Nuclear actin bundles in Amoeba, dictyostelium and human HeLa cells induced by dimethyl sulfoxide

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Printed in Sweden polyspermic because the cortical reaction is Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved disrupted [ 111. 0014-4827/79/070451-05$02.00/O In conclusion, the late phase in the activation potential is closely related to the cortical reaction in the fertilized egg cell. Nuclear actin bundles in Amoeba, It is evident that the event (or events) oc- Dictyostelium and human HeLa cells curring at this time prevent further sperm- induced by dimethyl sulfoxide egg interactions from occurring (compare FUKUI’ and HIRONOBU KATSUfig. 1 with the traces in fig. 2). However, YOSHIO MARU, Department of Biology, Faculty of Science, one cannot determine which phenomenon Osaka University, Toyonaka, Osaka 560, Japan is the cause of the other. Inversion of mem- Summary. In a previous study we demonstrated that brane potential (e.g., holding the membrane dimethyl sulfoxide (DMSO) induces the formation of bundles in the interphase nucleus of a potential at + 10 mV for 1 min by current microfilament cellular slime mold, Dictyostelium mucoroides [12], in injection) was not by itself able to elicit which the microfilaments bound rabbit skeletal muscle heavy meromyosin, forming an ‘arrowhead’ strucelevation of the fertilization membrane. ture, and that this binding could be reversed by Mg*+

I thank A. Monroy and B. E. Hagstrom for helpful suggestions. Some of the experiments were carried out in collaboration with B. Dale. This work has been supported in part by the CNR, Project on Biology of Reproduction.

References 1. Tyler, A, Monroy, A, Kao, C Y & Grundfest, H, Biol bull 111(1958) 153. 2. Steinhardt, R A, Lundin, L & Mazia, D, Proc natl acad sci US 88 (1971) 2426. 3. Steinhardt, R A, Shen, S & Mazia, D, Exp cell res 72 (1972) 195. 4. Ito, S & Yoshioka, K, Exp cell res 72 (1972) 547. 5. - Ibid 78 (1973) 191. 6. Jaffe, A L, Nature 261 (1976) 68. I. Dale, B, DeFelice, L J & Taglietti, V, Nature 275 (1978) 217. 8. Jaffe,‘L A & Robinson, K, Dev bio162 (1978) 215. 9. Harvey, E B, The American Arbaciu and other sea urchins. Princeton Univ Press, Princeton, N.J. (1956). 10. Lonning, S, Arbok, Univ Bergen 8 (1967) 1. 11. Hagstrom, B E & Allen, R D, Exp cell res 10 (1956) 14. Received June 28, 1978 Revised version received December 13, 1978 Accepted February 2, 1979

and ATP. In the present study, we show electron microscopic data demonstrating the occurrence of such microfilament bundles in the nucleus of Amoeba proteus and human HeLa cells, as well as in D. mucoroides. The similarities in the morphology and dimension of the microfilanets, as well as the snecific conditions by which they are induced, suggested that these microfilaments are actin. We present evidence that actin is involved in interphase nucleus of a variety of organisms, and that DMSO acts on the molecules to induce microtilament bundles specifically in the nucleus.

Actin is not only involved in skeletal and smooth muscle contraction, but may also have a contractile or skeletal function in non-muscle cell cytoplasm Cl]. In the 1970s especially in the latter half, various lines of study have been accumulated, demonstrating the occurrence of this protein in cell nuclei. Electron microscopic and immunofluorescence studies have suggested that actin is involved in the mitotic or meiotic apparatus of insect testis [2,3], higher plant cells [4] and mammalian cells [5, 61. There is an argument that this protein may be generating a mechanochemical force for chromosome movement as well as cytokinesis [7]. Biochemical evidence has also been ’ Current address: Department of Biology, Princeton University, Princeton, NJ 08544, USA. Exp Cell Res 120 (1979)

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presented showing that a considerable amount of non-histone protein of chromatin is actin in Amoeba [S], Physarum [9], Dictyostelium [lo] and rat liver nuclei [ll]. It was proposed that such nuclear actin might function in the regulation of gene replication and/or transcription through its contractile nature [9]. For the present, it is of importance to localize and characterize the nuclear actin in order to decide the true function(s) of this protein in cell nucleus. We have found that an aprotic solvent, dimethyl sulfoxide (DMSO), induces the formation of huge microfilament bundles in the interphase nucleus of a cellular slime mold, Dictyostelium. Such bundles were approx. 3 pm in length, 0.85 pm in width, and each microfilament was 6 nm in diameter. The chemical nature of the microfilaments was partially elucidated by our previous report [ 121,in which we presented data for the in situ identification of actin microfilaments originally reported by Ishikawa et al. [13]. We showed that the microfilaments could bind to rabbit skeletal muscle heavy meromyosin, forming an ‘arrowhead’ structure typical of F-actin. We further showed that this binding could be reversed by Mg2+ and ATP, presenting conclusive evidence that these microfilaments were actin. The present study was performed in order to ask whether or not the nuclear actin bundles could also be induced in other organisms. We found that the occurrence of such nuclear actin bundles was not limited to such lower organisms as Amoeba proteus or Dictyostelium mucoroides, but is also present in human HeLa cells. The present study presents a unique tool with which to study the mechanism of bundle formation of microfilaments in vivo, and it might also present a warning on the usage of DMSO for medical application. E-r/, Cell Res 120 (J979)

Materials and Methods Cells and cultures. D. mucoroides, Dm-7, was grown on Escherichia co/i on nutrient medium containing 1%

glucose and 1% .neotone. The cells were washed and . Incubated for 10 h at 22°C in Banner’s salt solution containing 10 mM NaCl, 10 mM KCI, and 3 mM CaCl, [14]. Then they were subjected to 10% DMSO in the salt solution for 30 min at 22°C. A. Droteus cells were grown by feeding Tetrahymena py$ormis in culture medium (6 mg KCl, 4 mg CaHPO,, 2 mg MgSO, in 1000 ml distilled water) [ 151. The cells were treated with 10% DMSO in the culture medium for 30 min at 22°C. Human HeLa S cells were cultured in a modified Earle’s BSS supplemented with 10% calf serum [16], and subsequently treated with 10% DMSO in the BSS for 30 min at 37°C. Electron microscorn.. . D. mucoroides and A. Droteus cells were initially fixed with a mixture of 1% paraformaldehyde and 1.25% glutaraldehyde in 25 mM Na-cacodylate buffer (pH 7.2) for 1 h at 0°C. The cells were post-fixed with 1% 0~0, in the buffer for 1 h at 4°C. HeLa cells were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 100 mM Nacacodylate buffer (pH 7.2) for-3 h at O”C, and then post-fixed with 1% 0~0, in the buffer for 1 h at 4°C. The cells were dehydrated through an ethanol series and embedded in Spurr’s resin. The thin sections were stained with 25% uranyl acetate in methanol and Reynolds’ lead citrate, and the electron micrographs were taken under a JEM 100-C electron microscope at 80 kV. Chemicals. DMSO (Nakarai, Merck), paraformaldehyde (Taab), glutaraldehyde (Lade),’ ds0, (Merck), cacodylate (Nakarai), and Spurr’s medium (Polyscience) were used. I

Results and Discussion As we have reported previously, the formation of nuclear actin bundles induced by DMSO was first revealed in D. mucoroides [12]. This induction was highly reproducible, irrespective of the developmental stages of the cells. The bundles were observed to form in between the initial 20-30 min of the treatment with 10% (v/v) DMSO in Banner’s [14] salt solution (fig. 1). More than 20% of DMSO was toxic to the cells, and furthermore less effective in inducing nuclear bundles. The treated cell nuclei reverted to the original morphology shortly after they were washed with salt solution, indicating that this induction of the bundles was reversible. It is noteworthy that the induction of the bundles occurred specifically in the nucleus, although this induction

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Fig. I. Electron micrographs showing the nuclear microfilament bundles of Dictvostelium mucoroides cells. Our previous study has demonstrated that these microfilaments were composed of actin. The cells were subjected to treatment with 10% DMSO in Bonner’s salt solution [13] for 30 min at 22°C before tixation. (a) Low-power micrograph showing a whole cell and a huge microtilament bundle in the nucleus (right side is marked); (&) high-power micrograph showing a wavy configuration of the microfilaments typical of F-actin. (u) x 8 800; (b) x24 600. Fig. 2. Nuclear microfilament bundles ofAmoeba proteus. The cells were treated with 10% DMSO in the culture medium of Prescott & James [ 141for 30 min at 22°C before fixation. (a) Low-power electron micrograph showing two microtilament bundles in the nu-

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cleus (marked). Note the evident honeycomb structure, typical of Amoeba nuclear envelope; (b) highDower microaranh showing one of the bundles in (a). The microfil~ments comp&ing the bundle are similar to those of Dictyostelium (a) x 8 800; (b) x 75 000. Fig. 3. Nuclear microfilament bundles of a HeLa S, cell. The activelv growing cells were treated with 10% DMSO in a mohi?ted Earle’s BSS [IS] for 30 min at 37°C. (a) Low-power electron micrograph showing two microfilament bundles in the nucleus (marked). The cytoplasmic microtilaments, intermediate filaments, and microtubules were ureserved intact; (b) Highpower micrograph showing one of the bundles in (a). The bundle was 0.9 wrn in length, 0.3 pm in width, and each microtilamknt was 6-nm in -diameter. (a) x 10 160; (b) x59200.

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no longer occurred once the cells were glycerinated or if the nuclei were isolated before treatment with DMSO [20]. We found that DMSO affects A. proteus cells by inducing the formation of the microtilament bundles in the nuclei. When actively growing amoeba were treated with 10% DMSO for 30-60 min, several microfilament bundles, ranging from 1 to 3 pm in length and from 0.25 to 1 pm in width, were induced in a nucleus (fig. 2). The bundles were most frequent in cells treated with the DMSO solution for 30 min at 22°C. The morphology and the dimension of the bundles as well as each microfilament involved closely resembled the bundles in Dictyostefium, suggesting that the nuclear microfilaments ofAmoeba are composed of actin. The nuclear microfilament bundles could also be induced in human HeLa S1 cells subjected to DMSO treatment. Exponentially growing cells were treated for 30-60 min at 37°C with lO-50% DMSO. DMSO was diluted with the modified Earle’s BSS. Huge microfilament bundles were induced in nuclei of more than half of the cells treated for 30 min with 10% DMSO (fig. 3). The scale of the nuclear bundles induced in HeLa cells was similar to those ofDicfyostelium or Amoeba, and each microfrlament was 6 nm in diameter. This induction of microfilament bundles occurred specifically in the nucleus, whereas the other filamentous structures in the cytoplasm (i.e., cortex microfilaments, 10 nm filaments in the vicinity of nucleus, and microtubules) were preserved intact after the treatment with DMSO. The present study demonstrated that DMSO works on living cells to induce the formation of huge microfilament bundles in the nucleus of human as well as protozoan cells. The optimum conditions for the inExp Cell Res I20 (1979)

duction of the bundles in D. mucoroides, A. proteus and HeLa cells were very similar except for the temperature: 30 min treatment with 10% DMSO is optimum at 22°C for D. mucoroides and A. proteus, and 37°C for HeLa cells. The idea that the microfilament bundles induced in HeLa and Amoeba nuclei are actin could be supported by the following facts: (a) the morphological and dimensional similarities between the microfilaments and those found in the nuclei of Dictyostelium; (b) the specificity of the conditions which induce the bundles. This induction of nuclear actin bundles will present a novel in vivo system for studying the mechanism of the bundle formation in microfilaments. Although we have no evidence for the occurrence of a low polymerized form of nuclear actin in the native state, the finding that DMSO works on cells to produce bundle formation of actin molecules, together with the fact that this protein is a major component of chromatin non-histone proteins [9-111, suggests that DMSO might affect the gene transcription of the cells. This suggestion is further supported by the interesting fact that DMSO stimulates Friend leukemic mouse cells and rat kangaroo myoblast cells to synthesize hemoglobin [17] and collagen [18] respectively, something they normally would not do. This possibility is also consistent with the idea that chromatin non-histone proteins may be involved in the regulation of gene activity in higher eukaryotic cells [19]. We might add that care should be taken in the medical application of this solvent; depending upon the circumstances, it could well have either beneficial or disastrous effects. We thank Mr Tetsuhiro Sakai. DeDartment of Phvsiology, Kinki University School of *Medicine, and I% Kiyoko Kuroda, Department of Biology, Osaka University, for kindly providing HeLa cells and Amoeba

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proteus, respectively. Our thanks are also due to Professor Yuji Tonomura and Mr Satoshi Ogihara of Department of Biology, Osaka University, for constructive criticisms of an earlier form of this manuscript. We are very grateful to Professor John Bonner of the Departments of Biology, Princeton University, for helpful comments on this manuscript, and for the hospitality of the Department during the preparation of this manuscript.

References 1. Clarke, M & Spudich, J A, Ann rev biochem 46 (1977) 797. 2. Behnke, 0, Forer, A & Emmerson, J, Nature 234 (1971) 408. 3. Cawadi, N, Nature 234 (1971) 410. 4. Forer, A & Jackson, W T, Cytobiology 12 (1976) 199. 5. Hinklev. R & Telser. A. EXD cell res 86 (1974) 161. 6. Cande,.W 2, Lazarides,‘G & Macintosh; J R,‘J cell biol72 (1977) 552. 7. Forer, A, Cold Spring Harbor conferences on cell proliferation (ed R Goldman, T Pollard & J Rosenbaum) vol. 3, pp. 1273-1293. Cold Spring Harbor Laboratory, New York (1976). 8. Rubin, R W, Goldstein, L & Ko, C, J cell biol 77 (1978) 698. 9. LeStourgeon, W M, Forer, A, Yang, Y, Bertram, J S & Rusch, H P, Biochim biophys acta 379 (1975) 529. 10. Pederson, T, Biochemistry 16 (1977) 2771. 1I. Douvas, A S, Harrington, C A & Bonner, J, Proc natl acad sci US 72 (1975) 3902. 12. Fukui, Y, J cell biol76 (1978) 146. 13. Ishikawa, H, Bishoff, R & Holtzer, H, J cell biol 43 (1969) 312. 14. Bonner, J T, J exp zoo1 106(1947) 1. 15. Prescott, D M & James, T W, Exp cell res 8 (1955) 256. 16. Miyamoto, H, Rasmussen, L & Zeuthen, E, Methods in cell biology (ed D M Prescott) vol. 13, pp. 15-27. Academic Press, New York (1976). 17. Friend, C, Sher, W, Holland, J G & Sato, T, Proc natl acad sci US 68 (1971) 378. 18. Miranda, A H, Nette, E G, Khan, S, Brockbank, K & Schonberg, M, Proc natl acad sci US 75 (1978) 3826. 19. Stein, G S, Stein, J S & Kleinsmith, L J, Sci Am 234 (1975) 64. 20. Fukui, Y & Katsumaru, H. In preparation. Received November 30, 1978 Revised version received January 3 1, 1979 Accepted February 2, 1979

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