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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 1 0 ~ 10 M (Whitaker and Patel, 1990), whereas chromosome stabilizing solutions usually contain 0.1 to 0.3 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
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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 . 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.


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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.

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

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)].


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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.


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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

compaction is directly dependent on Ca2+ concentration in the incubation medium (Zatsepina et al., 1983). However, Ca2+ concentrations (~ 10 ~ 4 ) 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 m (Whitaker and Patel, 1990). It is noteworthy that in isolated nuclei and chromosomes with Ca2+ concentrations of about 0.1-0.05 m 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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Fig. 5. Ultrathin sections of mitotic chromosomes (a),(c) and interphase nuclei (b),(d) in PK cells after 15 m i n 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


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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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