Cell Biology International 1998, Vol. 22, No. 7/8, 509-515
Article No. cb980291, available online at http://www.idealibrary.com on
THE ROLE OF Ca IONS IN RESTORATION OF THE STRUCTURE OF INTERPHASE
AND MITOTIC CHROMOSOMES IN PK LIVING CELLS AFTER HYPOTONIC
STRESS
A. A. AMCHENKOVA, I. M. BUZHURINA, L. A. GORGIDZE, I. V. KIREYEV, M. A. PANOV and
V. JU. POLYAKOV*
Department of Cell Physiology and Division of Electronic Microscopy, A.N. Belozersky Institute of
Physico-Chemical Biology, Moscow State University, Moscow, Russia
Received 13 August 1997; accepted 6 July 1998
The dynamics of mitotic chromosome and interphase chromatin recondensation in living PK
cells during their adaptation to hypotonic medium was studied. The recondensation process was
found to be slowed down by the modification of plasma membrane with low concentrations of
glutaraldehyde, while osmotic reactions of glutaraldehyde-treated cells remain unchanged. The
effect of glutaraldehyde can be rapidly reversed by the addition of Ca2+-ionophore A23187.
Intracellular Ca2+ measurements show that the adaptation to hypotonic shock is accompanied by
restoration of free Ca concentration, whereas the delay of chromatin condensation in
glutaraldehyde-treated cells is paralleled by the decrease of Ca level. The mechanisms implying the
role of low concentration of Ca2+ in chromatin compactization in vivo are discussed.
© 1998 Academic Press
KEYWORDS: chromosome condensation; intracellular calcium; hypotonic stress; glutaraldehyde.
INTRODUCTION
In a number of recent studies isolated mitotic and
interphase chromosomes are used for ultrastructural and biochemical analysis (Hancook et
al, 1977). The structural integrity of isolated
chromosomes or nuclei is usually maintained using
small concentrations of bivalent Ca2+ or Mg2+ ions
(Belmont et al., 1989). By changing the
concentrations of divalent cations in different
buffer systems, it is possible to obtain partially
decompacted chromosomes with different levels
of DNA packing (Zelenin et al., 1982). This
method was used to describe filamentous
elements—chromonemata (Zatsepina et al., 1983;
Belmont et al., 1989), globular structureschromomers (Zatsepina et al., 1983), loop domains
(Marsden and Laemmli, 1979) etc. in mitotic
chromosomes. The above-mentioned data should
be analyzed with due regard for concentrations of
*To whom correspondence should be addressed.
1065-6995/98/070509 + 07 $30.00/0
divalent cations being several orders higher than
physiological ones. According to some data, Ca2+
concentration in the living cell is about 10~ 1 0 M
(Whitaker
and
Patel,
1990),
whereas
chromosome stabilizing solutions usually contain
0.1 тм to 0.3 тм of CaCl2 (Laughlin et al, 1982).
In this connection a question arises about the
origin of structural complexes observed in
isolated
chromosomes
during
artificial
decondensation.
In the present work a model is suggested that
allows one to study structural organization of
mitotic and interphase chromosomes using
physiological Ca2+ concentrations.
Living cells of tissue culture with plasma membranes modified by glutaraldehyde are used as a
model. It has already been shown that hypotonic
treatment of a living cell causes complete
swelling of mitotic chromosomes and that, as
cells adapt to hypotonic medium, mitotic
chromosomes restore their structure (Zatsepina et
al., 1982; Smirnova et al., 1987). In this work the
behavior of mitotic chromosomes and their
ultrastructure in cells with
© 1998 Academic Press
510
Cell Biology International, Vol. 22, No. 718, 1998
modified membranes have been studied during
their adaptation to hypotonic medium and after
addition of a specific Ca2+ ionophore.
MATERIALS AND METHODS
Pig kidney epithelial cells were grown on
coverslips in 199 medium supplemented with
10% bovine serum and 4 µg/ml gentamycin. To
induce hypo-tonic swelling, cells were
incubated in Hanks' balanced salt solution,
diluted with distilled water to 70%.
Glutaraldehyde (Merck) was applied at a
concentration of 0.001% during entire
incubation in hypotonic medium. Ca2+-ionophore
A23187 was added to final concentration 1 им.
The reaction of living cells to hypotonic stress
was monitored under microscope ‘Opton-III’
using a thermostated chamber.
For electron microscopy the cells were fixed
in 2.5% glutaraldehyde in appropriate solution
and processed according standard technique.
Ultrathin sections were photographed in electron
microscope Hitachi HU-12.
Intracellular Ca2+ concentrations were measured
with spectrofluorometric analysis of the fura-2
after incubation of cells with fura-2-AM in the
presence of serum.
RESULTS
Light microscopy
Figure 1 shows photographs of mitotic cells in
controls [Fig. l(a)] and at different time after
hypotonic treatment [Fig. l(b),(c)]. Two minutes
after treatment, chromosomes are decondensed
and can not be seen under a phase-contrast
microscope [Fig. l(b)]. As cells adapt to
hypotonic medium chromosomes restore their
compact packing and in 15 min their density is
the same as in the control [Fig. l(c)]. When
placed into normal medium the adapting cells are
strongly compressed, i.e. hypo tonic medium
becomes 'normotonic' for them whereas normal
medium—hypertonic.
The initial response of cells with
glutaraldehyde-modified
membranes
is
analogous to that of the control ones. Mitotic
chromosomes are swollen and can not be seen
under a light microscope, the dimensions of cells
themselves increase. After a 15-min incubation in
hypotonic medium cells can be strongly
compressed in normotonic medium which,
thereby becomes 'hypertonic' for them.
Consequently the modification of plasma
Fig. 1. PK cells at different times after hypotonic
treatment: (a) control; (b) 2 min after treatment; (c) 15
min after treatment. Bar= 10 µm.
Cell Biology International, Vol. 22, No. 718, 1998
Fig. 2. PK cells after 15-min treatment by hypotonic
medium with glutaraldehyde (a) and after 15 min treatment
by hypo-tonic medium with glutaric aldehyde followed by
addition of Ca2+ ionophore A23187 (b). Bar=10 µm.
membranes by glutaraldehyde does not prevent
cellular membranes from adapting to changed
osmotic conditions [Fig. 2(a)]. Besides, cells probably remain completely viable, as indicated by
the fact that they cannot be stained with Trypan
blue. However, under these conditions mitotic
chromosomes remain decompacted and do not
restore their initial structure even after prolonged
incubation when the plasma membrane of such
cells has entirely adapted to the changed osmotic
conditions. Hence in these cells chromosomes
lose the ability to restore their structure under
hypotonic conditions. Structural restitution of
mitotic chromosomes can be carried out by
adding the selective Ca2+ ionophore A23187 to
cells treated with glutaraldehyde. In 15 min.
chromosomes
completely
restore
their
compactness [Fig. 2(b)].
511
Electron microscopy
The procedures described above were used for
electron microscopy of nuclei and chromosomes
restitution by the aid of Ca2+ ionophore.
Figure 3(a) demonstrates mitotic chromosomes
in cells fixed in situ. Chromosomes are observed
to have typical compact structure. Filamentous
elements, 100 nm in size—chromonemata—can
be observed on their periphery. Upon hypotonic
stress, mitotic chromosomes undergo considerable
decondensation [Fig. 3(b)]; after a 15 min
incubation of cells in 70% Hanks' solution
mitotic chromosomes recover their initial
structure [Fig. 3(c)]. The same changes can be
observed also in interphase nuclei [Fig. 4(a),(b)].
In cells with modified membranes, mitotic
chromosomes and interphase nuclei treated with
hypotonic solutions decondense as in normal
cells. However, in cells, treated with
glutaraldehyde, chromosomes do not recover
their structure [Fig. 5(a),(b)]. In spite of some
condensation observed in their 'axial' area they
are less compact than those in the control [Fig.
3(a)] and in untreated cells adapting to hypotonic medium [Fig. 3(c)]. Addition of ionophore
A23187 leads to complete restoration of the structure of nuclei and mitotic chromosomes. In interphase nuclei the chromomeric chromatin
structure,
nucleolus
and
clusters
of
interchromatin granules are restored [Fig. 5(d)].
Mitotic chromosomes also become compact and
their density becomes like in normal cells [Fig.
5(c)]. Filamentous chromonemata of about 100 nm
thick can be observed on the periphery of these
'restored' chromosomes as in the control [Fig.
5(c),(d)].
Fluorescent analysis
Fluorescent registration of the intracellular interaction between the Fura-2 and cytosolic Ca2+ was
undertaken without calibration of the intracellular
free Ca2+ concentration. Intracellular free Ca2+
concentration strictly declined after lowering of
the extracellular tonicity, but returned to the
initial level 5-10 min after incubation under
condition of reduced tonicity (Fig. 6). If we
added ionophore A23187 during this period, then
intracellular Ca2+ concentration increased sharply,
accompanied by chromosome condensation. If we
carried out hypo-tonic shock in the presence of
the glutaraldehyde, then intracellular free Ca2+
concentration declined to the same level as that
without glutaraldehyde, but this reduced level
lasted at least for 20 min (Fig. 6). In this condition
chromosome reconstruction was absent, but
ionophore A23187 increased
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Cell Biology International, Vol. 22, No. 718, 1998
Fig. 3. Ultrathin sections of mitotic chromosomes in PK cells after hypotonic treatment: (a) control: (b) 2 min after treatment; (c) 15
min after treatment. Bar=l µm.
Cell Biology International, Vol. 22, No. 718, 1998
Fig. 4. Ultrathin sections of interphase cells: (a) control, (b)
after 15min incubation in hypotonic medium. Bar=l µm.
the intercellular Ca2+ concentration and induced
the chromosomes condensation [Fig. 5(c),(d)].
Cell swelling and recovery to the initial volume
does not depend of the presence of
glutaraldehyde in the incubation medium.
DISCUSSION
In a number of studies, Ca2+ ions have been shown
to perform an important role in the regulation of
mitosis of many plants and animals (Hepler,
1989). Obviously, in a living cell many Ca induced effects are mediated by different enzymatic
systems, including proteinkinases (Planas-Silva
and Means, 1992). Ca + is likely to affect directly
the structure of chromatin and mitotic
chromosomes acting as a complex agent.
Therefore, the level of chromosome
513
compaction is directly dependent on Ca2+ concentration in the incubation medium (Zatsepina et
al., 1983). However, Ca2+ concentrations (~ 10 ~ 4
м) used to stabilize isolated structures are several
orders higher than that of free Ca2+ in the
cytoplasm (Whitaker and Patel, 1990).
Moreover, injection of approximately the same
concentration of Ca2+ into a living cell results in its
rapid death. The condensing effect of Ca2+ upon
the chromosome structure under conditions
similar to physiological remains to be studied.
The suggested model allows one to analyze the
effect of physiological Ca2+ concentrations on the
structural organization of interphase nuclei and
mitotic chromosomes. It may be supposed that
cells, treated with low concentrations of
glutaraldehyde lose their ability to restore
intercellular Ca2+ concentration, lowered by
hypotonic swelling of cells. It is well established
that Ca2+ injection into cells can be regulated by
transmethylation of phospha-idylethanolamine
molecules in the cytoplasmic layer of the plasma
membrane and their transloca-tion into the outer
layer of the cytoplasmic membrane (Godeau et
al., 1985). It is likely that modification of
phosphatidylethanolamine with glutaraldehyde
which occurs in the cytoplasmic membrane
prevents this process and accompanied activation
of the rapid Ca2+-channels. This supposition is
confirmed, in particular, by the fact that after
hypotonic treatment in a living cell with the
membrane modified by glutaraldehyde, the structure of swollen mitotic chromosomes and interphase nuclei may be completely restored using
selective Ca2+ ionophore A23187. According to
literature data Ca2+ concentration in the cytoplasm must not be over 180 mм (Whitaker and
Patel, 1990). It is noteworthy that in isolated
nuclei
and
chromosomes
with
Ca2+
concentrations of about 0.1-0.05 mм which well
exceed those in the cytoplasm, chromatin
remains in the state of complete diffusion. In this
connection a question arises why the condensing
effect of Ca2+ is observed at concentrations similar
to physiological. This phenomenon may have at
least two explanations.
(1) In a living cell mitotic and interphase
chromosomes contain specific Ca2+-binding proteins that are labile and easily extracted during
isolation of the material.
(2) Ca2+ effect is mediated by one of the
known enzymatic systems, for instance, by
protein kinase systems phosphorylating histones.
Experimental results indicating that the level of
chromosome compaction depends on the activity
of protein kinase phosphorylating histones HI
and H3 can testify for
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Cell Biology International, Vol. 22, No. 7/8, 1998
Fig. 5. Ultrathin sections of mitotic chromosomes (a),(c) and interphase nuclei (b),(d) in PK cells after 15 min treatment by
hypotonic medium: (a),(b) with glutaraldehyde; (c),(d) with glutaraldehyde followed by addition of Ca2+ ionophore A23187. Bar=l
µm.
Fig. 6. Intracellular Ca2+ concentration changes after hypotonic shock in the absence (a) and presence (b) of 0.001%
glutaraldehyde. ↓, hypotonic shock; ↑ , Ca2+-ionophore addition.
the above supposition. No matter what the mechanism of Ca2+ effect is, it is essential that macromolecular complexes formed under ionophore
effect in restored chromosomes and nuclei entirely
corresponds to analogous structures in cells fixed
in situ. In interphase nuclei globular
Cell Biology International, Vol. 22, No. 7/8, 1998
structures, chromomers, and in mitotic chromosomes, chromonema filaments are restored (Kireyev
et al., 1988). Similar structural complexes were
revealed in mitotic chromosomes and nuclei of
permeabilized cells at a gradual lowering of Ca2+
concentration in the incubation medium (Zatsepina
et al., 1983). Thus complete structural conformity
of macromolecular chromatin complexes in cells,
fixed in situ, in permeabilized cells and in
chromosomes, restored by calcium iono-phore, is
evidence for their structural origin. At the same time
these data contradict the observations that
'intermediate' levels of DNA compaction in mitotic
chromosomes are fibrillar elements of about 50-60
nm (Marsden and Laemmli, 1979; Adolph et al.,
1986). Obviously, in isolated chromosomes, treated
with high concentrations of divalent cations, the
level of compression of chro-monemas is higher
than that in chromosomes fixed in situ or
'reconstructed'
by
ionophore,
i.e.
under
physiological conditions.
ACKNOWLEDGEMENTS
This research is supported by the Russian State
Program 'Universities of Russia'. The authors would
like to thank Dr P. Avdonin for his assistance in
measuring intracellular free Ca2+.
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