Cajal-body formation correlates with differential coilin
phosphorylation in primary and transformed cell lines
Scoty M. Hearst1, Andrew S. Gilder1, Sandeep S. Negi1, Misty D. Davis1, Eric M. George1,
Angela A. Whittom1, Cory G. Toyota1, Alma Husedzinovic2, Oliver J. Gruss2and Michael D. Hebert1,*
1Department of Biochemistry, The University of Mississippi Medical Center, Jackson, MS 39216, USA
2Zentrum fur Molekulare Biologie der Universitat Heidelberg, DKFZ-ZMBH Alliance 69120 Heidelberg, Germany
*Author for correspondence (e-mail: firstname.lastname@example.org)
Cajal bodies (CBs) are subnuclear domains conserved in insects,
including Drosophila (Liu et al., 2006a; Liu et al., 2006b), yeast,
plants and mammals (reviewed by Gall, 2000; Matera, 2003; Cioce
and Lamond, 2005; Matera and Shpargel, 2006). CBs participate
in spliceosomal small nuclear ribonucleoprotein (snRNP)
biogenesis. Specifically, CBs contain small Cajal-body specific
RNAs (scaRNAs) that guide modification of the snRNA moiety of
the snRNP (Darzacq et al., 2002; Jady et al., 2003). The
modifications on the snRNAs are necessary for proper snRNP
function (Pan and Prives, 1989; Segault et al., 1995; Yu et al., 1998).
The CB also takes part in the assembly of spliceosomal
subcomplexes (Schaffert et al., 2004; Stanek and Neugebauer, 2004;
Xu et al., 2005; Stanek et al., 2008) and the final steps of U2 snRNP
biogenesis (Nesic et al., 2004). Other work has shown that CBs
participate in the biogenesis and delivery of telomerase to telomeres
(Jady et al., 2004; Lukowiak et al., 2001; Jady et al., 2006;
Tomlinson et al., 2006; Tomlinson et al., 2008). Interestingly, CBs
are mobile, contain basal transcription factors and can associate with
snRNA genes (e.g. genes encoding U2), histone gene clusters and
PML (promyelocytic leukemia) bodies (Gall, 2000; Ogg and
Lamond, 2002; Bongiorno-Borbone et al., 2008; Grande et al., 1996;
Sun et al., 2005). Finally, studies in Arabidopsis thaliana show that
certain steps in micro-RNA and small-interfering RNA biogenesis
might occur in plant CBs (Li et al., 2006; Pontes et al., 2006).
The marker protein for CBs is considered to be coilin (also known
as P80C) (Raska et al., 1990; Raska et al., 1991). It is also notable
that, in addition to the cytoplasm, the survival motor neuron protein
(SMN) localizes to CBs (Carvalho et al., 1999; Matera and Frey,
1998). SMN is a vital component in the cytoplasmic phase of snRNP
biogenesis (Meister et al., 2002; Massenet et al., 2002), and might
have a role analogous to its cytoplasmic functions in the CB by
ensuring that nuclear snRNPs remain functional after a splicing
reaction has taken place (Pellizzoni et al., 1998; Xu et al., 2005).
Phosphorylation impacts SMN activity and localization (Grimmler
et al., 2005; Petri et al., 2007). In particular, dephosphorylation of
SMN by the nuclear phosphatase PPM1G is needed for SMN
localization to CBs (Petri et al., 2007).
With the exception of scaRNAs, all of the factors enriched in
the CB also localize to other cellular compartments such as the
cytoplasm, nucleoplasm or nucleolus (Darzacq et al., 2002;Matera,
1999). For example, 70% of coilin is nucleoplasmic (Lam et al.,
2002). The fact that almost all the components of the CB can be
found in other locations in the cell makes the description of the
exact roles of the CB difficult. Indeed, any possible function ascribed
to the CB has to be reconciled with the reality that many cell types
(e.g. adult lung tissue) do not have CBs (Spector et al., 1992; Young
et al., 2001). Thus, the activities that take place within the CB can
probably also occur in the nucleoplasm. A key to understanding the
function(s) of the CB comes from observations showing that CBs
are most prominent in cells that are transcriptionally active, such
as neuronal and cancer cells (Matera, 2003). Moreover, inhibition
of transcription with actinomycin D or α-amanitin disrupts CBs
(Carmo-Fonseca et al., 1992). Active U snRNA transcription and
snRNP biogenesis is required for CB integrity (Shpargel and
Matera, 2005; Lemm et al., 2006; Girard et al., 2006). Clearly,
therefore, CB formation and activity are dynamic and balanced by
the transcriptional demands of the cell.
Cajal bodies (CBs) are nuclear structures that are thought
to have diverse functions, including small nuclear
ribonucleoprotein (snRNP) biogenesis. The phosphorylation
status of coilin, the CB marker protein, might impact CB
formation. We hypothesize that primary cells, which lack CBs,
contain different phosphoisoforms of coilin compared with that
found in transformed cells, which have CBs. Localization, self-
association and fluorescence recovery after photobleaching
(FRAP) studies on coilin phosphomutants all suggest this
modification impacts the function of coilin and may thus
contribute towards CB formation. Two-dimensional gel
electrophoresis demonstrates that coilin is hyperphosphorylated
in primary cells compared with transformed cells. mRNA levels
of the nuclear phosphatase PPM1G are significantly reduced
in primary cells and expression of PPM1G in primary cells
induces CBs. Additionally, PPM1G can dephosphorylate coilin
in vitro. Surprisingly, however, expression of green fluorescent
protein alone is sufficient to form CBs in primary cells. Taken
together, our data support a model whereby coilin is the target
of an uncharacterized signal transduction cascade that responds
to the increased transcription and snRNP demands found in
Supplementary material available online at
Key words: Coilin, SMN, Small nuclear ribonucleoproteins, Nuclear
Accepted 24 February 2009
Journal of Cell Science 122, 1872-1881 Published by The Company of Biologists 2009
Journal of Cell Science
Phosphorylation impacts CB formation
Coilin-knockout mice have been generated to better understand
the role of this protein and CBs (Tucker et al., 2001). Inbred strains
of coilin-knockout mice have significant viability defects (Tucker
et al., 2001). Cell lines derived from coilin-knockout mice do not
have typical CBs but instead have two kinds of ‘residual’ CBs
(Tucker et al., 2001; Jady et al., 2003). One kind of residual CB
contains scaRNAs (Jady et al., 2003) and the other contains
proteins such as fibrillarin and Nopp140 (Tucker et al., 2001). SMN
does not accumulate in either kind of residual CB, underscoring
the role of coilin in the formation of canonical CBs (Tucker et al.,
2001; Jady et al., 2003). Studies on Arabidopsis have identified a
coilin orthologue (Atcoilin) that, along with other loci, impacts CB
formation and size (Collier et al., 2006). Very recent work has shown
that a coilin orthologue is present in Drosophila melanogaster, and
this protein is required for normal CB organization (Liu et al., 2009).
Mutants lacking CBs in Arabidopsisand Drosophiladid not display
any obvious growth phenotypes, but it has been found that HeLa
cells depleted of coilin by RNAi proliferate more slowly than control
treated cells (Lemm et al., 2006) and are impaired in their ability
to splice an artificial reporter (Whittom et al., 2008). Consequently,
it is evident that coilin is not an essential protein and CBs are not
required for survival, yet their presence must be advantageous,
because genes encoding coilin and CBs are conserved in vertebrates,
flies and plants.
It is possible that coilin might serve as the scaffold of CBs and
bring together various factors necessary for a range of functions
into one nuclear subdomain, resulting in the most efficient platform
to prepare these factors for their activities. Additionally, coilin has
a role in the association of CBs with PML bodies (Sun et al., 2005)
and Gems (Hebert et al., 2001). Gems are subnuclear domains found
in some cell lines and fetal tissue, and are often found adjacent to
CBs (Liu and Dreyfuss, 1996; Young et al., 2000). Gems contain
SMN and associated proteins known as Gemins, but lack snRNPs
and coilin. In cell lines that normally lack Gems, reduction of coilin
by RNA interference abolishes CBs and induces Gem formation
(Lemm et al., 2006; Whittom et al., 2008). Post-translational
modification of coilin also has a role in whether or not a cell contains
Gems. Specifically, coilin contains symmetrically dimethylated
arginines that are important for direct interaction with SMN (Hebert
et al., 2001; Hebert et al., 2002) and the presence of Gems
correlates with a decrease in coilin methylation (Hebert et al., 2002;
Boisvert et al., 2002). Coilin also binds directly to several Sm
proteins of snRNPs (Hebert et al., 2001; Xu et al., 2005), suggesting
that direct coilin interaction with both SMN and snRNPs mediates
their localization to CBs. The interplay between coilin, SMN and
snRNPs at the CB might facilitate snRNP biogenesis and recycling.
It is unknown whether coilin in the nucleoplasm, where the majority
of the protein resides, contributes to its putative role in the CB or
possesses distinct nucleoplasmic-specific activities. For example,
a recent study has shown that coilin is recruited to centromeres in
response to damage or depletion of CENP-B, indicating that coilin
has an undefined role in some type of centromere-repair pathway
(Morency et al., 2007).
In addition to symmetrically dimethylated arginines, human coilin
is a known phosphoprotein (Carmo-Fonseca et al., 1993). During
mitosis, the level of phosphorylation on coilin increases (Carmo-
Fonseca et al., 1993). Cell cycle analysis reveals that CBs
disassemble during mitosis and reform in the cell cycle at early- to
mid-G1. However, throughout the cell cycle, coilin levels remain
constant (Andrade et al., 1993), giving rise to the hypothesis that
the phosphorylation status of coilin has a role in CB formation.
Support for this idea comes from studies showing that phosphatase
inhibitors alter CB localization (Lyon et al., 1997), as does mutation
of a critical serine in coilin (S202) to aspartate (Lyon et al., 1997;
Sleeman et al., 1998). Additionally, we have shown that coilin is a
self-interacting protein and this interaction is reduced in mitosis
when coilin is hyperphosphorylated (Hebert and Matera, 2000).
Furthermore, we have also shown that the C-terminus of human
coilin contains potential phosphoresidues that regulate the
availability of the N-terminal self-interaction domain (Shpargel et
al., 2003). Recent work using tandem mass spectrometry (MS/MS)
for the large-scale analysis of phosphoproteins has revealed that
coilin has at least eleven phosphorylated residues, with six of these
residing in the very C-terminus of coilin (Beausoleil et al., 2004;
Olsen et al., 2006; Beausoleil et al., 2006; Nousiainen et al., 2006)
(www.phosida.com). Hence, both cell biological and MS/MS
analyses support the hypothesis that coilin activity is regulated by
its phosphorylation status, which changes during the cell cycle.
Coilin might need to contain the proper phosphorylation pattern
in order for CBs to form during interphase and a different contingent
of phosphorylated residues to trigger CB disassembly during
mitosis. Transient expression of small nuclear ribonucleoprotein-
associated protein B(SmB; official symbolRSMB), but not coilin,
induces correspondingly transient CB formation in cells that
normally lack this structure (Sleeman et al., 2001). A possible
explanation for this finding is that the expression of Sm proteins
signals to the cell the need to upregulate the snRNP biogenesis
machinery. Part of this upregulation might include the formation
of CBs to efficiently modify the snRNA component of the newly
made snRNPs. To achieve CB formation, the phosphorylation status
of coilin might need to be altered. Thus, we hypothesize that coilin
is a target of an unknown signaling cascade that responds to
increases in the demand for splicing resources. The exact residues
on coilin that may be subjected to this putative phosphorylation
pathway are not known. Nor is it known whether some of the same
factors that modify SMN phosphorylation (e.g. PPM1G) can also
In this work, we provide evidence for this hypothesis by
demonstrating that coilin phosphomutants, particularly in the very
C-terminus, display altered localization, self-association and
mobility characteristics. We also show that coilin in a primary cell
line is hyperphosphorylated relative to that found in a transformed
cell line. This hyperphosphorylation correlates with decreased
PPM1G mRNA levels. Interestingly, expression of additional
PPM1G in primary cells induces CB formation. Hence, these data
support a role for coilin in the formation of CBs, and indicate that
hyperphosphorylated coilin in primary cell lines inhibits CB
Mutation of coilin phosphoresidues disrupts coilin localization
Tandem MS/MS analysis has shown that coilin has at least 11
phosphorylated residues (Fig. 1) (Beausoleil et al., 2004; Olsen et
al., 2006; Beausoleil et al., 2006; Nousiainen et al., 2006)
(www.phosida.com). Interestingly, 6 of the last 11 amino acids of
coilin are phosphorylated. To address the functional consequence
of these residues with regards to CB formation, two mutant coilin
cDNAs were generated in the GFP-coilin background. In the first,
the 6 residues subjected to phosphorylation were changed from
serines or threonines to alanines (C6A) (Fig. 1), mimicking a
dephosphorylated state. In the second mutant, the serines were
changed to aspartates and the threonines were converted to
Journal of Cell Science
glutamates (C6D/E) (Fig. 1), mimicking a phosphorylated state.
Upon expression of GFP-coilin, GFP-coilin(C6A) or GFP-
coilin(C6D/E) in HeLa cells (Fig. 2A), we observed that the vast
majority of GFP-coilin cells contained CBs, whereas only 47% of
GFP-coilin(C6A) cells displayed this normal phenotype (Fig. 2B).
The remaining cells had GFP-coilin(C6A) localization in a nucleolar
and CB pattern similar to that observed for proteins such as Nopp140
or fibrillarin (Fig. 2A,B). Fragments of coilin containing the first
248 or 315 residues also displayed this localization pattern (Hebert
and Matera, 2000). No obvious changes in localization were
observed in cells expressing GFP-coilin(C6D/E) (Fig. 2A,B). Thus,
mutation of the six C-terminal phosphoresidues in coilin to alanine
We next investigated the localization of GFP-tagged coilin
phosphomimics in which all 11 suspected phosphorylation sites were
converted to a phosphorylated or a dephosphorylated state. These
constructs (denoted as OFF for the dephosphorylated mimic and
ON for the phosphorylated mimic) were transiently transfected into
HeLa cells (Fig. 2A). For the OFF mutant, 40% of expressing cells
displayed a normal coilin localization pattern (Fig. 2B). However,
60% of OFF-expressing cells displayed GFP signal in nucleoli and
CBs, similarly to that observed with the C6A mutant. Cells
expressing the ON mutant displayed a high percentage of cells
(68%) with nucleoplasmic signal lacking coilin foci (Fig. 2B).
Detection of SMN in these ON-expressing cells demonstrates that
they also lack SMN foci, and are thus lacking in CBs (Fig. 2A).
Normal coilin localization pattern was observed in 32% of ON-
transfected cells (Fig. 2B). In summary, coilin mutants that mimic
dephosphorylation (C6A and OFF) displayed a significant
percentage of cells with nucleolar coilin localization. By contrast,
wild-type (WT) coilin and the mutants that mimic phosphorylation
(C6D and ON) did not display this phenotype. Interestingly, the
ON mutant had a high percentage of cells with nucleoplasmic signal
but lacking CBs (Fig. 2B).
An important caveat to the localization experiments described
above is the presence of endogenous coilin in HeLa cells. Through
interactions with other factors (e.g. SMN) and itself, endogenous
coilin might complicate the true CB formation potential of the
mutants studied here. Therefore, to further clarify coilin
phosphomutant localization, we transfected the various coilin
Journal of Cell Science 122 (11)
constructs into cells in which endogenous coilin was knocked down
using RNA interference. Previous work has shown that transiently
transfected coilin duplex siRNAs reduce coilin levels and disrupt
CBs (Lemm et al., 2006; Whittom et al., 2008). Additionally, the
level of GFP-tagged coilin (or mutants thereof) in coilin-knockdown
cells is approximately equal to the level of endogenous coilin in
cells treated with control siRNA (supplementary material Fig. S1).
By following this protocol, therefore, the impact of endogenous
coilin on the CB formation potential of the various phosphomutants
is greatly reduced. Compared with its localization in normal HeLa
cells, WT GFP-coilin expression in the coilin-knockdown
background increases the percentage of cells with only
nucleoplasmic localization (Fig. 2C). An increase in the percentage
of cells with nucleoplasmic localization was also observed for the
C6D mutant (Fig. 2C). By contrast, the localization of C6A, ON
and OFF mutants in the coilin-knockdown background did not vary
dramatically from that observed in normal HeLa cells (compare
Fig. 2B to 2C). We conclude from this analysis that endogenous
coilin facilitates the incorporation of GFP-tagged WT and C6D
mutant proteins into CBs.
Coilin phosphoresidues impact self-association and mobility
Coilin is phosphorylated on several residues during interphase, with
other residues becoming phosphorylated during mitosis (Carmo-
Fonseca et al., 1993; Beausoleil et al., 2004; Olsen et al., 2006;
Beausoleil et al., 2006). We have shown that coilin is a self-
associating protein and that this association is reduced when coilin
is hyperphosphorylated during mitosis (Hebert and Matera, 2000).
To address how these phosphomutations affect self-association,
if at all, extracts obtained from HeLa cells expressing WT,
C6A, C6D, OFF or ON GFP-coilin proteins were subjected to
immunoprecipitation with anti-GFP antibodies followed by western
blotting with anti-coilin antibodies (Fig. 3). We found that less
endogenous coilin is co-immunoprecipitated with the C6D mutant
(lane 6) compared with that recovered by the WT (lane 4) or the
C6A mutant (Fig. 3A, lane 5). We also observed a slight reduction
in the amount of endogenous coilin recovered by the ON mutant
compared with the WT (Fig. 3B, compare coilin signal in lane 1
with that in lane 2). No coilin was recovered when cells expressed
GFP only (Fig. 3B, lane 4). These results demonstrate that
phosphoresidues of coilin impact self-association.
To further characterize the role of coilin phosphoresidues, we
performed fluorescence recovery after photobleaching (FRAP) on
Cajal bodies in cells expressing GFP-tagged WT, C6A, C6D, OFF
or ON coilin proteins. Recovery curves were generated by double
normalization (supplementary material Fig. S2) and the time to half
maximal recovery (T50) was calculated for each protein (Fig. 4).
Compared with the WT, both the C6D and ON proteins had a faster
T50, suggesting that, in this time frame, there is a greater exchange
of the constitutively phosphorylated protein with an individual Cajal
body. By contrast, both the C6A and OFF proteins had a slower
T50 than the WT, implying that these proteins are less mobile.
WI-38 cells contain hyperphosphorylated coilin
Human primary foreskin fibroblasts cells (DFSF1), which
normally lack CBs, can be induced to form CBs by transient
expression of SmB, but only for a limited time (Sleeman et al.,
2001). Additionally, this same study demonstrated that fusion of
DFSF1 cells to HeLa cells leads to the formation of CBs in DFSF1
nuclei. Consequently, DFSF1 cells are capable of forming CBs if
the appropriate factors and/or signals are provided by HeLa cells.
Fig. 1. Known coilin phosphoresidues and mutations. Schematic of human
coilin showing the locations of the self-association domain and RG box.
Below is the C-terminal sequence from residue 562 to the end of the protein,
residue 576. Residues shown to be phosphorylated by tandem MS/MS analysis
are indicated in the schematic (T122, S271, S272, T303, S489) or by asterisks
in the sequence (S566, S568, T570, S571, S572, T573). Mutations of these six
C-terminal residues to alanine (C6A) or glutamate/aspartate (C6D/E, denoted
in subsequent text as C6D) are shown. The OFF and ON coilin mutants
contain mutations of all 11 phosphorylated residues.
Journal of Cell Science
Phosphorylation impacts CB formation
The fact that exogenous Sm protein, but not coilin or SMN,
expression can transiently induce CB formation in DFSF1 cells
(Sleeman et al., 2001) suggests that the cell is responding to a
direct need to upregulate snRNP biogenesis. We hypothesize that
part of this upregulation is the post-translational modification of
coilin by phosphorylation, rendering it in a conformation
conducive for CB formation. To test this idea, we used the WI-
38 primary cell line that has been shown to have CBs in only 2-
3% of cells (Spector et al., 1992). It is possible that CBs are rare
in the WI-38 primary cell line because coilin phosphorylation
levels and/or phospho-residues are different in this line compared
with that found in HeLa cells, which have CBs. To further support
this hypothesis, we conducted two-dimensional gel electrophoresis
experiments using western blotting to detect coilin. The predicted
pI of unphosphorylated coilin is 9.2 (www.phosphosite.org). As
a control experiment, we compared the pI of coilin from untreated
or phosphatase-treated HeLa lysate and observed that, as expected,
coilin is shifted to a higher pI when dephosphorylated (Fig. 5A).
It is also important to note that coilin in untreated HeLa lysate
focused close to the position corresponding to pI 7 on a pH 7-10
strip, in agreement with previous results demonstrating that coilin
is a phosphoprotein (Carmo-Fonseca et al., 1993). We then
assessed the position of focused coilin, β-tubulin and SMN from
both HeLa and WI-38 interphase cells on pH 5-8 strips. β-tubulin
was used as an internal standard to help gauge whether the pI of
coilin differed in the two lines. The pI of SMN was monitored to
determine whether SMN phosphorylation was also correlated with
CB formation, as we suspected was the case for coilin. The
predicted pI of unphosphorylated SMN is 6.1, whereas β-tubulin
is expected to be approximately 5.3. The focused position of β-
tubulin was unchanged in HeLa versus WI-38 extracts. By
contrast, the pI of coilin from HeLa differed compared with WI-
38 coilin (Fig. 5B,C). Specifically, coilin from HeLa cell extracts
was focused to several different pIs (arrows), implying that a range
of phosphoisoforms exist, and the majority of the protein migrates
to a more basic pI than that observed for WI-38 coilin. In fact,
using β-tubulin as an internal standard, we conclude that coilin
from WI-38 is more uniformly phosphorylated in this line
compared to coilin from HeLa (Fig. 5B,C), and its pI is consistent
with hyperphosphorylated coilin from HeLa mitotic lysate (Fig.
5D). SMN, a known phosphoprotein, was focused to several
distinct foci in both HeLa and WI-38 extracts, but the overall
migration of these foci did not appear to differ in the two lines
relative to β-tubulin (Fig. 5E,F). Thus the lack of CBs in the
Fig. 2. Coilin phosphomutants impact localization. (A)HeLa cells expressing wild-type (WT) GFP-coilin or GFP-coilin mutants (C6A, C6D, OFF or ON) were
fixed and GFP (green), SMN (red) and DAPI (blue) signals were detected. The right column shows the overlay of all three images (Merge). Some CBs are labeled
with arrows. Arrowheads indicate nucleolar staining observed in cells expressing C6A and OFF. Scale bars: 10μm. (B)Quantification of WT and coilin mutant
localization in HeLa cells. At least 100 cells were counted for each construct. (C)Quantification of WT and coilin mutant localization in coilin-knockdown HeLa
cells. At least 50 cells were counted for each construct.
Journal of Cell Science
primary cell line WI-38 correlates with an increase in the degree
of phosphorylation of coilin.
WI-38 cells contain reduced PPM1G phosphatase message
The kinases and phosphatases responsible for coilin phosphorylation
have not been clearly identified. Moreover, the kinases that
phosphorylate SMN are likewise unclear. However, recent work
has defined PPM1G as the phosphatase that governs SMN
localization to CBs (Petri et al., 2007). Specifically, knockdown of
PPM1G in HeLa cells results in the loss of SMN from CBs, but
does not drastically alter coilin localization to CBs, or abolish CBs,
although numerous small coilin foci were observed (Petri et al.,
2007). It is possible that the small coilin foci induced upon
knockdown of PPM1G indicates that this phosphatase also
modifies coilin, and the small foci might contain relatively
hyperphosphorylated coilin compared with that found in normal
CBs. Since WI-38 cells only very rarely contain CBs, and coilin is
hyperphosphorylated in this line compared with that found in HeLa
cells, we suspected that PPM1G activity might be reduced in WI-
38 compared with HeLa cell extracts. To explore this possibility,
we conducted qRT-PCR on PPM1G mRNA levels from both lines
and found that, relative to actin, PPM1G levels are significantly
reduced (approximately 50%) in WI-38 compared with HeLa
Journal of Cell Science 122 (11)
extracts (Fig. 6A). Consistent with previous results comparing the
expression levels of SMN in transformed versus primary lines
(Sleeman et al., 2001), we found that SMN message levels are
significantly reduced in WI-38 cells (Fig. 6A). By contrast, coilin
levels showed a slight but statistically insignificant increase in WI-
38 (Fig. 6A). Incubation of recombinant PPM1G with mitotic HeLa
cell lysate results in a shift of coilin on SDS-PAGE consistent with
its dephosphorylation (Fig. 6B, compare the mobility of coilin in
lane 4 to that in lane 5). Thus, at least in vitro, coilin is a substrate
for PPM1G. Taken together, these results suggest that reduced
PPM1G and SMN levels contribute to the lack of CBs in WI-38
cells. The most straightforward interpretation of this data is that
reduced PPM1G levels in WI-38 cells leads to hyperphosphorylated
coilin promoting CB disassembly. Reduced SMN levels in WI-38
might also contribute to an environment in which CB formation is
not favored, although correspondingly reduced PPM1G levels
might result in no change in the overall phosphorylation of SMN
compared with that observed in HeLa cells.
PPM1G phosphatase expression in WI-38 cells induces CBs
To determine whether PPM1G expression could induce CBs in a
primary cell line, YFP-PPM1G was transfected into WI-38 cells,
followed by detection of CBs using anti-coilin antibodies. Most of
the cells overexpressing YFP-PPM1G did not have CBs (Fig. 7A,
row a). However, approximately 30% of these cells had clear CBs
(Fig. 7A, rows b and c, arrows) and these CBs contained both SMN
and snRNPs (our unpublished observations). Interestingly, cells
clearly overexpressing YFP-PPM1G, as evidenced by cytoplasmic
localization in addition to nuclear accumulation (Fig. 7A, row c),
nearly always had at least one CB. Since CBs are normally found
in only 2-3% of WI-38 cells (Spector et al., 1992), we conclude
that PPM1G has an important role in the regulation of CB formation.
To further validate the ability of PPM1G to induce CBs in primary
cells, we also scored WI-38 cells transiently transfected with a
catalytically inactive form of PPM1G, D496A (Murray et al., 1999).
To reduce possible overexpression artifacts, we only scored cells
with nuclear YFP-PPM1G (or YFP-inactive PPM1G) localization
and found that 17% of YFP-PPM1G-expressing cells had CBs
compared with 5% of YFP-inactive PPM1G-expressing cells (Fig.
7C). WI-38 cells expressing GFP-coilin, GFP-SmB or GFP alone
were also scored. Neither GFP-coilin nor GFP-SmB expression
triggers significant CB formation (Fig. 7B). A previous study has
shown that YFP-SmB expression for 2 hours can induce CBs in
another primary cell line, but CBs are absent after 16 hours of
expression (Sleeman et al., 2001). At neither time point did GFP-
coilin expression induce CBs (Sleeman et al., 2001). Thus, our
results for FP-SmB and GFP-coilin, obtained after 24 hours of
expression, are consistent with previously published findings.
In stark contrast to the failure of GFP-SmB and GFP-coilin to
form CBs, expression of GFP alone induces CB formation
significantly above the 2-3% of WI-38 cells that normally have CBs
(Fig. 7B). In fact, expression of GFP alone yields the highest
percentage of cells with CBs amongst the constructs tested, with
approximately 30% of transfected cells displaying CBs (Fig. 7C).
In summary, expression of YFP-PPM1G induces CB formation in
a primary cell line more than inactive YFP-PPM1G, GFP-coilin
and GFP-SmB, suggesting that this phosphatase participates in the
regulation of CB formation. The induction of CB formation in cells
expressing GFP alone is unexpected, and might be a compensatory
response by the cell to accommodate the transcription and
processing of the GFP message. It should be pointed out that only
Fig. 3. Coilin phosphomimics reduce coilin self-association. HeLa cells were
transfected with wild-type (WT) GFP-coilin or mutants thereof (C6A, C6D,
OFF, ON), followed by immunoprecipitation with anti-GFP antibodies and
western blotting with anti-coilin antibodies. TCL, total cell lysate; IP,
immunoprecipitation. The positions of GFP-tagged and endogenous coilin are
shown. (B)Immunoprecipitation reactions are shown for WT, OFF and ON
coilin constructs. Lane 4 is a negative control in which cells were transfected
with empty GFP vector and the lysate was treated as described above.
Fig. 4. FRAP analysis of wild-type and phosphomutants of coilin. FRAP
analysis was performed on Cajal bodies in cells expressing wild-type GFP-
Coilin as well as cells expressing ON, OFF, C6A and C6D mutants. After
alignment, recovery curves were generated by double normalization and the
time to half maximal recovery (T50) was calculated for each. The mean ± s.e.
value of T50from at least five cells is displayed.
Journal of Cell Science
Phosphorylation impacts CB formation
low to moderately expressing GFP cells were scored (similarly to
that shown in Fig. 7B), and the GFP message does not contain an
intron, and thus is not spliced.
Our previous findings, and data from other groups, support a model
whereby the phosphorylation status of human coilin impacts CB
formation. Central to this model are the observations that coilin
hyperphosphorylation during mitosis correlates with reduced self-
interaction and CB disassembly (Carmo-Fonseca et al., 1993; Hebert
and Matera, 2000). Thus CBs might share a common feature with
the nucleolus and the nuclear membrane in that increased
phosphorylation of vital proteins in these compartments promotes
their disassembly. There is also indirect evidence supporting the
ideas that proper coilin phosphorylation is required to form CBs,
and coilin hyperphosphorylation during mitosis triggers CB
disassembly. First, the overexpression of human coilin in HeLa cells
does not result in the formation of more CBs. One interpretation
of this result is that the CB has a limited number of coilin-binding
sites. We do not favor this interpretation, because our work shows
that the overexpression of human coilin in HeLa cells in fact
abolishes CBs, as assessed by SMN staining (Hebert and Matera,
2000). It is possible that newly overexpressed coilin does not contain
the proper composition of phosphoresidues for CB localization, but
is able to disrupt CBs by binding to and titrating out endogenous
coilin in CBs. MS/MS analysis has confirmed our initial belief that
the C-terminus of human coilin contains several phosphoresidues
(Shpargel et al., 2003; Nousiainen et al., 2006; Olsen et al., 2006).
Second, our studies into the CB formation potential of human,
mouse and frog coilins in both human and mouse cell lines
demonstrate that these coilins all contain an intrinsic nuclear body
formation potential, but this potential is subject to increasing layers
of regulation from frog, to mouse, to human (Shpargel et al., 2003).
We suspect that phosphorylation of coilin is a major contributor to
the regulation of CB formation and number in humans.
Additionally, two previous studies provide compelling indirect
evidence that coilin phosphorylation changes upon transformation
or in response to the RNP biogenesis needs of the cell. It should
be pointed out, however, that neither of these papers contains direct
data concerning changes in coilin phosphorylation. In the first study,
the Spector group showed that cells of limited passage number have
the fewest CBs, immortalized cells contain an intermediate number
of CBs, and transformed cells have the greatest number of CBs
(Spector et al., 1992). Most importantly, it was found that an
immortalized cell line (Ref-52) had a dramatically higher frequency
of CBs upon transformation (24% of Ref-52 cells have CBs
compared with 99% of transformed Ref-52 cells). Therefore,
transformation correlates with CB formation, and we believe that
changes in the phosphorylation state of coilin underlie CB formation.
Fig. 5. IEF of endogenous coilin from HeLa and WI-38 cells. (A)Lysate from HeLa cells was untreated or treated with CIP, followed by IEF (pH 7-10 IPG strip),
SDS-PAGE and western blotting. Coilin was detected using anti-coilin antibodies. (B)Interphase HeLa cell lysate was subjected to IEF (pH 5-8 IPG strip), SDS-
PAGE and western blotting. Coilin and β-tubulin were detected on the same blot using appropriate antibodies. (C)Lysate from interphase WI-38 cells was
subjected to IEF (pH 5-8 IPG strip), SDS-PAGE and western blotting, followed by the detection of coilin and β-tubulin. (D)Mitotic HeLa cell lysate was subjected
to IEF (pH 5-8 IPG strip), SDS-PAGE and western blotting, followed by the detection of coilin and β-tubulin. In panels E and F, HeLa and WI-38 extracts were
treated as described for B and C, except that β-tubulin and SMN were detected. Representative gels are shown. Note that interphase HeLa coilin contains more
phosphoisoforms (arrows) than found in WI-38, and coilin is hyperphosphorylated in WI-38 (arrow) relative to HeLa.
Fig. 6. Quantitative PCR analysis of coilin, SMN and PPM1G expression in
HeLa and WI-38 cell lines and dephosphorylation of coilin by PPM1G.
(A)Coilin, SMN and PPM1G expression levels relative to β-actin are shown.
HeLa values for each message of interest are normalized to 100%. Error bars
represent percentage error about the mean. The difference between relative
coilin levels in HeLa compared with WI-38 is not significant (P=0.25).
However, there is a significant decrease in the relative expression levels of
SMN (P=0.0023) and PPM1G (P=0.000058) in WI-38 compared with HeLa
cells. (B)Lysate from mitotic HeLa cells was untreated or treated with CIP or
recombinant His-tagged PPM1G, followed by SDS-PAGE, western blotting
and detection of coilin using appropriate antibodies.
Journal of Cell Science
The other study that suggests that coilin phosphorylation impacts
CB formation is from the Lamond group (Sleeman et al., 2001). In
this study, transient overexpression of SmB in a primary cell line
that does not normally contain CBs is sufficient to induce the
Journal of Cell Science 122 (11)
appearance of correspondingly transient CBs (Sleeman et al.,
2001). Conversely, the overexpression of coilin in this cell line does
not induce CBs. We hypothesize that the transient expression of
Sm proteins in primary cell lines triggers a signal transduction
cascade that changes the phosphorylation status of coilin from a
CB-restrictive to a CB-permissive state. When Sm protein levels
decrease, we suspect that coilin is restored to a CB-restrictive
phosphorylation state and CBs disperse.
The data presented here provide evidence supporting the
hypothesis that coilin phosphorylation impacts CB formation and
may be part of an unknown signaling pathway initiated in response
to the increased demand for snRNPs. Importantly, we demonstrate
that coilin is hyperphosphorylated in the primary WI-38 line
compared with that found in the transformed HeLa line (Fig. 5).
We also show that PPM1G mRNA levels are reduced in WI-38
compared with HeLa cells (Fig. 6A). Since PPM1G is a known
phosphatase of SMN and can dephosphorylate coilin in vitro (Fig.
6B), our results indicate that this phosphatase directly, or indirectly,
impacts coilin phosphorylation levels and thus explain in part why
transformed cells have CBs whereas primary cells lack these
structures. Other work shown here reveals that CBs can be induced
to form in the WI-38 primary cell upon expression of YFP-PPM1G
but inactive PPM1G, GFP-coilin and GFP-SmB do not form CBs
(Fig. 7). The finding that GFP alone is the most efficient of the
constructs tested at inducing CBs is extremely interesting (Fig. 7),
although the mechanisms underlying this observation are not
entirely obvious. We speculate that CBs are triggered to form in
GFP expressing cells owing to the increased transcription demand
imposed on the cell by the vector. Since CB formation is balanced
by the level of transcription, as demonstrated by studies using
transcription inhibitors such as actinomycin D (Carmo-Fonseca et
al., 1992), it is possible that WI-38 cells expressing GFP alone are
inducing CBs to accommodate the flux of GFP mRNA through the
RNA-processing pathway, despite the fact the GFP message is not
spliced. Clearly, more studies will be necessary to understand these
findings and assess whether they are coupled to changes in coilin
Tandem MS/MS analyses by several groups have found that at
least 11 residues of coilin are phosphorylated (Fig. 1) (Beausoleil
et al., 2004; Olsen et al., 2006; Beausoleil et al., 2006; Nousiainen
et al., 2006) (www.phosida.com). Three of these residues, T122,
S489 and S566, have been shown to be phosphorylated during
mitosis. It is unclear, however, as to the exact contingent of
phosphorylated amino acids of coilin during interphase and mitosis.
To address this issue, we generated mutations in the C-terminal
phosphoresidues of coilin (Fig. 1). We found that a mutant
mimicking a constitutively dephosphorylated state (C6A) disrupts
normal coilin localization in half of the transfected cells (Fig. 2)
and co-immunoprecipitates endogenous coilin (Fig. 3A). By
contrast, a constitutively phosphorylated-like mutant (C6D/E)
localized normally, yet had greatly reduced amounts of co-
immunoprecipitated coilin. These findings suggest that
phosphorylation of coilin C-terminal residues impacts self-
association, yet does not affect the ability of the mutant protein to
incorporate into CBs. However, the ON mutant, which contains D/E
changes in all 11 suspected phosphoresidues, shows a majority of
transfected cells displaying only nucleoplasmic localization (Fig.
2A) and a faster recovery in CBs compared with WT or OFF mutant
coilins, as assessed by FRAP analysis (Fig. 4). Thus, it appears that
hyperphosphorylated coilin is more mobile and more nucleoplasmic
than WT or OFF coilin. However, this interpretation is complicated
Fig. 7. Expression of PPM1G in WI-38 cells induces CBs. (A)WI-38 cells
expressing YFP-PPM1G were fixed and YFP (green), coilin (red) and DAPI
(blue) signals were detected. The right column shows the overlay of all three
images (Merge). The majority of YFP-PPM1G-expressing cells (70%) did not
have CBs (row a), but 30% of cells exhibited CBs (rows b and c, arrows).
Approximately half of the cells with CBs strongly overexpressed YFP-
PPM1G, which accounted for 14% of the 36 cells scored (row c, note also
cytoplasmic localization of YFP-PPM1G). In this particular cell, a nuclear
aggregate of YFP-PPM1G was detected (arrowhead) next to a CB (arrow).
Scale bars: 2μm. (B)GFP-coilin, GFP-SmB and GFP alone expression in WI-
38 cells. Merged images are shown and an arrow indicates the location of a CB
in a cell expressing GFP alone. Scale bar: 2μm. (C)Induction of CBs in WI-
38 cells. The constructs shown were transfected into WI-38 cells. After 24
hours, the cells were processed and scored for CBs. The percentage of cells
with CBs is shown. Note that only cells with nuclear YFP-PPM1G (or
inactive-PPM1G) were scored and, for all constructs, high-expressing cells
were excluded. Cells counted: YFP-PPM1G active=132, YFP-PPM1G
inactive=128, GFP-coilin=50, GFP-SmB=31 and GFP alone=44.
Journal of Cell Science
Phosphorylation impacts CB formation
by the fact that endogenous coilin (which is assumed to be properly
phosphorylated) can interact with the mutant proteins in HeLa cells.
In summary, the work presented here strongly suggests that coilin
phosphorylation impacts the organization of the nucleus with
regard to CB formation in HeLa cells. Furthermore, in the WI-38
primary line that does not contain many CBs, coilin is
hyperphosphorylated relative to that found in HeLa cells, which
have CBs. This hyperphosphorylation correlates with reduced
PPM1G mRNA levels. Transformation, therefore, might increase
snRNP demand and signal to the cell the need to form CBs to
efficiently generate snRNPs. To achieve CB formation in primary
cell lines, we propose that SMN and PPM1G expression have to
be elevated coupled with a dephosphorylation of coilin. Our finding
that PPM1G expression in WI-38 cells induces CBs (Fig. 7) supports
this belief. Recent findings elegantly demonstrate that a wide range
of CB components, including coilin and SMN, can trigger CB
formation at an artificial gene stably integrated into the HeLa
genome (Kaiser et al., 2008). This study supports a self-assembly
model wherein cooperation amongst CB components is necessary
for CB formation. We suggest that an additional requirement for
the CB self-assembly model is that coilin must be in the correct
phosphorylation state. The next step towards deciphering the
putative signaling pathway we propose will be to identify the
repertoire of phosphatases and kinases that control coilin
phosphorylation. We cannot detect a direct interaction between
recombinant bacterially expressed coilin and PPM1G (our
unpublished observations), so it is possible that PPM1G does not
directly act on coilin, or that the interaction is transient and weak.
Previous work has shown that phosphatase inhibitors alter CB
localization (Lyon et al., 1997), although the exact phosphatase(s)
responsible is unknown. With regards to kinases that modify coilin,
we have shown that CDK2–cyclin-E and casein kinase 2 can
phosphorylate coilin in vitro (Liu et al., 2000; Hebert and Matera,
2000); therefore, these kinases will be obvious targets of our future
investigations. It will also be important to determine whether the
phosphorylation of coilin influences its symmetrical dimethylation
and thus interaction with SMN.
Materials and Methods
Cell lines, cell culture, DNA constructs and transfection
HeLa and WI-38 cells were obtained from the American Type Culture Collection.
All cells were cultured and imaged as previously described (Sun et al., 2005). Where
indicated, HeLa cells were treated with 0.4 μg/ml nocodazole for 16 hours to arrest
cells in mitosis. The GFP-coilin clone has been described previously (Hebert and
Matera, 2000). The GFP-coilin construct was used as a template to generate C-terminal
mutations of residues known to be phosphorylated. This was accomplished using the
QuikChange Mutagenesis kit (Stratagene, La Jolla, CA) and appropriate mutagenesis
primers (supplementary material Table S1). Mutations were verified by sequencing.
YFP-PPM1G and inactive YFP-PPM1G has been described previously (Petri et al.,
2007; Murray et al., 1999). DNA was transfected into HeLa and WI-38 cells using
either SuperFect (Qiagen), Lipofectamine 2000 (Invitrogen), or FuGene 6 according
to the manufacturer’s directions. HeLa stable cell lines expressing GFP-coilin, GFP-
coilin(ON) or GFP-coilin(OFF) were generated by G418 selection. For studies in
coilin-knockdown cells, HeLa cells were transfected with coilin duplex siRNAs
(Whittom et al., 2008) for 48 hours, followed by transfection with the various coilin
constructs for 24 hours. GFP-coilin signal can still be detected, albeit faintly, in the
coilin-knockdown cells given that the EGFP vector has a CMV promoter. Cells were
verified for endogenous coilin knockdown by assessing if SMN was present in CBs.
Coilin knockdown was measured at the protein level by western blotting and
comparing GFP-coilin (or mutants thereof) and endogenous coilin to tubulin after
treatment with control or coilin siRNA.
Antibodies, immunofluorescence, coimmunoprecipitation and
Immunofluorescence, western blotting and image acquisition were performed as
previously described (Sun et al., 2005). Monoclonal antibodies against SMN were
from BD Biosciences. Coilin polyclonal antibody H-300 was purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Tubulin monoclonal antibody TUB 2.1 was
purchased from Sigma (St Louis, MO). Lysates were generated by resuspending cells
in a modified RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA,
1% sodium deoxycholate and 1% NP-40), followed by brief sonication. Cell debris
was removed by centrifugation for 10 minutes at full speed in a 4°C microcentrifuge.
The samples were then subjected to western blotting or immunoprecipitation. For
immunoprecipitation, samples were first pre-cleared with 20 μl 50% protein-G-
Sepharose (GE Healthcare) for 1 hour at 4°C with gentle inversion. After the
incubation, the beads were collected by centrifugation at 3000 r.p.m. in a
microcentrifuge for 5 minutes and the supernatant was placed in a new tube. To the
supernatant was added 2 μg of monoclonal antibodies to GFP (Roche), followed by
incubation for 1 hour at 4°C with gentle inversion. After the incubation, 30 μl of
50% protein-G-Sepharose was added to the samples and they were incubated for an
additional 1 hour at 4°C with gentle inversion. The samples were then centrifuged
at 3000 r.p.m. for 5 minutes and the beads were washed with 1 ml RIPA. The procedure
was repeated two more times, after which the beads were resuspended in 20 μl 5?
SDS loading buffer and subject to SDS-PAGE and western blotting as described (Sun
et al., 2005). Coilin (endogenous or fused to GFP) was detected using a polyclonal
antibody (H-300) from Santa Cruz Biotechnology.
Fluorescence recovery after photobleaching (FRAP)
FRAP experiments were performed on a Zeiss 510 Meta confocal LSM. Images were
collected with the 488 line of an argon laser (30 mW output, detection 500-575 nm)
with a Plan-Apochromat ?63/1.4 oil DIC objective lens. During imaging, cells were
maintained in Labtek II chambered coverslips (Nunc) and maintained at 37°C with
an objective heater and heated stage equipped on the microscope. For each scan,
three pre-bleach images were taken, and a single 2 μm spot containing a Cajal body
was bleached with the 488nm line at 100% transmission. Images were obtained by
scanning at 3% transmission at 3 second intervals and fluorescence recovery in the
bleached area was monitored until the plateau was reached. Z-stacks were aligned
with the stackreg plugin for ImageJ and resulting normalized FRAP recovery curves
were subjected to double normalization as previously described (Phair et al., 2004a;
Phair et al., 2004b). The resulting recovery curves were plotted with Origin 6.1
(Microcal). The recovery curves for each cell were fitted to a double exponential
equation and the time necessary to reach half-maximal recovery (T50) was determined
as previously described (Phair et al., 2004a; Kimura and Cook, 2001).
Two-dimensional gel electrophoresis
Lysates were prepared from mitotic HeLa, interphase HeLa or WI-38 by resuspending
cell pellets in 2-D solubilization buffer containing 9.5 M urea, 2% NP-40 and 2% β-
mercaptoethanol. The cells were then disrupted using the Sonic Dismembranator,
Model 100 (Fisher Scientific, Pittsburg, PA), with an output of 1 for three pulses of
5 seconds each, with cooling on ice between each pulse. After sonication, the samples
were centrifuged for 5 minutes at 17,000 g. Immobilized pH gradient (IPG) strips
(Bio-Rad, Hercules, CA) were used according to the manufacturer’s protocol. Briefly,
125 μl sample was used to rehydrate 7 cm pH 7-10 or pH 5-8 IPG ready strips for
12-15 hours before isoelectric focusing (IEF). Following IEF using a Protean IEF
cell (Bio-Rad) for 10,000 volt-hours at 50 μA per strip with rapid ramping, the strips
were equilibrated for 20 minutes with equilibration buffer containing 6 M urea, 2%
SDS, 0.05 M Tris-HCl pH 8.8, 20% glycerol, and 2% β-mercaptoethanol, followed
by SDS-PAGE (10%) and Western blot analysis. Images were obtained using the
Bio-Rad Molecular Imager ChemiDoc XRS system and processed as previously
described (Sun et al., 2005). For treatment with calf intestinal alkaline phosphatase
(CIP), cell pellets were first lysed in RIPA as described above, followed by the addition
of 10 μl of 10 U/μl CIP from New England Biolabs (Ipswich, MA) in 1? New
England Biolabs buffer 2. The reactions were incubated for 1 hour at 37°C, followed
by a buffer change into 2-D solubilization buffer using an YM30 Microcon filter unit
(Millipore Corporation, Bedford, PA) according to the manufacturer’s instructions.
cDNA synthesis and quantitative real-time PCR
cDNA was synthesized from RNA isolated from HeLa and WI-38 cells using the
iScript cDNA Synthesis kit from Bio-Rad according to the manufacturer’s protocol.
Samples were incubated at 25°C for 5 minutes followed by 42°C for 30 minutes and
then 85°C for 5 minutes using a PCT-200 Peltier Thermal Cycler (MJ Research). For
qRT-PCR, cDNA and primers outlined in supplementary material Table S2 were added
to the Brilliant SYBR green QPCR Master Mix kit (Stratagene, La Jolla, CA) and
the reactions were subjected to a 10 minute incubation at 95°C, followed by 40
amplification cycles (95°C for 30 seconds, 50°C for 30 seconds, 72°C for 1 minute)
and a dissociation curve-analysis step. The reactions were conducted using a
MX3000P or a MX3005P real-time PCR system (Stratagene). Amplification rates,
Ct values and dissociation curve analyses of products were determined using MxPro
(version 4.01) software. Relative expression was determined using the 2(–ΔΔCt)method
(Livak and Schmittgen, 2001). Three independently isolated RNA samples were used
for each cell line, and each sample was conducted in triplicate. Student’s t-test was
used to determine statistical significance (a P-value of less than 0.05 is considered
Journal of Cell Science
1880Journal of Cell Science 122 (11)
In vitro phosphatase assay
Mitotic HeLa cells were lysed and sonicated in RIPA buffer. The lysate was untreated,
treated with 5 μl of 10 U/μl CIP from New England Biolabs (Ipswich, MA), or treated
with 2.6 μM recombinant His-tagged PPM1G in 1? New England Biolabs buffer 2
for 1 hour at 37°C, followed by SDS-PAGE, western blotting, and detection of coilin
using appropriate antibodies.
This work was supported by NIGMS grant 1R01GM081448-01A1
to MDH. We thank the NIH supported MFGN INBRE Program of the
National Center for Research Resources (RR 016476) for the use of
the confocal microscope at the University of Southern Mississippi.
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