and incubations were at 4°C.
Results
Fig. 1.
The PNC is predominantly present in human cancer cells.
PNCs were detected by immunolabeling using antibody SH54.
The left column shows the presence of the PNC in human cancer
cells (arrowheads), including HeLa (epithelia carcinoma), MG63
(osteosarcoma), MDA-MB-468 (mammary adenocarcinoma), and
WI-38 VA13 (human lung fibroblast cells WI-38 transformed by
SV40 T). The right column shows that the PNC is generally not
present in normal human diploid cells including Detroit 551 (skin
fibroblast), Wacar (skin fibroblast), HNME (human normal mammary epithelium), and WI-38 (lung fibroblast). Bar, 10 µm.
[View Larger Version of this Image (38K GIF file)]
Fig. 2.
The percentage of cells that contain the PNC (PNC
prevalence) is correlated with the transformed phenotype. The
histogram indicates the statistical evaluation of PNC prevalence
among human cancer cells and normal diploid cells. 500 cells
from each cell line were examined.
[View Larger Version of this Image (49K GIF file)]
Fig. 3.
The PNC dissociates at prophase and reforms at late telophase. The horizontal rows show different stages of mitosis. The
left column shows the immunolabeling of the PNC with antibody
SH54, the center column the immunolabeling of fibrillarin with
human anti-fibrillarin antibody, and the right column DNA staining by Dapi. The dissociation of the PNC at prophase (D) appears to be a gradual event. A concentrated PTB labeling is still
spatially linked with the partially dissociated nucleolus (D and E,
arrows). Both PTB (G) and fibrillarin (H) are diffusely distributed in metaphase cells. The earliest detectable PNCs (J, arrows)
in the daughter cell nuclei are associated with nucleolar regions
(K, arrows). Bar, 10 µm.
[View Larger Version of this Image (68K GIF file)]
)
was amplified by PCR and inserted into the pEGFP-C1
vector (Clontech Laboratories, Inc.) generating a fusion
protein in which PTB is at the COOH-terminal end of GFP.
The construct was transiently transfected into HeLa cells,
and the expression of GFP-PTB was examined. Proteins
from cells transfected with either GFP-PTB or GFP alone
were analyzed by Western blot using the SH54 antibody
(Fig. 4 A). Cells transfected with the GFP-PTB construct
expressed the fusion protein of expected size (84 kD) and
the endogenous PTB (57 kD; Fig. 4 A, lane a). Cells transfected by GFP alone showed only the endogenous PTB protein (Fig. 4 A, lane b). To examine if the subcellular localization of the fusion protein is similar to that of the endogenous PTB, the intranuclear localization of the fusion
protein and endogenous PTB was compared (Fig. 4 B). 24 h
after transfection, cells were fixed, immunolabeled with
the SH54 antibody, which recognizes both endogenous
PTB and GFP-PTB, and subsequently probed with Texas
red-conjugated secondary antibody (Fig. 4 B, a, red signal). GFP-PTB alone emitted a green signal (Fig. 4 B, b).
Comparisons between a and b reveal that the GFP-PTB
fusion protein and the endogenous PTB were colocalized
to the same nuclear regions, which included both the PNC
(Fig. 4 B, a and b, arrows) and the nucleoplasm. To exclude the possibility that the formation of the PNC may be due to the overexpression of the GFP-PTB fusion protein,
we examined cells that do not contain a PNC, as determined by immunolabeling. We found that the expression
of GFP-PTB at a high level (roughly evaluated by the intensity of the green fluorescence) did not induce the formation of the PNC (Fig. 4 B, c and d, left cell).
Fig. 5.
The PNC is a dynamic structure with small movements over time when observed in living cells. This figure illustrates the observation of a single cell through time as indicated in all frames. The PNC changes its shape and position over a 3-h period. In the insets, the PNC appears to contain substructures that are reorganized through time. Bar, 10 µm.
[View Larger Version of this Image (38K GIF file)]
). The results of these experiments revealed that
the PNCs are in direct contact with the surface of the nucleolus (Figs. 6 and 7), and occasionally, they extend into
the nucleolus (Fig. 6 C). In addition, the PNCs are irregular in shape. A clear margin of the structure was not observed at this level as compared to what was revealed at
the light microscopic level. The immunolabeling of PTB in
the PNC appeared to be heterogeneous, with some areas
more intensely stained than others. Higher magnification micrographs (Fig. 6, B and C) or stereo images from 1-µm-
thick sections viewed at 300 kV (Fig. 7 B) revealed strandlike structures across the PNC (Figs. 6 and 7 B).
Fig. 6.
The PNC is spatially associated with the nucleolus
when examined at high resolution by electron microscopy. Photooxidation pre-embedding immunocytochemical labeling of the
PNC with SH54 demonstrates that the PNC is in direct contact
with the nucleolus (A-C), and PTB is heterogenously distributed
in the PNC (B and C). The arrowheads indicate the PNC.
[View Larger Version of this Image (141K GIF file)]
Fig. 7.
Stereo pair images of 1-µm thick sections observed at
300 kV using intermediate voltage electron microscopy reveal in
three dimensions that the PNC is directly linked to the surface of
the nucleoli (A). Images at higher magnification show strand-like structures. Bars: (A) 4 µm; (B) 1 µm.
[View Larger Version of this Image (119K GIF file)]
Fig. 8.
Fluorescence and electron microscopic examination of
the PNC in the same cell. The nuclear region corresponding to
the PNC in the fluorescence micrograph was examined in poststained, 80-nm-thick sections. Arrowheads indicate the corresponding nuclear region from fluorescence to electron microscopic images. The PNC is composed of electron-dense, thick,
short strands measuring 80-180 nm diam. These strands are surrounded by less electron-dense areas. Some of the strands are in
direct contact with the surface of the nucleolus. Bars: (A) 10 µm;
(B) 2 µm; (C) 1 µm.
[View Larger Version of this Image (155K GIF file)]
; Ghetti et al., 1992
). Nine mutants (Fig. 9)
containing various numbers of RRMs from either the NH2
or COOH terminus of the protein were made. The inserts
in all mutant constructs were verified by restriction enzyme analysis (data not shown). The constructs were transiently transfected into HeLa cells, and the expression of
the mutant proteins was examined by fluorescence microscopy. All mutant fusion proteins were expressed at a detectable level, as monitored by the expression of GFP. The
subcellular localization of these mutants was examined
(Fig. 10) and compared to the localization of endogenous
PTB (Table I). The result of the analysis demonstrated
that only two mutant fusion proteins, PTB-1 and -4, both
of which contain three RRMs, were localized to the PNC
in transfected cells (Fig. 10 and Table I). Mutants with a
combination of two RRMs, including combinations of RRMs 1 and 2, 3 and 4, or 2 and 3, as well as mutants with only
one RRM were not able to target the fusion protein to the
PNC (Fig. 10 and Table I). These observations suggest
that a minimum of three RRMs are required for GFP-
PTB to be efficiently targeted to the PNC.
Fig. 9.
Deletion mutants were generated to analyze the sequence requirement for PTB to be localized to the PNC. The mutants are designed according to the four RNA recognition motifs
of the protein.
[View Larger Version of this Image (16K GIF file)]
Fig. 10.
The subcellular
localization of a series of deletion mutants of GFP-PTB
fusion proteins. Deletion of
either RRM 1 or 4 does not
affect the localization of the
fusion protein to the PNC
(GFP-PTB 1 and 4, arrows).
However, deletion mutants
that contain fewer than
three RRMs result in the inability to target the fusion
proteins to the PNC (other
frames). Deletion mutants
that contain RRM1 show a
predominantly nuclear localization (top row), whereas
mutants missing RRM1 show
a much more cytoplasmic localization (other frames).
The mutant GFP-PTB with
RRMs 3 and 4 shows that the
cytoplasmic localization becomes dominant over the nuclear localization. Bar, 10 µm.
[View Larger Version of this Image (129K GIF file)]
To examine if the expression of the mutant GFP-PTBs interfered with the localization of endogenous PTB in the PNC, the location of the endogenous PTB was examined by immunolabeling with SH54 (summarized in Table I). The result showed that the expression of the wild-type GFP-PTB and GFP-PTB mutants did not affect the localization of the endogenous PTB in the PNC (Table I). In addition, the expression of these fusion proteins did not appear to affect the subcellular distribution of the nucleolar protein, fibrillarin, or splicing factors such as SC35 (Table I).
In addition to the requirement of three RRMs for the localization of GFP-PTB to the PNC, we have also observed some interesting features of these mutant proteins with respect to their subcellular localization. The GFP- PTB fusion proteins with an intact NH2 terminus gave rise to a predominantly nuclear localization. The cytoplasmic localization of these proteins was hardly detectable (Fig. 10, top row and Table I). In contrast, deletion of the NH2terminal RRM resulted in a much more prominent cytoplasmic localization of these mutant proteins (Fig. 10 and Table I). Particularly, the mutant PTB-5, which contained two RRMs at the COOH terminus, showed a predominantly cytoplasmic localization (Fig. 10). However, neither RRM3 nor RRM4 alone is sufficient to direct a strong cytoplasmic localization (Fig. 10). These findings suggest that the NH2 terminus of PTB may be responsible for the nuclear localization of this protein.
We have examined a large number of human cancer and
normal diploid cells and have found that the presence of
the PNC correlates with the transformed phenotype. Our
findings agree with and further extend unpublished data
described in Matera et al. (1995), stating that the PNC is
found predominantly in transformed cells. The correlation
between the PNC and cancer cells suggests that the formation of the PNC may be the result of oncogenic transformation. Further support for this suggestion comes from
the controlled observation of a pair of cell lines in which
PNC prevalence increased >25-fold from WI-38 cells, normal human lung fibroblasts, to their transformed derivative, WI-38 VA13 cells. In addition, we found that PNC
prevalence varies tremendously among different cancer cell lines examined, suggesting that PNC prevalence may
correspond to the degree of malignancy. This possibility is
supported by the examination of breast cancer cells, in
which PNC prevalence appears to be 5-fold higher in
MDA-MB-468 cells, derived from a breast metastatic carcinoma that induces tumors in nude mice, than in Hs 578T
cells, derived from a breast ductal carcinoma that does not
induce tumors in nude mice. Investigations are currently underway to examine the correlation between the PNC
and human cancer tissues.
The development of cancer is clearly a multi-step process (Knudson, 1971), a chain of events in which the consequence of one change unleashes a cascade of subsequent
alterations. Although the function of the PNC is unknown,
its preferential presence in cancer cells suggests that the
formation of the PNC is part of an evolving process that
transforms normal cells into cancer cells. The formation of
the PNC may result from initial abnormalities and in turn
may promote additional changes during the progression of
cancer. However, it is also possible that the formation of these nuclear structures merely represents a cellular response to overall physiological changes that occur during
cancer formation.
Observations in living and fixed cells at the light and
electron microscopic levels have demonstrated a close association between the PNC and the nucleolus. The association begins during nucleologenesis at late telophase and
extends to prophase before nucleolar dissociation. Such a
close interaction suggests that the PNC may be linked to
nucleolar activities. It has been documented that nucleoli
undergo significant changes during carcinogenesis (for review see Busch, 1981), including increases in the expression of certain nucleolar proteins as well as alterations in
the number and shape of nucleoli. Immunocytochemical
labeling of nucleolar proteins has provided useful markers
in cancer diagnosis and prognosis (Busch, 1981
, 1990
).
However, the functional significance of the changes in nucleoli during carcinogenesis remains unclear. In normal
cells, the function of the nucleolus has long been shown to
be primarily involved in the biogenesis of preribosomal
particles (for reviews see Busch and Smetana, 1970
; Hadjiolov, 1985
; Scheer and Benavente, 1990
); more recently,
studies in the yeast system revealed that nucleoli may also
play a role in the processing and transport of poly(A)+
RNA (Schneiter et al., 1995
; Tani et al., 1995
). During carcinogenesis, nucleolar activities may change to accommodate the changes in cellular physiology. The spatial association between PNCs and the nucleolus as well as the
correlation between PNCs and oncogenic transformation
raise the possibility that the PNC may participate in the
changes in nucleolar activities that occur during carcinogenesis. However, only when structural components and
the function of the PNC are better understood, can its role
in carcinogenesis be addressed.
The PNC is an electron-dense structure consisting of
multiple strands, some of which are directly linked to the
surface of the nucleolus. These thick strands may correspond with strand-like substructures observed in living
cells (Fig. 5). As compared to the coiled body, which is
densely packed with coiled fibrils (Ramon y Cajal, 1903),
the PNC is loosely packed with thick strands that are surrounded by areas of lesser electron density. The PNC seems to bear some resemblance to a structure described by Cohen et al. (1984; Chung et al., 1984). These investigators
identified protuberances that develop at the nucleolar periphery in estrogen-stimulated nerve cells (Chung et al.,
1984
; Cohen et al., 1984
). Examination by electron microscopy showed these structures to be electron dense without
well defined margins. Similar to the PNC, they are linked
to the surface of the nucleolus by strands of electron-dense material (Cohen et al., 1984
). Sodium tungstate staining
and DNase digestion on resinless sections suggested that
they contain DNA (Chung et al., 1984
; Cohen et al., 1984
).
Studies are underway to determine if these protuberances
and the PNC are the same structure.
Using deletion mutagenesis we have analyzed the sequence requirement for PTB targeting to the PNC. We
have found that at least three RRMs at either the COOH
or NH2 terminus are necessary and sufficient for the truncated fusion proteins to be localized to the PNC. It is unlikely that a specific signal involving only a small stretch of
conserved sequences is responsible for the localization of
the protein to the PNC. Analysis of translational regulation by PTB in virus-infected human cells has previously shown that three RRMs are essential and sufficient for an
efficient binding of PTB to the internal ribosomal entry
sites of viral RNA transcripts (Kaminski et al., 1995). Our
finding that three RRMs are needed for PTB to be localized to the PNC suggests that the localization of PTB in
the PNC may be due to its binding to polypyrimidine tract
containing RNA(s). In addition, we have recently found
that the presence of PTB in the PNC is sensitive to RNase A treatment (data not shown), further supporting the idea
that the presence of PTB in the PNC is dependent upon its
RNA binding. Our observations agree with and augment
the finding and suggestion by Matera et al. (1995)
that several small RNA polymerase III transcripts containing pyrimidine-rich sequences, including multiple Y RNAs, RNase P,
and MRP RNAs, are present in the PNC and that PTB
may therefore bind these RNAs. PTB is an RNA-binding
protein preferentially recognizing pyrimidine-rich sequences and has been reported to be involved in multiple
cellular functions including pre-mRNA splicing (Patton et
al., 1993
; Gozani et al., 1994
; Singh et al., 1995
), splice site
selection in alternative pre-mRNA splicing (Lin and Patton, 1995
), RNA polyadenylation (Lou et al., 1996
), and
translational regulation of certain viral RNA transcripts (Hellen et al., 1994
; Kaminski et al., 1995
; Witherell et al., 1995
). Its capability of shuttling between the nucleus and
cytoplasm suggests that PTB may also be involved in RNA
transport (Pinol-Roma and Dreyfuss, 1992; Michael et al.,
1995
). PTB appears to participate in these functions through
the binding to pyrimidine-rich RNA sequences. Thus, it is
possible that PTB serves as a bridge between the pyrimidine tract RNAs and different macromolecules in fulfilling different cellular functions. However, the association and
function of PTB in the PNC remains to be determined.
In summary, we have extensively characterized the PNC both in fixed and living cells using PTB as a probe. We have found that the PNC is a dynamic structure that is in direct contact with the nucleolus. The association initiates at the beginning of the cell cycle and ends at prophase. The PNC appears to be an electron-dense structure. It is composed of multiple strands, each measuring 80-180 nm diam. Some of the strands are in direct contact with the surface of the nucleolus. Deletion mutagenesis of GFP- PTB indicates that at least three RRMs, either NH2 or COOH terminal are required for PTB to be localized to the PNC, suggesting that the role of PTB in the PNC may involve its binding to polypyrimidine tract-containing RNAs. Furthermore, the presence of the PNC is closely associated with oncogenic transformation. Studies are underway to further analyze the structural components and functional characteristics of the PNC.
Received for publication 10 February 1997 and in revised form 21 March 1997.
1. Abbreviations used in this paper: GFP, green fluorescent protein; PML, promyelocyte; PNC, perinucleolar complex; POD, PML oncogenic domain; PTB, polypyrimidine tract binding protein; RRM, RNA recognition motif.