Cell Biology International Reports, Vol. 6, No. 8, August 1982

DETECTION OF SEQUESTERED CALCIUM DURING MITOSIS IN MAMMALIAN CELL CULTURES AND IN MITOTIC APPARATUS ISOLATED FROM SEA URCHIN ZYGOTES

Gerald Schatten* Heide Schatten Calvin Sinerly

Department of Biological Science Florida State University

Tallahassee, Florida 32306 USA

ABSTRACT

Chlorotetracycline, which fluoresces in the presence of divalent cations in membrane environments, has been employed to localize sequestered calcium in dividing cells and isolated mitotic apparatus. HeLa and CHO cells during division have cytoplasmic granules that fluoresce in the presence of chlorotetracycline with spectral characteristics similar to those of calcium cheiates. Mitotic apparatus isolated from dividing sea urchin zygotes fluoresce with punctate sources in the presence of chlorotetra- cycline. The intensity of fluorescence is markedly reduced by membrane disruption with detergents and is unaffected by exogenous calcium chelators. It is concluded that membranes found in asso- ciation with the mitotic apparatus, revealed by electron microscopy, can sequester cvtoplasmic calcium ions. It is proposed that this sequestration, which would result in altered cytoplasmic calcium concentrations, may participate in the regulation of micro- tubule stability during division.

INTRODUCTIOH

The role of cytoplasmic calcium ions in regulating motility during cell division (reviewed by Mazia, 1961, Inoue, 1981, and Forer and Zimmernan, 1982) appears quite attractive on the basis of recent experinents demonstrating the sensitivity of spindle birefringence to calcium in vivo (Kiehart, 1981) and in vitro -- (Salmon and Segall, 138C). Models predicting the spexficle of calcium ions during mitosis have also enjoyed substantiation from recent experimental evidence including the demonstration that calmodulin increases the lability of microtubules to calcium ions (Marcum et al., 1978) and calnodulin antibody specifically stains spindle microtubules (Welsh et al., 1979). The importance of membranes surrounding the mitotic apparatus in this alleged calcium

*,to whom correspondence should be addressed

0309-l 651182/080717-08/$03.00/O 0 1982 Academic Press Inc. (London) Ltd.

718 Cell Biology International Reports, Vol. 6, No. 8, August 1982

Fipure 1: Chlorotetracycline fluorescence microscopy of dividing HeLa (A) and CHO (B) cells. Note both punctate diffuse fluorescence. 1,250 x.

regulation of cytoplasmic calcium has been reviewed by Harris (1078) and Paweletz (1981) and is supported by the careful electron microscopy of Harris (1975), Hepler (1988), and Paweletz (1978). The demonstration that isolated mitotic apparatus can sequester exogenous 45Ca++ (Silver et al., 1980) has provided support for the existence of a calcium-activated ATPase, present in isolated mito- tic apparatus (Maria et al., 1972; Petzelt, 1972; reviewed by Petzelt, 1980). Followin? the discovery that chlorotetracycline will form fluorescent chelates in membrane environments and can therefore be utilized as a vital probe to study sequestered calcium ions (reviewed by Caswell, 1975), Ilolniak et al. (138C) have demon- strated chlorotetracycline fluorescence surrounding the chromosomes in the plant iiaenanthTas during division. In this study mammalian ccl1 cultures during cell division and mitotic apparatus isolated from sea urchin zygotes are Shown to fluoresce with spectral pro- nerties sinilar to those of calcium chelates. This fluorescence is sensitive to membrane-disrupting detergents but is insensitive to Jivalent ion chelators, which would not be expected to cross intact t:embranes. It is concluded that nembrane found in association with the mitotic apparatus of animal cells can sequester endogenous Ca++ and may therefore participate in the cytoplasmic regulation of the events during mitosis.

;&'ERIhLS :,ilD KSTHODS

b,itotic IieLa and CHO cells were cultured by standard means, and sea urchin (Stron;;ylocentrotus purpuratus, Lytechinus varic,cztus) zygotes obtained by routine procedures.

Cell Biology International Reports, Vol. 6, No. 8, August 1982

Figure 2: Chlorotetracycline fluorescence of sea urchin mitotic apparatus isolated with hexylene glycol (A) and glycerol-DMSO (B). In the isolated mitotic apparatus both punctate and diffuse staining patterns are apparent. 900 x.

Mitotic Apparatus Isolation

Isolation of mitotic apparatus of the sea urchin Lytechinus variegatus was performed in the absence of any detergents using a modified homogenization medium of Forer and Zimmerman (1974) com- posed of 50% (v/v> glycerol, 10% (v/v> dimethylsulfoxide (DMSO), 5 mM tigC12, 10 mM MES, 100 mM EGTA, pH 6.8, or the hexylene glycol (12%) procedure of Kane (1962). Following insemination the fer- tilization coats were removed by brief treatment with 10 mM DTT, 1 mg/nl pronase , pH 8.1, washed three tines and permitted to develop with constant stirring. At metaphase the zygotes were suspended in the homogenization buffer and lysed by vortex mixing. Unbroken zygotes were removed by centrifugation at 200 x g for 5 min. The isolated mitotic apparatus were collected by centrifugation at 1000 x g for 15 min. and washed three times.

Fluorescence Microscopy and Fluorometry

The living cell cultures and the isolated mitotic apparatus were exposed to 100 IJM chlorotetracycline for 15 minutes, washed free of exogenous chlorotetracycline, and examined using fluorescent epi-illumination and appropriate filters (Zeiss). Fluorometric analyses were performed using a Hitachi-Perkins Scanning Fluorescence Spectrophotometer (MPF-2a).

Scanning Electron Microscopy

Scanning electron microscopy was performed on isolated mitotic apparatus. Following fixation in 2.5% glutaraldehyde, the isolated mitotic apparatus were affixed to polylysine-coated cover slips, ethanol dehydrated and dried at the critical point in C02.

720 Cell Biology International Reports, Vol. 6, No. 8, August 1982

a I I 1

b loo- I 7 IEOmM MOO,. IOpM CTC

80 - in mm &OH

r _ Emhrbn al 49Ot-m

f 60-

9 5 40-

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C

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Figure 3: Fluorometric analysis of chlorotetracycline fluorescence.

a. Chlorotetracycline in 70% methanol. In the absence of divalent cations, chlorotetracycline in a non-aquaeous environment does not exhibit fluorescence.

b. Chlorotetracycline-magnesium chelates. In the presence of magnesium CTC fluoresces with maxima at 397 nm and 285 nm.

C. Chlorotetracycline-calcium chelates fluoresce with maxima at 397 nm and 305 nm.

d. Chlorotetracycline treated mitotic apparatus isolated with hexylene glycol fluoresce with a secondary maximum near 300 nm, similar to calcium-CTC chelates.

e. Chlorotetracycline treated dividing HeLa cell suspensions fluoresce with a secondary maximum near 300 nm, similar to calcium-CTC chelates.

Cell Biology International Reports, Vol. 6, No. 8, August 1982 721

The platinum-carbon-coated specimens were examined in the field emission scanning electron microscope (Coates and Welter).

RESULTS AND DISCUSSION

Chlorotetracycline Fluorescence of Cell Cultures

The pattern of chlorotetracycline fluorescence is demonstrated in figure la on HeLa cells and in figure lb on CHO cells. Numerous punctate fluorescent sources, at times associated with the peri- nuclear regions, are detected. Fluorometric analysis of the HeLa cells (figure 3e) demonstrates a 1600% increase over background with virtually all the fluorescence derived from the cells since the cell-free supernatant does not fluoresce.

Fluorescence studies on isolated mitotic apparatus (figure 2) demonstrates the fluorescent image of mitotic apparatus isolated in hexylene glycol or DMSO-glycerol. Again numerous punctate sources are detected by chlorotetracycline fluorescence. The fluorescence intensity of the isolated mitotic apparatus is over 2% tines greater than background and is reduced by over 70% following the addition of 1% triton X-100. The addition of 10 mM EGTA reduces the fluorescence intensity by only 3.4%. These results indicate that the fluorescence is not due solely to non-aqueous charac- teristics of the isolation medium. It is sensitive to membrane integrity as evidenced by the effects of triton and is not sen- sitive to aqueous calcium chelators, indicating that the sequestered ions are sequestered in an internal store.

Fluorometric Analysis

Chlorotetracycline will fluoresce in the presence of either magnesium or calcium in membrane environments, as depicted in figures 3a, b, c. In figure 3a chlorotetracycline in the absence of divalent cations exhibits little fluorescence. In figure 3b in the presence of magnesium the fluorescence intensity increases greatly, with a maximum at 397 nm and a secondary maximum at 285 nm. In contrast chlorotetracycline-calcium chelates fluoresce at 397 nm with a secondary maxinum at 305 nm (figure 3~). Importantly, in figure 3d of mitotic apparatus isolated from sea urchins and in figure 3e of HeLa cell cultures a secondary maximum at approximately 305 nm is found. The analysis of secondary maxima permits the separation of magnesium from calcium fluorescence. In both the living mammalian cell cultures (figure 3e) and the iso- lated mitotic apparatus (figure 3d) the fluorescence observed is due primarily to calcium and not magnesium (figure 3b).

722 Cell Biology International Reports, Vol. 6, No. 8, August 1982

Figure 4: Scanning electron microscopy of isolated mitotic apparatus.

a. survey view; 1,500 X. b. higher magnification demonstrating astral nicrotubules and

vesicular components; 15,000 X.

Scanning Electron tiicroscopy

Scanning electron microscopy (figure 4a, b) of isolated mito- tic apparatus demonstrates the surface features of the isolated organelle in which numerous membranes and vesicular components can be detected with microtubules projecting from the asters. The membranes are detergent sensitive and appear associated with the entire mitotic apparatus as small vesicular components.

CONCLUSION

In this study scanning electron microscopy demonstrates numerous membranes associated with the mitotic apparatus. Chlorotetracycline fluorescence in dividing cells and isolated mitotic apparatus demonstrates the presence of punctate vesicular sources that are detergent sensitive and fluoresce with spectral properties similar to those of calcium chelates. It is concluded that the mitotic apparatus in aninal cells can sequester endogenous cytoplasmic calcium ions. It is proposed that fluctuations in cytoplasmic calcium concentration, produced by the selective

Cell Biology International Reports, Vol. 6, No. 8, August 1982 723

sequestration and release of this divalent cation from these membrane stores , play an important role in regulating microtubule stability and in effecting chromosome movements during cell division.

ACKNCWLEDGMENTS

We wish to thank Professor Penny Gilmer for the generous use of her fluorometer. The support of this research by the William J. Thayer Research Grant from the American Cancer Society and from the National Institutes of Health (research grant HD 12913, research career development award HD 00363) is gratefully acknowledged. Portions of this research were presented at the seventeenth con- ference of the American Society for Cell Biology and the Second International Conference on Cell Biology (Schatten and Schatten, 1977; Schatten and Schatten, 1980).

REFERENCES

Caswell, A. H. (1975) Methods of measuring intracellular calcium. International Review of Cytology, 56, 145-181.

Forer, A., and Zimmerman, A. (1974) Characteristics of sea-urchin mitotic apparatus isolated using a dimethyl sulfoxide/glycerol medium. The Journal of Cell Science, 16, 481-497.

Forer, A., and Zimmerman, A. (1982) Cellular Biodynamics: Mitosis and Cytokinesis. Academic Press, New York.

Harris, P. (1975) The role of membranes in the organization of the mitotic apparatus. Experimental Cell Research, 32, 323-333.

Harris, P. (1978) Triggers, triger waves, and mitosis: A new model. In: J. R. Jeter, I. L. Cameron, G. M. Padilla and A. PI. Zimmerman (eds.). Monographs on Cell Biology. Cell Cycle Regulation. pp. 75-104. Academic Press, New York.

Hepler, P. K. (1980) Membranes in the mitotic apparatus of barley cells. The Journal of Cell Biology, 86, 490-499.

, Inoue, S. (1981) Cell division and the mitotic spindle. The

Journal of Cell Biology, 91, 1315-1475.

Kane, R. E. (1962) The mitotic apparatus: Isolation by controlled PH. The Journal of Cell Biology, 12, 47-55.

Kiehart, D. E. (1981) Studies on the in vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium-sequestering system. The Journal of Cell Biology, 88, 604-617.

724 Cell Biology International Reports, Vol. 6, No. 8, August 1982

Mazia, D. (1961) Mitosis and the physiology of cell division. In: J. Brachet and A.E. Mirsky (eds.). The Cell III. pp. 77-412. Academic Press, New York, London.

Mazia, D., Petzelt, C., Williams, R.O. and Meza, I. (1972) A Ca++-activated ATPase in the mitotic apparatus of the sea urchin egg (isolated by a new method). Experimental Cell Research, 70, 325-332.

Marcum, J.M., Dedman, J., Brinkley, B.R. and Means, A. (1978) Control of microtubule assembly-disassembly by calcium- dependent regulator protein. Proceedings of the National Academy of Sciences of the USA, 75, 3771-3775.

Paweletz, N. (1978) Membranes in the mitotic apparatus of mammalian cells. In: E.R. Dirksen, D.M. Prescott and C.F. Fox (eds). Cell Reproduction: In Honor of Daniel Mazia. pp. 515-524. Academic Press, New York.

Paweletz, N. (1981) Membranes in the mitotic apparatus. Cell Biology International Reports, 5, 323-326.

Petzelt, C. (1972) Ca 2+ -activated ATPase during the cell cycle of the sea urchin Strongylocentrotus purpuratus. Experimental Cell Research, 70, 333-339.

Petzelt, C. (1980) Biochemistry of the mitotic spindle. Inter- national Review of Cytology, 60, 53-92.

Salmon, E.D. and Segall, R.R. (1980) Calcium-labile mitotic spindles isolated from sea urchin eggs (Lytechinus variegatus). The Journal of Cell Biology, 86, 355-365.

Schatten, G. and Schatten, 8. (1980) Calcium sequestration by the mitotic apparatus in mammalian cell cultures and sea urchin zygotes studied with the fluorescent probe chlorotetracycline (CTC). European Journal of Cell Biology, 22, 314.

Schatten, H. and Schatten, G. (1977) The mitotic apparatus: High resolution scanning electron microscopy of the surface. The Journal of Cell Biology, 75, 284a.

Silver, R.B., Cole, R.D. and Cande, W.Z. (1980) Isolation of mitotic apparatus containing vesicles with calcium sequest- ering ability. Cell, 19, 505-516.

Welsh, J.J., Dedman, J.R., Brinkley, B.R. and Means, A.R. (1979) Tubulin and calmodulin. Effects of microtubule and micro- filament inhibitors on localization in the mitotic apparatus. The Journal of Cell Biology, 81, 624-634.

Wolniak, S.M., Helper, P.K. and Jackson, W.T. (1980) Changes in the associations of endomembranes and spindle microtubules during mitosis. The Journal of Cell Biology, 87, 236a.

Received: 14th April 1982 Accepted: 4th May 1982