University of Dundee, MSI/WTB Complex, School of Life Sciences, Dow Street, Dundee DD1 5EH, UK
* Author for correspondence (e-mail: a.i.lamond{at}dundee.ac.uk)
Accepted 29 January 2003
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Summary |
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Key words: SMN, Coilin, Nucleus, Cajal bodies, Nucleolus, Mitosis
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Introduction |
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The nuclear bodies termed gems (gemini of Cajal bodies) are defined by the
presence of the protein SMN (Liu and
Dreyfuss, 1996). As their name suggests, they are closely related
to Cajal bodies (CBs), which are usually defined by the presence of the marker
protein p80-coilin (Andrade et al.,
1991
; Raska et al.,
1991
). In many cell types, both in vitro and in vivo, the two
structures co-localise. CBs and gems appear as different structures, however,
in some cell lines and in many fetal tissues
(Matera and Frey, 1998
;
Young et al., 2000
;
Sleeman and Lamond,
1999b
).
Splicing small nuclear ribonucleoproteins (snRNPs, U1, U2, U4/U6 and U5)
are subunits of spliceosomes. They are ribonucleoproteins comprising an snRNA
core together with the common, Sm, proteins and several proteins unique to the
different snRNPs. Four of the five spliceosomal snRNAs (U1, U2, U4 and U5) are
transcribed by RNA polymerase II. Following their transcription they are
exported into the cytoplasm where they undergo modification of their 5'
termini to form a trimethylguanosine (TMG) cap. The core, Sm, proteins are
also assembled in a ring structure around the snRNA
(Kambach et al., 1999a;
Kambach et al., 1999b
). Only
once these modifications are complete are the nascent snRNPs re-imported into
the nucleus where further maturation occurs before they function in splicing
(Fischer et al., 1993
;
Hamm et al., 1990
;
Lehmeier et al., 1994
;
Lerner and Steitz, 1979
;
Lührmann et al., 1990
;
Mattaj, 1986
;
Nagai and Mattaj, 1994
;
Raker et al., 1996
). SMN has a
role in the cytoplasmic maturation of snRNPs, most probably in the assembly of
the Sm core (Fischer et al.,
1997
; Pellizzoni et al.,
1999
; Yong et al.,
2002
), with a possible additional role in the re-import process
(Narayanan et al., 2002
;
Massenet et al., 2002
). Thus,
it is probable that the SMN complex is associated with snRNPs throughout the
cytoplasmic stages of their biogenesis. Upon reentry into the nucleus, CBs are
the first sites of accumulation of newly assembled snRNPs
(Carvalho et al., 1999
;
Sleeman and Lamond, 1999a
),
suggesting that the CB may have a function in later stages of snRNP
modification or assembly. Furthermore, small nuclear RNAs capable of
functioning as guide RNAs for 2'-O-methylation and pseudouridylation
have recently been identified as CB components
(Darzacq et al., 2002
). Studies
in cell lines containing separate CBs and gems demonstrate that this putative
role in the nuclear maturation of snRNPs specifically involves CBs that also
contain SMN (Sleeman et al.,
2001
). Thus SMN and coilin may both have roles in nuclear stages
of snRNP maturation. Coilin has been implicated as a molecular link,
recruiting SMN, its associated proteins and possibly snRNPs into the CB
(Hebert et al., 2001
).
The presence, in some cell lines, of nuclear bodies containing coilin in
the absence of any SMN-complex proteins suggests that coilin-positive bodies
and coilin itself may also have other roles within the nucleus. Conversely,
the presence of gems containing SMN and its associated proteins in the absence
of coilin, suggests that SMN may also have roles independent of coilin. The
dynamic behaviour of CBs has been studied in animal and plant cells using a
variety of resident proteins as markers, including SmD1
(Sleeman et al., 1998), coilin
(Platani et al., 2000
;
Platani et al., 2002
),
fibrillarin (Snaar et al.,
2000
) and U2B''
(Boudonck et al., 1999
). These
studies demonstrate that CBs are dynamic structures within nuclei, showing a
variety of movements including the joining of CBs, separation of large CBs
into smaller CBs and spatial interactions of CBs with each other, with
nucleoli and probably also with chromatin. In this study, we have established
several cell lines that stably express GFP-SMN and CFP-SMN to examine the
dynamics of SMN-positive CBs that will probably be involved in snRNP
maturation and to compare the dynamics of the CB proteins SMN and coilin both
in interphase cells and through mitosis.
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Materials and Methods |
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Establishment of stable cell lines
Stable cell lines were established using G418 selection of HeLa cells
following transfection using Effectene transfection reagent (Qiagen) as
described previously (Sleeman et al.,
2001).
Cell culture and transfection assays
Cells were grown in Dulbecco's modified Eagles' medium (DMEM) supplemented
with 10% fetal calf serum and 100 U/ml penicillin and streptomycin (Life
Technologies). Medium used to maintain stable cell lines also contained 200
µg/ml G418 (Life Technologies). For immunofluorescence assays, cells were
grown on coverslips and transfected (if necessary) using Effectene
transfection reagent (Qiagen) according to the manufacturer's instructions.
For the preparation of cell lysates, cells were grown in 10-cm diameter
dishes. For live cell microscopy, cells were grown on 32 mm coverslips
(Intracel).
Cell fixation and immunostaining
Cells were grown on glass coverslips and fixed for 10 minutes in 3.7%
paraformaldehyde in 37°C PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM
EGTA, 2 mM MgCl2, pH 6.9). Following a 10-minute permeabilisation
with 1% Triton X-100 in phosphate-buffered saline (PBS), cells were blocked
with 1% donkey serum for 10 minutes and then incubated with primary antibodies
for 30 minutes, washed, and incubated with secondary antibodies (Jackson Labs)
for 30 minutes. If required, cells were stained with DAPI (0.3 µg/ml;
Sigma). After a final set of washes, cells were mounted in Vectashield medium
(Vector Labs). Primary antibodies used were Y12 anti-Sm
(Pettersson et al., 1984), 204
anti-coilin (Bohmann et al.,
1995
), MANSIPI anti-SIP1/Gemin2 (a gift from G. Morris) and Ab1
anti-TMG (Calbiochem).
Preparation of cell lysates, immunoblotting and
immunoprecipitation
Cells were washed twice with ice-cold PBS and then lysed in 0.5 ml of
ice-cold 50 mM Tris-HCl pH 7.5; 0.5 M NaCl; 1% (v/v) Nonidet P-40; 1% (w/v)
sodium deoxycholate; 0.1% (w/v) SDS; 2 mM EDTA plus Complete protease
inhibitor cocktail (Roche, one tablet per 25 ml). The lysate was passed
through a Qiashredder column (Qiagen) to break up the DNA and then cleared by
centrifugation for 15 minutes at 4°C and 13,000 rpm. Lysates were
electrophoresed on an 8% SDS-polyacrylamide gel and transferred to
nitro-cellulose membranes for immunoblotting. Immunoprecipitation using
anti-green fluorescent protein (GFP) antibodies (Roche) was performed as
described previously (Trinkle-Mulcahy et
al., 2001). Primary antibodies used were anti-GFP mouse monoclonal
(Roche), MANSMA1 anti-SMN (Young et al.,
2000
) and MANSIP1 anti-Gemin2/SIP1 (a gift from G. Morris).
FACS analysis
Cells were harvested by trypsinisation and fixed in 70% ethanol for 3 hours
at 4°C. Cells were stained with propidium iodide (25 µg/ml) containing
RNase A (100 µg/ml). Fluorescence was measured using a FACScan (Becton
Dickinson). Data analysis was performed using Cell Quest software (Becton
Dickinson).
Microscopy of fixed cells
Immunostained specimens were examined using a Zeiss 100x NA 1.4
PlanApo objective. Images were recorded on a Zeiss DeltaVision Restoration
microscope (Applied Precision) equipped with a 3D motorised stage and a Roper
Scientific Micromax camera containing a Sony Interline 1300 CCD. For each
cell, optical sections separated by 200 nm were recorded using a binning of
2x2. Images were restored using a constrained iterative deconvolution
algorithm using an empirically measured point-spread function.
Microscopy of live cells
For live cell microscopy, cells were grown on glass coverslips and mounted
in phenol-red free medium in a closed, heated chamber (Bioptechs FCS2).
Imaging and restoration were performed essentially as for fixed cells, but
using a 500 nm separation of optical sections. Time points were taken between
3 minutes and 5 minutes apart.
Photobleaching analyses
Short time-course fluorescence recovery after photobleaching (FRAP)
analyses were performed using a Zeiss 510 confocal laser scanning microscope
equipped with an argon-krypton laser. Cells were maintained at 37°C in a
closed chamber (Helmut Saur POC). Using a 63x, 1.4NA PlanApo lens
(Zeiss) and the 488 nm laser line, image acquisition was performed using 10%
of laser power. Photobleaching was performed using 80% of laser power and 100
iterations. Following bleaching, images were collected at 4- or 5-second
intervals using 10% laser power. A pinhole setting of 1.32 Airy units was used
for image collection. Quantitation was performed using the LSM510 software.
Long time-course FRAP analyses were performed using a Zeiss 410 confocal laser
scanning microscope. A series of optical sections were taken using a 488 nm
laser at 1/30 of full power. Bleaching was performed using the laser at full
power, using 6 iterations, each with a dwell time of 0.24 milliseconds per
pixel. Z-series of images were then collected at 5-minute intervals using 1/30
of laser power. Fluorescence loss in photobleaching (FLIP) analyses were
performed using bleaching settings as above, with series of images collected
before the initial bleach and after each bleaching event. Quantitation was
performed using SoftWorx software (Applied Precision) as previously described
(Sleeman et al., 2001).
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Results |
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We analysed the expression levels of the tagged proteins in the stable cell lines. Lysates of total protein from cell lines CFPSMNE8.8 (Fig. 2A) and GFP-SMNE18.6 (data not shown) were separated by SDS PAGE, transferred to nitrocellulose and probed with anti-SMN and anti-FP antibodies (see Materials and Methods). Both antibodies show a single band of the expected size for FP-SMN (68 kDa). Detection with anti-SMN antibodies shows a second band, representing endogenous SMN (38 kDa), and demonstrates that CFP-SMN is expressed at a lower level than endogenous SMN (Fig. 2A, left-hand panel). Similar analysis of total protein lysates from the parental HeLa cell line and from HeLa cells transiently transfected with a plasmid expressing CFP alone show a single band representing endogenous SMN (Fig. 2A, left-hand panel). Probing with anti-FP antibodies shows a single band in the lysate from cell line CFP-SMNE8.8 representing CFP-SMN (Fig. 2A, right-hand panel), confirming that the CFP signal seen in the cell line is full-length CFP-SMN. No free CFP or truncated CFP fusion proteins are detected.
|
We next tested whether the stably expressed FP-fusion proteins can be immunoprecipitated with anti-FP antibodies and whether they interact with the core SMN-complex protein SIP1/Gemin2. Both GFP-SMN (Fig. 2B) and CFP-SMN (data not shown) can be efficiently immunoprecipitated using antibodies to GFP. The left-hand panel, detected with anti-SMN, shows total lysate from cell line GFP-SMNE18.6 (input) and the material immunoprecipitated using anti-FP antibodies. The right-hand panel shows duplicate samples detected using anti-SIP1/Gemin2 antibodies. This confirms that a large proportion of the SIP1/Gemin2 from this cell line is co-immunoprecipitated with GFP-SMN. Thus, FP-SMN is in a complex with endogenous SIP1/Gemin2, a core member of the SMN complex, in the stable cell lines.
We next analysed the growth properties of the stable cell lines to see whether expression of FP-SMN affects cell cycle progression. The growth rate of each FP-SMN cell line is similar to that of the parental HeLa cell line (data not shown). Furthermore, FACS analysis (Fig. 2C) demonstrates that, in both stable cell lines, a similar proportion of cells are in G1, S and G2 stages of the cell cycle as in the parental HeLa cell line.
We conclude that constitutive low-level expression of FP-SMN in stable HeLa cell lines does not prevent cell cycle progression or alter their growth rate. Furthermore, the FP-tagged proteins maintain in vivo interactions shown by the endogenous SMN protein.
Over-expression of FP-SMN induces cytoplasmic structures containing
SMN complex and core snRNP proteins
We observed that, both following transient transfection of cells with
plasmid vectors encoding FP-SMN, and during early stages of the establishment
of the stable cell lines, some cells expressed high levels of FP-SMN as judged
by fluorescence microscopy. These cells did not survive the selection process
and no cell lines expressing high levels of FP-SMN were obtained, suggesting
that over-expression of SMN may be toxic. Closer examination of HeLa and MCF-7
cells over-expressing SMN showed that they had large accumulations of FP-SMN
in the cytoplasm, coupled with either an absence of nuclear CBs
(Fig. 3B,E) or a large number
of very small nuclear CBs (Fig.
3H,K,N). The cytoplasmic FP-SMN accumulations also contained the
SMN complex protein SIP1/Gemin2 (Fig.
3A-F, arrows), and the core snRNP, Sm proteins
(Fig. 3G-I). They did not
contain either TMG-capped snRNA (Fig.
3J-L, arrows) or the CB protein, coilin
(Fig. 3M-O). The small nuclear
bodies seen contain Sm proteins (Fig.
3G-I), TMG-capped snRNA (Fig.
3J-L, arrows) and coilin (Fig.
3M-O, arrows). Thus, whereas constitutive low expression of FP-SMN
has no obvious deleterious effects on cells, transient over-expression appears
toxic and results in the accumulation of both the SMN-complex and Sm proteins
in the cytoplasm, and an additional disruption of nuclear bodies.
|
To confirm that this effect was caused by increased levels of FP-SMN, cells from line GFP-SMNE18.6 were transiently transfected with plasmid pECFP-SMN expressing CFP-SMN (Fig. 4). This resulted in cytoplasmic accumulations of both GFP-SMN (A) and CFP-SMN (B) specifically in the transfected cells.
|
CBs labelled with either FP-SMN or FP-coilin show similar dynamics
during interphase
Time-lapse analyses were performed to examine the dynamic behaviour of CBs
containing SMN in interphase cells (see Materials and Methods). Analysis of
the movements of Cajal bodies demonstrates that CBs are dynamic structures in
cell line GFP-SMNE18.6. In addition to constant small movements, CBs were seen
to join together (Fig. 5A),
separate to form two bodies (Fig.
5B) and move in close proximity to each other without joining
(Fig. 5C). This mirrors the
dynamic behaviour previously reported using a stable HeLa expressing
GFP-coilin under a tetracycline-inducible promoter
(Platani et al., 2000).
Similar dynamic behaviour was also seen for CBs in a stable cell line derived
from the same parental HeLa cell line expressing YFP-coilin under a
constitutive promoter (cell line EYFP-CoilinE1.1.1) (J.E.S. and A.I.L.,
unpublished). The frequency of each type of movement is shown in
Fig. 5D. Using GFP-SMN as a
marker, small CBs are also occasionally observed to appear or disappear within
the nucleoplasm. These data demonstrate that the dynamic properties of CBs are
not dependent upon, or apparently affected by, the presence of different
labelled marker proteins.
|
Differential dynamics of GFP-SMN and YFP-coilin within Cajal
bodies
To compare the dynamics of SMN and coilin proteins within CBs, FRAP
experiments were performed on cells from line GFP-SMNE10.3 and from line
YFP-coilinE1.1.1. Fluorescence signal was bleached from individual CBs
(Fig. 6A,B), using conditions
that bleached the signal from the entire structure, and single confocal
sections recorded at either 4- or 5-second intervals during recovery. The
YFP-coilin signal returned rapidly to CBs, with a plateau of 50% recovery
reached by 40 seconds. Half of this recovery was attained within the first 10
seconds (Fig. 6A). Similar
results were obtained in cells transiently transfected with GFP-coilin (data
not shown). In contrast, GFP-SMN showed no appreciable return to Cajal bodies
over a 1 minute recovery period (Fig.
6B). To investigate further the dynamics of SMN in CBs, a longer
time course was performed, collecting a series of confocal sections through
the cell over a 1 hour recovery period
(Fig. 6C). In this time,
GFP-SMN did show an appreciable return to CBs, regaining almost half of the
original signal within 1 hour (Fig.
6C). Interestingly, although no appreciable loss of signal from
CBs because of the imaging was seen in a neighbouring cell, a significant loss
of signal was seen from unbleached CBs in the nucleus containing the bleached
CB. This apparent FLIP effect suggests that at least some of the recovered
GFP-SMN was recruited from neighbouring CBs. In parallel FLIP experiments, a
region of the cytoplasm was repeatedly bleached and the resulting effect on
the fluorescence of GFP-SMN in CBs monitored using serial confocal sections
collected after each bleaching event (Fig.
6D). Each bleaching event took 5 seconds, with an imaging time of
approximately 10 seconds at each time point. Fluorescence signal was rapidly
lost from CBs in bleached cells, with half of the signal lost after 2 to 4
bleaching events.
|
There appears to be a rapid exchange of SMN from CBs into the cytoplasm,
because bleaching of the cytoplasmic GFP-SMN signal results in rapid loss of
GFP-SMN from CBs. Considering that it takes 1 hour for recovery of
50% GFP-SMN signal in CBs following direct photobleaching of CBs, the
rapid loss of GFP-SMN signal from CBs after bleaching the cytoplasm is
unlikely to be a result of a rapid incorporation into CBs of bleached GFP-SMN
molecules from the cytoplasm. Instead, the data suggest that the nuclear pool
of SMN undergoes relatively rapid transport back to the cytoplasm.
Dynamics of Cajal body markers through mitosis
It is known that both snRNPs and p80 coilin are present in bodies during
mitosis. However, there are at least two classes of snRNP-containing bodies in
mitotic cells, some of which co-localise with p80-coilin and some of which do
not (Ferreira et al., 1994). In
addition, the co-localisation of coilin with SMN has been seen to persist
through mitosis, although coilin re-enters the nucleus earlier than SMN, which
is targeted to pre-formed Cajal bodies
(Carvalho et al., 1999
). The
ability to follow live cells expressing GFP-SMN during mitosis allows a direct
analysis of the dynamics of these mitotic CB remnants. The top three rows of
Fig. 7 show three separate
cells from line GFPSMNE10.3 undergoing mitosis. During prophase, metaphase,
anaphase and telophase, a small number of GFP-SMN-positive bodies are seen (Ai
to v and Bi). During late telophase, however, a large number of GFP-SMN bodies
form rapidly (Bii to v, Ci and ii). As the daughter cells begin to flatten
out, these bodies rapidly disappear, prior to the appearance of the normal
interphase distribution of GFP-SMN in a diffuse cytoplasmic pool with a small
number of nuclear bodies (Ciii to v). Cells expressing YFP-coilin were also
followed through mitosis (Fig.
7D,E). In marked contrast to the behaviour of GFP-SMN, YFP-coilin
is seen in a small number of bodies only as far as anaphase (Di and ii).
During telophase, YFP-coilin is rapidly imported into the forming daughter
nuclei (Diii to v). As the daughter cells flatten out, nuclear bodies can be
seen to form either distant from (arrow) or close to (arrowhead) nascent
nucleoli. Because it is recognised that SMN is targeted to pre-formed nuclear
CBs that already contain coilin (Carvalho
et al., 1999
), the initial appearance of coilin-positive bodies
can be concluded to be the initial formation of nuclear CBs in the daughter
nuclei. It is, therefore, interesting to note that they can form in close
proximity to a nucleolus, but do not always form at the nucleolar periphery,
supporting the view that there may be several different mechanisms for CB
formation.
|
Mitotic bodies containing SMN differ in their composition from those
caused by GFP-SMN over-expression in interphase cells
Immunostaining of cells from line GFPSMNE10.3 with antibodies to coilin
demonstrates that both SMN and coilin are present in the same bodies during
mitosis until late telophase (Fig.
8A-F). The association persists as coilin begins to enter the
newly formed nuclei (Fig. 8G-I)
but is subsequently lost as coilin continues to concentrate in the nuclei and
GFP-SMN begins to form the numerous, smaller cytoplasmic bodies seen in the
time-lapse studies. Further analysis of the composition of the mitotic CB
remnants demonstrates that they contain both Sm proteins
(Fig. 9A-H) and TMG-capped RNA
(Fig. 9I-L). The presence of
TMG-capped RNA and coilin sets these structures apart from the cytoplasmic
accumulations of SMN-complex proteins that result from the over-expression of
GFP-SMN in interphase cells (Fig.
3). It is also interesting to note that bodies are present in
mitosis that contain TMG-capped RNA in the absence of SMN or coilin
(Fig. 9I arrowheads). These may
represent snRNAs that had been transcribed but not yet begun assembly into
snRNPs prior to the onset of mitosis.
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Discussion |
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At the cellular level, the reduction in expression of the SMN protein in
SMA is associated with a failure of the SMN complex to assemble into nuclear
bodies (Coovert et al., 1997;
Lefebvre et al., 1997
;
Frugier et al., 2000
). The
restriction of the disease phenotype to motor neurons, despite a systemic
decrease in SMN expression, has led to the suggestion that most human cells
express SMN in excess of their needs, whereas motor neurons express only the
amount they require. This would make motor neurons uniquely sensitive to
depletion of the protein (Wang and
Dreyfuss, 2001
). Observation of cells expressing high levels of
FP-SMN in this study, however, suggest a more complex regulation of SMN and
its associated proteins. Over-expression of FP-SMN in HeLa or MCF-7 cells
leads to the appearance of cytoplasmic accumulations of FP-SMN (Figs
3,
4), which also contain the
SMN-complex protein SIP1/Gemin2 and Sm proteins. Importantly, TMG-capped RNA
is absent from these accumulations, suggesting that they may represent SMN
complexes blocked at an early stage of snRNP assembly, similar to the
cytoplasmic block previously shown to be induced by expression of a myc-tagged
deletion mutant of SMN (SMND
27)
(Pellizzoni et al., 1998
).
Furthermore, over-expression of SMN leads to a disruption of CBs within the
nucleus, where we observed a proliferation of small bodies containing SMN,
coilin, Sm proteins and TMG-capped snRNAs. This suggests that, although coilin
is required for the recruitment of SMN and Sm proteins to CBs
(Tucker et al., 2001
;
Hebert et al., 2001
), the
level of SMN expression may have a role in determining the size and number of
CBs formed.
FP-SMN provides a specific marker for CBs that accumulate newly
imported snRNPs
CB dynamics have been studied using GFP-U2B'' in plant cells
(Boudonck et al., 1999) and
GFP-SmD1 (Sleeman et al.,
1998
), GFP-fibrillarin (Snaar
et al., 2000
) and GFP-coilin in mammalian cells
(Platani et al., 2002
;
Platani et al., 2000
).
Time-lapse analysis of FP-SMN cell lines in interphase reveals similar types
of movements and interactions between CBs as seen previously using other
GFP-tagged proteins as markers to label CBs
(Fig. 5). Fusions between
bodies (A), separations of bodies into two (B) and close interaction between
bodies (C) were all seen with a similar frequency as in cells from the same
parental HeLa line expressing YFP-coilin. In addition to establishing FP-SMN
as a useful marker for following the movement of CBs in living cells, this
also suggests that low level over-expression of either FP-SMN or FP-coilin
does not, in itself, alter CB dynamics. The CBs involved in the accumulation
of new snRNPs on re-entry into the nucleus are specifically those that contain
SMN-complex proteins (Carvalho et al.,
1999
; Sleeman and Lamond,
1999a
). Indeed, the transient expression of tagged core snRNP, Sm,
proteins leads to the formation of CBs in primary cells that do not normally
contain them (Sleeman et al.,
2001
). In light of the existence, in some cell types, of
coilin-positive CBs that lack SMN, we can now be certain, using the FP-SMN
cell lines, that the CBs studied are specifically those containing SMN-complex
proteins implicated in snRNP maturation.
GFP-SMN and YFP-coilin show markedly different dynamics of
interaction with CBs
Despite the similarities in the dynamic behaviour of CBs seen using either
FP-SMN or FP-coilin as markers, FRAP experiments demonstrate that the flux of
these two proteins through CBs are different. Thus, whereas YFP-coilin reaches
a plateau of recovery of signal to a bleached CB within one minute, with most
of this recovery seen in the first 10 seconds, GFP-SMN shows minimal recovery
over this time period, with 1 hour required to recover 50% of the original
GFP-SMN fluorescence. At first sight, this may seem intuitive as, judged by
immunofluorescence microscopy, there appears to be a more substantial
nucleoplasmic pool of coilin than of SMN. However, previous studies using
GFP-fibrillarin, which also has a small nucleoplasmic pool, showed a rapid
recovery of the tagged protein following bleaching of either a nucleolus
(Phair and Misteli, 2000) or
CBs (Snaar et al., 2000
).
Furthermore, the recovery of YFP-coilin to CBs was also rapid in cells in
which the nucleoplasmic pool of YFP-coilin was perceived by microscopy to be
extremely low (data not shown). The rate of recovery of YFP-coilin to CBs was
similar to that previously reported for the recovery of the splicing factor,
ASF/SF2 to speckles (Phair and Misteli,
2000
; Kruhlak et al.,
2000
). It has been demonstrated that coilin has the ability to
self-associate (Hebert and Matera,
2000
). The presence of the coilin self-interacting domain is both
necessary and sufficient for the localisation of coilin to CBs. In contrast,
SMN, although also capable of self-interaction
(Lorson et al., 1998
),
requires the presence of coilin for its recruitment into CBs
(Tucker et al., 2001
;
Hebert et al., 2001
). SMN and
SmB have been demonstrated to compete for binding to coilin, leading to the
suggestion that coilin may also have a role in the dissociation of the
SMN-snRNP complex (Hebert et al.,
2001
). Our current observations show a rapid turnover of coilin in
CBs and a much slower turnover of SMN. This is consistent with the idea of a
simple molecular interaction between coilin molecules leading to the
incorporation of coilin into the CB, with a more complex, multi-molecular
interaction being required for the association and disassociation of SMN from
the CB.
FLIP experiments demonstrate that repeated bleaching of the cytoplasmic
pool of GFP-SMN leads to a rapid loss of GFP-SMN from nuclear CBs. Taken
together with the slow recovery of GFP-SMN into nuclear CBs, with concomitant
loss of signal from neighbouring CBs, this suggests that the GFP-SMN in CBs is
able to return to the cytoplasm, so that depletion of the cytoplasmic pool
also directly depletes the nuclear pool of SMN. Although it is increasingly
probable that SMN accompanies newly formed snRNPs from the cytoplasm into the
nucleus via CBs (Carvalho et al.,
1999; Sleeman et al.,
2001
; Narayanan et al.,
2002
), less is known about any return transport of SMN to the
cytoplasm.
Dynamics of Cajal body markers through mitosis
In addition to differences in the flux of coilin and SMN through CBs, the
dynamic behaviour of the two proteins during their re-entry into the nucleus
following mitosis is also different. From prophase through to telophase,
coilin and SMN co-localise in a small number of mitotic CB remnants (MCBs).
These structures differ from the cytoplasmic SMN accumulations resulting from
over-expression of FP-SMN in that they contain both coilin and, in most cases,
TMG-capped RNA, in addition to SMN and Sm proteins. Rather than representing
SMN-snRNP complexes blocked in their assembly, as the interphase cytoplasmic
accumulations most probably do, we suggest that these structures may represent
a way to preserve essential SMN-snRNP processing complexes during mitosis. A
similar phenomenon has been reported for rRNA processing complexes during
mitosis (Dundr et al., 2000).
In this case, partially processed rRNA is seen to co-localise with several of
its processing components (fibrillarin, nucleolin and B23) through mitosis in
nucleolus-derived foci (NDFs). In contrast to NDFs, MCBs do not contain
detectable levels of fibrillarin, nucleolin or B23 (J.E.S. and A.I.L.,
unpublished), so appear to be separate structures. It is probable, therefore,
that the partial preservation of RNA processing complexes during mitosis is a
feature common to snRNAs and rRNAs and, importantly, common to CBs and
nucleoli, whose roles in these processing events may be related and linked to
each other. Nucleoli and CBs contain many common factors, such as fibrillarin,
Nopp140 and snoRNAs, suggesting that the two structures have co-operative
roles in ribosome production, with the CB involved in the maturation of
macromolecular complexes, such as snoRNPs, essential for rRNA processing in
the nucleolus (reviewed by Terns and
Terns, 2001
; Gall,
2000
). Events required for the maturation of snoRNPs may well be
similar to those required for the maturation of splicing snRNPs, in which the
CB probably participates. Furthermore, under certain conditions, coilin and
snRNP proteins are found inside nucleoli
(Ochs et al., 1994
;
Malatesta et al., 1994
;
Lyon et al., 1997
;
Sleeman et al., 1998
). It is
probable that the nucleolus has roles in processes other than rRNA processing
(reviewed by Pederson, 1998
).
The morphological link between nucleoli and CBs has been evident from their
initial description as nucleolar accessory bodies
(Ramon-y-Cajal, 1903
). The
similarities in their molecular composition and potential joint roles in the
processing of many RNA species are becoming increasingly apparent (reviewed by
Gall, 2000
).
Mitotic CBs containing coilin and snRNP proteins have been reported
previously (Ferreira et al.,
1994) and the co-localisation of SMN and coilin in these
structures has been described (Carvalho et
al., 1999
). Here we present the first dynamic analysis of MCBs,
which reveals striking differences in the re-entry of coilin and SMN into the
forming daughter nuclei. The MCBs are extremely mobile within the cell, making
tracking of individual bodies problematic using a frequency of imaging that
can be tolerated by mitotic cells (Fig.
7A,D). This is in marked contrast to the behaviour of nuclear CBs
during interphase, in which the basic spatial arrangement of CBs is often more
stable (Fig. 5) and may be
because of the huge rearrangements of cellular structure that occur during
mitosis. These data are, therefore, suggestive of a role for rearrangements of
areas of the nucleus, perhaps including the re-organisation of chromatin, for
large movements of interphase CBs.
At the end of telophase, GFP-SMN rapidly forms a large number of punctate
cytoplasmic structures of various sizes. This event is usually concomitant
with a perceived decrease in the amount of diffuse GFP-SMN. These highly
mobile structures persist for approximately 20 minutes, then rapidly
disappear, immediately prior to the re-appearance of GFP-SMN in nuclear CBs.
In contrast, YFP-coilin is rapidly lost from MCBs early in telophase, when the
protein is imported into the forming daughter nuclei. Initially showing a
`clumped' distribution, the YFP-coilin gradually becomes more diffuse as the
nuclei flatten out. In early G1, CBs re-form within the daughter nuclei.
Because coilin is observed in CBs before SMN
(Carvalho et al., 1999) (J.E.S.
and A.I.L., unpublished), this most probably represents the initial formation
of nuclear CBs. It is interesting, therefore, to note that CB formation is
seen both in proximity to (Fig.
7E, arrowhead) and distant from
(Fig. 7E, arrow) nascent
nucleoli. Electron microscopy studies of CBs in close proximity to nucleoli
have shown CBs to have a close structural relationship to nucleoli, appearing
either to fuse with or bud from nucleoli. Although not ruling out the
possibility that CBs can form at the nucleolar periphery, our current data
indicate that CBs can form within the nucleoplasm at some distance from
nucleoli.
In summary, we have established stable HeLa cell lines expressing SMN fused to fluorescent proteins and compared them with cells stably expressing FP-tagged coilin. The two CB proteins, SMN and coilin, although showing similar distributions within the nucleus, show very different rates of flux through nuclear CBs, presumably related to differences in the complexity of the interactions that cause their accumulation in CBs. The dynamic behaviour of the two proteins through mitosis and during the reformation of nuclear structures following mitosis show further differences between SMN and coilin, but reveal parallels in the behaviour of the nucleolar rRNA processing machinery and the snRNA processing machinery normally resident in CBs.
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