3601
Journal of Cell Science 107, 3601-3614 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
Nuclear calmodulin/62 kDa calmodulin-binding protein complexes in
interphasic and mitotic cells
Manuel Portolés1, Magdalena Faura1, Jaime Renau-Piqueras1,*, Francisco José Iborra1, Rosana Saez2,
Consuelo Guerri2, Joan Serratosa3, Eulalia Rius4 and Oriol Bachs4
1Centro de Investigación, Hospital “LA FE”, Avda. de Campanar 21, 46009 Valencia, Spain
2Instituto Valenciano de Investigaciones Biomédicas, Amadeo de Saboya 4, 46010 Valencia, Spain
3Departament de Farmacologia i Toxicologia, Consejo Superior de Investigaciones Científicas, Jorge
Girona Salgado, 18-26,
08034 Barcelona, Spain
4Departament de Biologia Cel.lular i Anatomia Patològica, Universitat de Barcelona, Casanova 143, 08036 Barcelona, Spain
*Author for correspondence
SUMMARY
We report here that a 62 kDa calmodulin-binding protein
(p62), recently identified in the nucleus of rat hepatocytes,
neurons and glial cells, consists of four polypeptides
showing pI values between 5.9 and 6.1. By using a DNAbinding overlay assay we found that the two most basic of
the p62 polypeptides bind both single- and double-stranded
DNA. The intranuclear distribution of calmodulin and p62
was analysed in hepatocytes and astrocyte precursor cells,
and in proliferating and differentiated astrocytes in
primary cultures by immunogold-labeling methods. In
non-dividing cells nuclear calmodulin was mostly localized
in heterochromatin although it was also present in euchromatin and nucleoli. A similar pattern was observed for p62,
with the difference that it was not located in nucleoli.
p62/calmodulin complexes, mainly located over heterochromatin domains were also observed in interphasic
cells. These complexes remained associated with the
nuclear matrix after in situ sequential extraction with
nucleases and high-salt containing buffers. In dividing
cells, both calmodulin and p62 were found distributed over
all the mitotic chromosomes but the p62/calmodulin aggregates were disrupted. These results suggest a role for
calmodulin and p62 in the condensation of the chromatin.
INTRODUCTION
The nuclear concentration of CaM has been recently quantified in a few different cell types (Pujol et al., 1989; Vendrell
et al., 1991) and it has been shown that the nuclear CaM
content can be modulated by hormones and growth factors. In
adrenal cortex cells the nuclear location of CaM becomes very
pronounced after stimulation with ACTH (Harper et al., 1980).
Simmen et al. (1984) have reported that estrogens induced the
transient association of CaM with the nuclear matrix of chicken
liver cells. It has also been demonstrated that when rat hepatocytes are proliferatively activated in vivo by partial hepatectomy, a fourfold increase in the total nuclear CaM concentration is produced (Serratosa et al., 1988; Pujol et al., 1989).
Moreover, an increased association of CaM with the nuclear
matrix is also induced after the proliferative activation of rat
liver cells (Serratosa et al., 1988; Pujol et al., 1989).
CaM function requires CaM targets, i.e. CaM-binding
proteins. Therefore, the identification of nuclear CaM-binding
proteins, their specific location in the nucleus and the study of
their changes during cell proliferation and differentiation are
crucial aspects for the understanding of the roles of CaM in
nuclear function. During the last few years several reports have
shown the presence of CaM-binding proteins in the nuclei of
different cell types. The nuclear detection of α-spectrin,
Calmodulin (CaM), the major calcium-binding protein in
smooth muscle and non-muscle cells is a universal regulator of
the calcium signal (Klee and Vanaman, 1982; Means et al.,
1982). CaM is present in all eukaryotic organisms although its
levels vary in the different cell types. The primary structure of
CaM has been highly conserved throughout evolution, suggesting that it is involved in the regulation of processes that
are fundamental to cell life. Intracellularly, CaM is present in
the cytosol but it has also been found associated with several
cellular structures such as plasma membrane, cytoskeleton and
nuclei (Harper et al., 1980; Bachs and Carafoli, 1987; Bachs
et al., 1992). The finding of CaM in the nucleus of many cell
types has prompted, during the last few years, the investigation of different aspects of the role of CaM in the regulation
of nuclear functions. The evidence reported to date have
indicated that DNA replication, DNA repair, the transcription
of several genes and the phosphorylation and dephosphorylation of nuclear proteins could be modulated by CaM (Boynton
et al., 1980; Chafouleas et al., 1984; Sahyoun et al., 1984;
White, 1985; Rasmussen and Means, 1989; Kapiloff et al.,
1991, Bosser et al., 1993).
Key words: calmodulin, hepatocyte, p62, astrocyte
3602 M. Portolés and others
myosin light chain kinase (MLCK) and caldesmon, CaMbinding proteins that are components of the actin motility
systems activated by Ca2+ and CaM, indicates that an intranuclear contractile system is present in the nuclei of rat liver cells
(Bachs et al., 1990). α-Spectrin (Vendrell et al., 1991) and
recently actin and MLCK (Pujol et al., 1993) have also been
observed in the nuclei of rat neurons. The presence of CaMdependent protein kinase II and calcineurin in the nuclei of
neurons (Sahyoun et al., 1984; Pujol et al., 1993) indicates that
nuclear CaM could also regulate the phosphorylation and
dephosphorylation of nuclear proteins.
A 62 kDa CaM-binding protein (p62) showing low affinity
for CaM has been recently identified in the nuclei of rat liver
cells (Bachs et al., 1990). It has been partially purified by
affinity chromatography using CaM-Sepharose columns, and
polyclonal antibodies that specifically recognize p62 have been
obtained (Bachs et al., 1990). This protein has been found to
be also present in rat neurons and glial cells (Vendrell et al.,
1991). However, the possible functions of p62 are at present
unknown. In order to gain insight into the nuclear role of p62
we analysed the ability of p62 to bind DNA and its precise
intranuclear location in differentiated and proliferating cells.
We report here that p62 is a DNA-binding protein and that it
is mainly localized in the heterochromatin of interphasic cells
and in the chromosomes during mitosis. We also report here
that p62/CaM complexes are present in interphasic cells
whereas these complexes are disrupted during mitosis.
MATERIALS AND METHODS
Astrocyte cultures
Primary cultures of astrocytes from 21-day-old rat fetuses were
prepared from brain hemispheres as described (Renau-Piqueras et al.,
1989; Gómez-Lechón et al., 1991). These cultures grew rapidly for 710 days (proliferative period) and after that a decrease in cell proliferation occurs (differentiation period) (Renau-Piqueras et al., 1989;
Guerri et al., 1990). All the experiments were done in triplicate on 7and 21-day cultures. The purity of astrocyte cultures was assessed
using a mouse anti-glial fibrillary acidic protein (GFAP) monoclonal
antibody and fluorescence microscopy (Renau-Piqueras et al., 1989;
Saéz et al., 1991).
Isolation of nuclei and nuclear matrix
The procedure for the isolation of nuclei from astrocytes was that
described by Thompson (1973). To obtain rat liver cell nuclei 3-10 g
of liver were homogenized in 40 ml of STM buffer (250 mM sucrose,
5 mM MgSO4, 50 mM Tris-HCl, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.5 g/ml aprotinin (Sigma). The
homogenate was filtered through four layers of cheescloth and then
centrifuged at 800 g for 10 minutes at 4°C. This step was repeated
once more with the pellet. The pellet was resuspended with 30 ml of
RSB buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2,
1 mM PMSF and 0.5 g/ml aprotinin); then Nonidet P-40 to a final
concentration of 0.5% was added and the samples vortexed for 40
seconds. The suspension was centrifuged at 800 g for 5 minutes at
4°C and the step was repeated twice more. Finally, the pellet, which
corresponded to the nuclei, was washed three times with RSB buffer
and resuspended in STM buffer.
Nuclear matrix fractions were obtained from isolated liver cell
nuclei as previously described (Bachs et al., 1990). In some experiments nuclear matrix fractions were extracted with 20 mM Tris-HCl,
pH 7.4, 0.1 mM MgCl2, 10% sucrose, 2% Triton X-100 and 0.5 mM
PMSF for 10 minutes at 4°C. Then, the samples were centrifuged at
22,000 g for 10 minutes. The pellet was subsequently extracted with
the same buffer containing 6 M urea instead of Triton X-100 for 10
minutes at 4°C and then centrifuged at 22,000 g for 10 minutes. The
pellet was then collected and analysed by gel electrophoresis.
Electrophoresis and immunoblotting
Samples were separated on Laemmli type (Laemmli, 1970) SDSpolyacrylamide mini slab gels (8 or 10%). Then, the proteins were
transferred to immobilon-P membranes for 2 hours at 60 V (Towbin
et al., 1979). The sheets were subsequently subjected to western
blotting according to Bachs et al. (1990) using affinity-purified polyclonal antibodies against p62 (1:200 dilution). Samples were also
subjected to two-dimensional electrophoresis according to O’Farrell
(1975).
DNA-binding assay
DNA-binding experiments were performed as described by Hakes and
Berezney (1991). Immobilon P membranes containing proteins transferred from one- or two-dimensional gels were incubated in TNMT
buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, and
0.5% Tween-20) for 2 hours at room temperature. Genomic DNA
from salmon, labeled with 32P by random primer (5×106 cpm/ml),
were incubated with the blots in TNMT buffer overnight at room temperature. Unbound DNA was removed by washing the blots in TNMT
buffer for 1 hour with four changes of buffer. The filters were allowed
to dry and then exposed to X-ray films (Kodak).
Protein determination
The protein content of the fractions was measured according to the
method of Lowry et al. (1951) using bovine serum albumin as a
standard.
Tissue processing for electron microscopy
Rat liver (Wistar rats of 150-200 g) was fixed by perfusion with 0.5%
glutaraldehyde/3% formaldehyde as described (Roht et al., 1985) and
embedded in Lowicryl K4M (Bendayan et al., 1987). Proliferating and
differentiated astrocytes in primary cultures were fixed as monolayers with 0.5% glutaraldehyde/4% formaldehyde in 0.1 M PIPES
buffer, pH 7.3, for 60 minutes at 4°C, detached from the plastic using
a rubber policeman, incubated for 60 minutes in 50 mM ammonium
chloride and embedded in Lowicryl K4M (Bendayan et al., 1987;
Renau-Piqueras et al., 1989). Astrocytes growing as monolayers were
fractioned as described by Fey et al. (1984). The nuclear matrix-intermediate filament fractions were fixed in 0.5% glutaraldehyde/4%
formaldehyde in 0.1 M cacodylate buffer, pH 7.4, dehydrated and
embedded in Epon. Gestational day 16 (E16) rat embryos were
perfused intracardially with cold 0.5% glutaraldehyde/4% formaldehyde in 0.1 M PIPES buffer, pH 7.2. Brains were then removed by
dorsal craniectomy, and immersed in the same fixative solution for 4
hours. After washing with the buffer, brains were postfixed in 1%
OsO4 for 45 minutes at 4°C, dehydrated in ethanol and embedded in
LR White. Osmication of the tissues was used to make possible the
location of the different cell layers in the cortical wall.
Immunogold labeling
Ultrathin sections (80 nm) mounted on parlodion-coated nickel grids
were floated for 30 minutes on 0.1% BSA-Tris buffer (20 mM TrisHCl, 0.9% NaCl, pH 7.4 containing 0.1% BSA, type V) supplemented
with 5% heat-inactivated fetal calf serum (FCS), and then transferred
to droplets of 0.1% BSA-Tris buffer containing 1% FCS and a rabbit
anti-p62 polyclonal antibody (1/125 dilution) obtained as previously
described (Bachs et al., 1990) or an anti-CaM polyclonal antibody
(1/8,000 dilution) developed in goat (Sigma C-6784). After several
assays the dilutions 1/125 for p62 and 1/8000 for CaM, which
minimize the background staining, were chosen. The sections were
incubated in a moist chamber for 2 hours at 37°C. After three rinses
Calmodulin and p62 in hepatocytes and astrocytes 3603
(10 minutes each) with 0.1% BSA-Tris buffer, the grids were placed
on droplets of 0.1% BSA-Tris buffer containing 0.05% Tween-20, 5%
FCS, and a goat anti-rabbit IgG-gold complex (10 nm, Sigma, 1/10
dilution), or a rabbit anti-goat IgG-gold complex (5 nm, Sigma, 1/10
dilution) for p62 and CaM, respectively. The incubation time was 60
minutes at room temperature as above. After two rinses (10 minutes
each) with 0.1% BSA-Tris buffer and a rinse in bidistilled water, the
sections were air dried and finally counterstained with uranyl acetate
for 30 minutes. In some cases double labeling using anti-p62 and antiCaM antibodies, anti-GFAP (1/25 dilution) and anti-p62, or antiGFAP and anti-CaM were carried out.
Controls were incubated without the first antibody. In some cases,
0.05% Tween-20 was used in all solutions to reduce possible nonspecific charge attraction to antibody. The latter control, but with
sections previously floated on 0.5 M ammonium chloride for 60
minutes, was also followed. Controls of clumping of gold particles in
the IgG-gold complexes were routinely performed.
Serial coronal semithin sections of fetal brain, from olfatory bulbs
through occipital poles, were used for categorization of cellular localization in the developing cerebral wall. Ultrathin sections from the
dorsal domain were selected (Austin and Cepko, 1990). In this domain
the following zones can be observed: (a) germinal matrix; (b) subventricular zone; (c) intermediate zone; (d) cortical plate; and (e)
marginal zone. Micrographs of cells from the germinal matrix and the
sub-ventricular zone were taken. Double labeling with anti-GFAP and
anti-p62 or with p62 and CaM antibodies was also carried out in these
cells.
EDTA regressive staining
EDTA regressive staining (Bernhard, 1969; Risueño and Moreno
Diaz de la Espina, 1979) was performed on sections of astrocytes or
liver previously processed for the immunocytochemical demonstration of p62. The sections were floated in distilled water for several
minutes and then for 20 minutes in 20% uranyl acetate. After washing
in water, the sections were treated with EDTA (1/10) for 5 seconds,
washed in water, stained with lead citrate for 15 seconds, rinsed and
air-dried.
Quantitative evaluation
Quantitative analysis of micrographs was carried out as previously
described (Renau-Piqueras et al., 1989; Iborra et al., 1992) and the
results were expressed as number of gold particles/µm2. The
procedure used to select samples and micrographs has been previously
described in detail (Cruz-Orive and Weibel, 1981; Renau-Piqueras et
al., 1987, 1989). The minimum sample size for each parameter considered was determined by the progresive mean technique (confidence
limit ± 10%) (Salpeter and McHenry, 1973; Williams, 1977). All the
quantitative analyses were carried out using a BASIC programme
developed in our laboratory (De Paz et al., 1990). All the quantitative
data, both biochemical and immunocytochemical, were statistically
analysed by Student’s t-test and the ANOVA test using the Statistic
program (version 3.1).
RESULTS
Binding of DNA to p62
Nuclear matrix from rat liver cells was extracted with 2%
Triton X-100 and subsequently with 6 M urea as described in
Materials and Methods. Then, the residual fraction was
subjected to two-dimensional gel electrophoresis. In these gels,
p62 appeared as four spots showing pI values between 5.9 and
6.1 (Fig. 1B). The three most basic spots showed the same Mr,
whereas the most acidic polypeptide had a slightly lower Mr.
Fig. 1C shows that all the spots reacted with polyclonal antibodies raised against the protein purified from one-dimensional
Fig. 1. DNA overlay and western blotting with anti-p62 antibodies
on nuclear matrix from rat liver cells. Nuclear matrices from rat liver
cells were sequentialy extracted with 1% Triton X-100 and 6 M urea.
The proteins of the residual insoluble fraction were separated on
two-dimensional gels, then transferred to Immobilon P membranes
and incubated with 32P-labeled genomic DNA (double-stranded) (A)
or subjected to western blotting using specific anti-p62 antibodies
(C). A gel was also stained with Coomassie Blue (B). The
arrowheads indicate the positions of two p62 polypeptides that bind
[32P]DNA.
gels (Bachs et al., 1990). The different spots were electroeluted
from the two-dimensional gels and then, antibodies against the
high Mr spots or the low Mr polypeptide were raised independently. The antibodies were used for immunoblotting experiments, which showed that both antibodies reacted against all
the four spots (data not shown), indicating that the high and
3604 M. Portolés and others
Fig. 2. Immunological identification of p62 in nuclear matrices from
astrocytes in primary cultures. Samples of nuclear matrices (60 µg)
from astrocytes (4, 7, 14 and 21 days in culture) were
electrophoresed in 8% SDS-PAGE gels, transferred to nitrocellulose
sheets, and subjected to western blotting using the antibody against
p62.
To analyse the ability of p62 to bind DNA, [32P]DNA
overlay experiments were carried out. As showed in Fig. 1A
double-stranded genomic [32P]DNA bound to the two most
basic spots of p62 (arrowheads), but not to the others. The
intensity of the binding was higher in the most basic protein.
Similar results were obtained when experiments using singlestranded DNA were carried out (data not shown).
To look for the presence of p62 in astrocytes, immunoblot
experiments on nuclear matrix fractions prepared from astrocytes at different times after culturing (4, 7, 14 and 21 days)
were carried out. As shown in Fig. 2, the anti-p62 antibodies
recognized only one band at the 62 kDa level in all the samples.
Two-dimensional western blot experiments also revealed that
p62 from astrocytes displays a polypeptide pattern similar to
that from hepatocytes (data not shown).
the low Mr polypeptides are immunologicaly related and that
they are possibly variants of the same protein.
Immunolocalization of p62 and CaM
The intracellular localization of p62 and CaM was analysed
in differentiated (21 days) and proliferating (7 days) astro-
kDa
Fig. 3. Immunocytochemical localization of p62 in 21-day astrocytes. (A) 21-day astrocytes in primary cultures were processed for
immunocytochemical co-localization of GFAP (5 nm particles) and p62 (10 nm particles). Anti-GFAP-binding sites are over intermediate
filaments, whereas anti-p62 is located mainly over condensed chromatin. (B) 21-day astrocytes were processed for the visualization of RNP
structures using the EDTA technique. Then, they were subjected to immunogold labeling using anti-p62 antibodies. Perichromatin granules
(PG), interchromatin granules (IG), perichromatin fibrils (PF) and interchromatin fibrils (IF) were seen by this method. Bars, 0.5 µm.
Calmodulin and p62 in hepatocytes and astrocytes 3605
cytes. Cells from both populations, when observed in the
electron microscope, showed a flat profile with a cell body
containing the nucleus and long processes filled with intermediate filaments, which were stained with anti-GFAP
antibody (Fig. 3A). The nucleus was elongated, with a scant
amount of condensed chromatin (CC), mainly in contact with
the nuclear envelope. Stereological analysis demonstrated no
significant differences between the volume density of CC
from proliferating and differentiated astrocytes. In most cells,
the nucleus contained one or more well developed nucleoli.
Fig. 4. Immunolocalization of p62 in
proliferating and differentiated
astrocytes in primary cultures.
Proliferating (A) or differentiated (B)
astrocytes were subjected to
immunogold labeling using anti-p62
antibodies. In all cases, gold particles
are mainly over condensed chromatin.
No labeling of RNP structures is
observed (arrows). Bar, 0.5 µm.
3606 M. Portolés and others
Moreover, nuclear bodies, perichromatin and interchromatin
granules as well as perichromatin fibrils were seen within the
nucleus. These ribonucleoprotein structures (RNP) were
clearly seen when EDTA regressive staining was used (Fig.
3B).
Cells from both cell populations showed a similar distribution of p62. Gold particles were found over both cytoplasm and
nucleus. In the cytoplasm, no specific labeling was seen associated with any cytoplasmic component. In the nucleus,
labeling was mainly located over CC, although it could also be
observed in euchromatin (Fig. 4). In addition to single gold
particles, groups of particles were also observed, mainly
located in the heterochromatinic regions. Very few particles
were seen associated with RNP structures (Fig. 4A and B,
arrows), but labeling was never observed in the nuclear pores.
Labeling was specific as shown by the absence of gold particles
under control conditions (data not shown). Like p62, CaM was
present in cytoplasm and nucleus. Nuclear CaM was mainly
located in the CC. However, in contrast to p62, CaM was also
detected over some RNP (Fig. 5).
The intracellular distribution of p62 was also studied in liver
cells. As shown in Fig. 6, p62 appeared mainly over nuclei and
Fig. 5.
Immunolocalization of
CaM in differentiated
astrocytes in primary
cultures. 21-day
astrocytes were subjected
to immunogold labeling
using specific anti-CaM
antibodies. It can be
observed that the labeling
is preferentially located
over condensed
chromatin (A). Some
RNP structures were also
labeled with gold
particles (arrows) (B,C).
Bar, 0.25 µm.
Calmodulin and p62 in hepatocytes and astrocytes 3607
there mostly located over CC regions. As in astrocytes, the
labeling consisted of both single particles and groups. The
intranuclear distribution of CaM in liver cells was similar to
that of astrocytes (data not shown). In addition analysis of p62
labeling on purified nuclear envelopes revealed that this protein
was absent from this nuclear component. In the nucleus, as for
p62, isolated or groups of gold particles appeared located
mainly over the CC. Moreover, only CaM was also found over
the nucleolus and some RNP particles (data not shown). Obser-
vation of nuclear matrix preparations showed that p62
appeared to be located over the matrix network.
Quantitative analysis was carried out in both differentiated
and proliferating astrocytes. The results revealed that the level
of p62 in the nuclei was higher than in the cytoplasm or the
nucleoli and that the amount of this protein was higher in proliferating than in differentiated cells (Fig. 7A). Most of the p62
(80%) was located over the heterochromatin without any difference in location between peripheral and central CC (Table
Fig. 6. Immunolocalization of p62 in
rat hepatocytes. Sections of rat livers
were subjected to immunogold
labeling using anti-p62 antibodies.
(A) The gold particles were
preferentially located over
condensed chromatin. (B) The
nucleoli and other RNP-containing
structures lacked labeling (arrow).
Bar, 0.25 µm.
3608 M. Portolés and others
Table 1. Particle density of anti-p62 and anti-CaM binding
sites over nuclear components (perinuclear, central and
total heterochromatin and total euchromatin) in
proliferating and differentiated astrocytes
Proliferating
astrocytes
(7 days)
Differentiated
astrocytes
(21 days)
p62
Heterochromatin:
total
central (%)
perinuclear (%)
Euchromatin
71.4±15.0
49.9±23.4
50.1±20.5
28.6±5.7***
82.0±27.1
48.1±23.1
51.9±20.2
18.0±7.9***
CaM
Heterochromatin:
total
central (%)
perinuclear (%)
Euchromatin
83.0±33.6
50.1±25.0
49.9±27.9
17.0±10.3***
66.8±24.0
50.5±18.2
49.5±32.7
33.2±22.2**
As shown, particles are located preferentially over heterochromatin. The
anti-p62 and anti-CaM labeling patterns over differentiated cells were similar
to those of proliferating cells. Results, expressed as particle density (number
of gold particles/µm2) percentage are the mean ± s.d. of three different
experiments (***P<0.001, **P<0.005). Central and perinuclear
heterochromatin results are expressed as percentage of total heterochromatin.
Fig. 7. Particle density of (A) anti-p62-binding sites and (B) antiCaM-binding sites in the nucleus (N), nucleolus (Nu) and cytoplasm
(Cy) of 7- and 21-day astrocytes. The anti-p62- and anti-CaMbinding sites were quantified as described in Materials and Methods.
Results, expressed as number of gold particles/µm2 (particle
density), are the mean ± s.d. of three different experiments. *P<0.05;
**P<0.005; and ***P<0.001. Statistical differences between 7 and
21 days of culture are shown in parenthesis.
1). No significant differences between the amount of CaM in
the nucleus and in the cytoplasm of proliferating astrocytes
were observed. Nevertheless, in differentiated cells the amount
of nuclear CaM was 2-fold higher than that of the cytoplasm.
The amounts of nuclear CaM in both cellular populations were
similar. However, the amount of cytoplasmic CaM was 2-fold
lower in differentiated cells (Fig. 7B). Like p62, the levels of
CaM in the CC were higher than in the euchromatin (Table 1).
The presence of p62 and CaM in nuclear matrix preparations
obtained by in situ extraction with nucleases and high-salt containing buffers was also studied. The nuclear matrix sections
showed a morphology similar to that reported by Fey et al.
(1984). In these sections both p62 and CaM appeared to be dis-
tributed on some filaments of the nuclear matrix network
(Fig. 8).
The intranuclear distribution of p62 and CaM was also
analysed in astrocyte precursor cells (GFAP positive cells).
These cells are continuosly dividing in the brain of rat embryos
at gestational day 16 (E16). Therefore, they constitute a good
model for the analysis of the distribution of p62 and CaM during
mitosis. Fig. 9 (A and B) shows sections of the germinal matrix
of E16 rat cortex in which mitotic figures can be easily observed.
In interphasic cells, the intranuclear distribution of p62 and CaM
was similar to that of astrocytes in primary cultures (Fig. 10A).
During mitosis, p62 and CaM were found mostly over chromosomes (Fig. 10B). The density of p62 gold particles over the
chromosomes was 27.2±6.6 particles/µm2, whereas over the
remaining cell area was 2.3±0.5 particles/µm2.
Immunoco-localization of p62 and CaM
The co-localization of p62 and CaM in quiescent and dividing
cells was analysed by incubating ultrathin section simultaneously with anti-p62 and anti-CaM antibodies. The results
revealed that in quiescent cells two different types of aggregates of gold particles could be distinguished: (1) groups of 5
nm particles, corresponding to CaM aggregates; and (2)
complexes formed by 10 nm particles (p62) surrounded by 5
nm gold particles (Fig. 11A and C). The p62/CaM complexes
were mainly localized over CC in cultured astrocytes, hepatocytes and brain cortical astroglia cells (Fig. 11A and C). These
p62/CaM aggregates were also observed in nuclear matrix
preparations (Fig. 11D), indicating a strong interaction of both
proteins with this nuclear structure. In astrocytes the ratio of
anti-CaM/anti-p62-binding sites was 9.7±3.2 and the mean
distance between 10 nm and 5 nm gold particles in the
p62/CaM aggregates was of 17.6±3.1 nm. This distance was
calculated between the 10 nm particles and the small particles
forming the first annulus. In mitotic cells from the germinal
layer of E16 brains, both CaM and p62 were located over chro-
Calmodulin and p62 in hepatocytes and astrocytes 3609
Fig. 8. Immunolocalization of p62 and CaM in nuclear matrices from astrocytes. Ultrathin sections of 15-day astrocytes in primary cultures
were extracted with nucleases and subsequently with a high-salt containing buffer as described in Materials and Methods. Then the sections
were processed for immunocytochemical localization of p62 (A) or CaM (B). In these preparations both p62 and CaM appeared to be
distributed over filaments of the nuclear matrix network. Bars, 0.25 µm.
mosomes. However, the double-labeling pattern revealed that
only single gold particles corresponding to CaM or p62 were
present on the chromosomes and work in progress suggests that
these complexes are modulated during mitotic phases. Thus, in
contrast to non-dividing cells, no p62/CaM aggregates were
found in mitotic chromosomes (Fig. 11B). The distribution of
both CaM and p62 was over all the chromosome and no
specific accumulations were found over any specific chromosomal region (data not shown). No labeling was found over
microtubules near the chromosome.
DISCUSSION
Recently, the detection of a low-affinity CaM-binding protein
of 62 kDa in the nucleus of rat liver cells and neurons has stimulated studies on the presence and the precise intranuclear
localization of this protein in different cell types (Bachs et al.,
1990; Vendrell et al., 1991; Bosser et al., 1993). p62 is a highly
insoluble CaM-binding protein, which cannot be extracted
from the nuclei by treatment with nucleases, high-salt containing buffers or 6 M urea. Thus, after these treatments p62
still remains associated with the residual nuclear matrixlamina, indicating a close association between p62 and this
nuclear structure.
Not much is known about the identity of p62. However, this
protein does not correspond to the 62 kDa glycoprotein
component of the nuclear pores (Starr et al., 1990; Finlay et
al., 1991), since they have different binding capacities for the
lectin wheat-germ agglutinin (O. Bachs, unpublished results)
3610 M. Portolés and others
Fig. 9. Low-magnification electron micrographs of E16 cerebral wall. In (A) several post-mitotic cells of the sub-ventricular zone, next to the
germinal matrix, are seen. In (B) a mitotic cell of the germinal matrix is shown. V, ventricular surface. Bar, 2 µm.
and, moreover, p62 is not present in the nuclear pores as
revealed by the immunogold-labeling experiments reported
here. p62 is also distinct from a 62 kDa protein described by
Fields and Shaper (1988) in the metaphase chromosomes of rat
hepatocytes, since two-dimensional electrophoresis revealed
that p62 is clearly more acidic (pI 5.9-6.1) than this chromosomal protein, which shows a pI of 7.0-7.2. p62 consists of
four different polypeptides. The three more basic polypeptides
show the same Mr, which is slightly higher than that of the most
acidic protein. Partial proteolysis experiments revealed that the
high Mr spots correspond to the same protein whereas the most
acidic polypeptide has a slightly different peptidic profile (P.
James, personal communication). However, the finding that
antibodies generated against the higher Mr polypeptides recognized all four proteins, as well as the antibodies raised
against the low Mr protein, strongly suggest that all polypeptides are variants of the same protein.
In addition to the p62 proteins found in rat liver, the presence
of two proteins with similar molecular mass has been described
in sea urchin embryos and human cells, revealed using an antip230 obtained from the protozoan Polyplastron m. The former
protein was associated with the mitotic apparatus whereas the
second one appears to be located in the centrosome (Dinsmore
and Sloboda, 1988; Moudjon et al., 1991). The results
presented here indicate that the p62 used in our work is not
associated with these mitotic components.
An important characteristic of p62 is that it is able to bind
to single- and double-stranded DNA. However, not all the p62
polypeptides are able to bind DNA, indicating that the binding
is in some way regulated. The finding that the binding capacity
decreases from the basic to the acidic polypeptides suggests
that the ability of p62 to bind DNA could be decreased by
phosphorylation of the protein. Since p62 is a CaM-binding
protein it is possible that the association of CaM with p62 could
also regulate the binding capacity of p62 to DNA. However,
these possibilities still remain to be explored. The findings that
p62 is strongly associated with the nuclear matrix and that it
is a DNA-binding protein suggest that p62 could be involved
in the binding of chromatin to the nuclear matrix. Since
chromatin associates with the nuclear matrix at specific
sequence sites termed matrix-attached regions (MAR)
(Cockerill and Garrard, 1986), which are involved in the maintaining of the loop structure of the chromatin, the possibility
that p62 could bind to MAR sequences needs to be investigated.
Immunocytochemical studies by optical microscopy
indicated that p62 is distributed intranuclearly, following a
pattern similar to that showed by the heterochromatin (Bachs
et al., 1990; Vendrell et al., 1991). The immunogold-labeling
experiments reported here confirm that in non-dividing cells
p62 is mainly localized within the nuclei and that mostly of the
nuclear p62 is associated with the heterochromatin. Nevertheless, it is also present in the euchromatinic regions and to a
very small extent in the nucleolus. Part of p62 forms aggre-
Calmodulin and p62 in hepatocytes and astrocytes 3611
Fig. 10. Distribution of
anti-p62-binding sites in
sub-ventricular glial cells
and mitotic cells from the
germinal matrix.
(A) Anti-p62-binding
sites in sub-ventricular
glial cells. Most of the
gold particles are over
condensed chromatin.
(B) Anti-p62-binding
sites in mitotic cells of
germinal matrix. The
gold particles are mainly
over the chromosomes.
Note the absence of
labeling over
microtubules (arrow).
Bars, 0.5 µm.
gates, which are mainly localized in the heterochromatin.
These data support the hypothesis that p62 could be involved
in structural roles related to the maintenance of the condensed
state of the chromatin. The finding that the aggregates of p62
are mostly observed in the heterochromatin suggests that its
possible role in the condensation of the chromatin could be
mediated by the formation of these aggregates.
The intranuclear distribution of CaM as analysed by
immunogold labeling is similar to that reported by Wong et al.
(1991) using an in vitro binding assay with 125I-CaM in com-
3612 M. Portolés and others
Fig. 11. Co-localization of CaM and p62 in astrocytes. 7-day astrocytes in primary cultures (A), mitotic cells of germinal layer (B) and
hepatocytes (C,D) were subjected to immunogold labeling using anti-CaM (5 nm gold particles) and anti-p62 (10 nm gold particles). In glial
interphasic cells and in hepatocytes CaM and p62 co-localize in the cell nucleus (A,C). In mitotic cells, the gold particles corresponding to
CaM and p62 are located over the chromosomes but no p62/CaM complexes were found (B). In nuclear matrix sections, the p62/CaM
complexes were found in this nuclear component (D). Bar, 0.25 µm.
bination with ultrastructural autoradiography. The CaM pattern
is also similar to that shown by p62, with the exception of the
location in the nucleolus and the RNP. CaM is present in both
nuclear structures whereas the amount of p62 in both nucleolus
and RNP is very low. These results indicate that CaM could
be involved in nuclear functions in both nuclear structures that
are not mediated by p62. Like p62, CaM also forms aggregates
that are mostly visualized in the heterochromatinic regions.
Interestingly, the double-labeling experiments using anti-p62
and anti-CaM antibodies showed that most of the aggregates
of p62 also contain CaM, indicating a clear association of both
proteins mainly in the heterochromatinic regions. Nevertheless, not all the p62 forms aggregates with CaM and not all the
CaM is associated with p62. Since several other CaM-binding
Calmodulin and p62 in hepatocytes and astrocytes 3613
proteins have been detected in the nuclei of different cell types
(Sahyoun et al., 1984; Simmen et al., 1984; Bachs and Carafoli,
1987; Bachs et al., 1990; Vendrell et al., 1991), it is possible
that nuclear CaM could also form aggregates with other CaMbinding proteins. The co-localization of p62 and CaM suggests
that they are involved in the same roles in the heterochromatinic regions.
Surprisingly, the analysis of the distribution of CaM in
mitotic cells revealed that CaM is associated with the mitotic
chromosomes and distributed over all the chromosomes. The
detection of CaM in the mitotic chromosomes deserves to be
emphasized, since until now it has been generally accepted that
during mitosis CaM was associated with the microtubules of
the mitotic spindle but absent from the chromosomes (Fields
and Shaper, 1988; Starr et al., 1990). However, it should be
mentioned that most of the work on CaM distribution during
mitosis has been carried out using immunofluorescence techniques on permeabilized cells and this method has possibly
limited the observation of CaM in the structure of the chromosomes. Thus, the results reported here indicate that the
function of CaM during cell division is not limited to the organization and function of the mitotic spindle, and that unknown
roles for CaM at the chromosomal level and possibly related
to the condensation of the chromatin should be considered.
p62 is also distributed over all the chromosomes during
mitosis, like CaM. It should be emphasized that, whereas in
non-dividing cells p62 and CaM are co-localized to form
aggregates mainly in the condensed chromatin, during mitosis
the CaM/p62 aggregates disappear, although both proteins still
remain present over all the chromosomes. These results
suggest that during, or perhaps shortly before, mitosis,
CaM/p62 complexes disaggregate. These findings also suggest
that the disruption of CaM/p62 complexes could play a role in
the regulation of the condensation of the chromatin during
mitosis. The distribution of p62 and CaM in the chromosomes
does not correlate at all with the possible connection sites of
microtubules with the chromosomes at the kinetochore level
and also supports a role for both proteins in chromatin condensation.
Magdalena Faura and Francisco José Iborra are fellows of the
Spanish Ministerio de Sanidad y Consumo (F.I.S.S.), and Ministerio
de Educación y Ciencia (P.F.P.I.), respectively. This work was
partially supported by grants SAL89-898, SAL91-968, SAL91-0020C02 and SAF92-0283 from CICYT of the Spanish Ministerio de
Educación y Ciencia, and 90/0897-2 from FISS of the Spanish Ministerio de Sanidad. We thank Dra. Mari Carmen Risueño for her
excellent help in the EDTA procedure. We also express our gratitude
to Ms Inmaculada Monserrat, Ms María Teresa Huerta and Ms
Epifania Belenchon for technical assistance.
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(Received 10 May 1994 - Accepted 26 July 1994)