Migration of newly synthesized RNA during mitosis

Experimental Cell Research 65 (1971) 340-344

MIGRATION

OF NEWLY

SYNTHESIZED

RNA DURING

MITOSIS

I. Embryonic Cells of the Loach (Misgurnus fossilis L.) A. A. NEYFAKH Institute

of Developmental

and A. A. KOSTOMAROVA

Biology, Academy of Sciences of the USSR, Moscow V-133, USSR

SUMMARY The behavior of newly synthesized nuclear DNA-like RNA during mitosis was studied by means of autoradiography in early loach (Misgurnus fossilis) embryos at late blastula stage. It is demonstrated that in interphase cells labeled RNA is localized in the nucleus before onset of mitosis. With onset of mitosis labeled RNA enters cytoplasm and after completion of the cell division returns to the nuclei of the daughter cells.

Synthesis of RNA occurs in nuclei during the whole period of interphase. With time, and subsequent to its synthesis, RNA partly migrates from the nucleus to the cytoplasm and partly is broken down in the nucleus [2]. During mitosis, as the process of chromosome condensation proceeds, RNA synthesis either ceases completely in the nuclei [6] or decreases sharply [7]. The onset of new RNA synthesis is revealed in late telophase [12]. As shown by autoradiography, at the onset of mitosis, in late prophase, the nuclear membrane is dispersed and RNA enters the cytoplasm. Metaphase chromosomes contain relatively little RNA [12, 131. However, the fate of nuclear RNA after mitosis remains unknown. It follows from the data of Rao & Prescott [14] that in amoeba about 25 % of labelled nuclear RNA apparently returns to the nuclei after mitosis. Yet, the authors do not give any definite answer to the question, emphasizing the difference between the behaviour of nuclear RNA and that of nuclear Exptl Cell Res 65

protein; the latter was shown to return entirely to the nuclei after mitosis in amoeba. When an amoeba nucleus containing labelled RNA was transplanted to a nonlabelled amoeba, a certain share of the label could be detected in the nonlabelled nucleus of the recipient [3]. This fact points to the possibility of RNA transfer from cytoplasm to nucleus. The present paper describes the behavior of newly synthesized nuclear RNA during mitosis in loach (Misgurnis fossilis) embryos. In order to learn whether labelled RNA returns to the nuclei of the daughter cells or remains in the cytoplasm, the following two conditions should be fulfilled: (1) during the period of experimentation all cells of the embryo should divide at least once; (2) RNA synthesis should be blocked after mitosis. The latter condition is necessary because the labelling of the nuclei of daughters cells might be due to new RNA synthesis from labelled precursor pool material made avail-

Migration of RNA during mitosis. Z 341 able by cytoplasmic RNA turnover. In order to fulfill the first condition the mitotic activity of cells was stimulated by transferring embryos at optimal temperature after having kept them in the cold [IO]. To block RNA synthesis in the nuclei of the daughter cells, actinomycin D was used.

where n is the cell number counted in the longitudinal sections (the shape of the blastoderm was taken as a hemisphere).

Cell number

RESULTS

After the completion of incubation with 3Huridine (at the moment of addition of actinomycin, table 1, stage 1) the average number MATERIALS AND METHODS of cells in the blastoderm was 2 300f200. After 60 min incubation with “cold” uridine The loach blastoderms were isolated from the yolk [S] at mid-late blastula stage and incubated in doubleand actinomycin (table 1, stageII), the average strength Holtfreter solution with 3H-uridine (20 ,U cell number in the blastoderm was 5 100k 150. Ci/ml, specact. 25 mCi/mM) for 2 h at low temperature (5°C). Two hours after incubation with SH- These data show that during 60 min of incubauridine the blastoderms were raised to 21°C which is the optimal temperature for loach development [4]. tion with “cold” uridine and the inhibitor The incubation was continued in the presence of the each cell in the blastoderm divided at least precursor for further 15 min. The blastoderms were once. transferred to the medium containing unlabelled uridine (100 y/ml) and actinomycin D (25 y/ml). Thus the labelled interphase nuclei seen in Thus, further incorporation of the label into RNA the preparations after 60 min incubation with was suppressed by combined action of the inhibitor and of pool dilution. the inhibitor, would represent the nuclei of Blastoderms were detected at the following stages the second generation of cells which had synof the experiment: (1) after 2 h incubation with the precursor at 5°C (table 1) stage 0; (2) after 15 min thesized RNA during the incubation with heating at 21°C (table 1) stage 1; and (3) every 5-10 3H-UdR before mitosis. min of cultivation with non-labeled uridine and actinomycin D. The last determination was made at the 75th min of incubation at 21”C, i.e. 60 min from the inception of actinomycin treatment. Blastoderms were fixed with Carnoy fluid and embedded in paraffin wax by the routine method. All blastoderms were stained totally with carmine and sections 5-7 pm thick were stained with Carrachi haematoxylin. The sections were covered with liquid radiosensitive emulsion (type “M”, NIIChimPhoto, Moscow) and incubated for 15 days at 4°C. Amidol was used as developer. Almost 100 % of the label in the sections was RNase-sensitive [9], thus representing the incorporation of the label into RNA. At late blastula stage there is still no ribosomal RNA synthesis [l] so that uridine incorporation largely reflects DNA-like RNA synthesis [5]. The number of silver grains was counted over the nuclei and cytoplasm in interphase cells and over the chromosomes and cytoplasm in metaphase cells. Sixty interphase cells and 25 metaphase cells were analysed for each observation point. Distribution of the label is shown in fig. 1a-d. In order to learn whether all blastoderm cells have entered mitosis, the cell number in blastoderm was counted at the moment of washing away the label and adding actinomycin and after 60 min incubation with the inhibitor. The cell number in blastoderm (N) was determined according to the formula:

Change of RNA synthesis Table 1 shows that during 2 h incubation at 5°C approximately the same amount of RNA is synthesized as during 15 min incubation at 21°C (40 grains over a cell area after incubation for 2 h at 5’C and 85 grains over a cell area after additional incubation for 15 min at 21°C). After addition of the actinomycin D, RNA synthesis was suppressedand during further 60 min incubation the extent of labelling remains almost constant (table 1). The grain number per cell volume can be derived from the grain number per cell area in sections. Proceeding from the assumption that the volume of each daughter cell is half that of the mother cell one can calculate the total amount of labelled RNA per cell volume in daughter cells. From such a calculation it was found that the total amount of labelled RNA in a daughter cell accounted for 59 % ExptI Cell Res 65

342 Table

A. A. Neyfakh & A. A. Kostomarova 1. Distribution

of the labelled RNA in interphase and dividing cells Stage of experiment Oa

Interphase Grain count per Grain count per Grain count per Per cent of label Per cent of label

nucleus cytoplasm cell area per nucleus per cytoplasm

4022 40 100 -

Ib Interphase

Metaphase

75t4 lo+1 85 88 12

12*2 56+3 68 18 82

IIC Interphase 49+3 15+2 76% 26

a 120 min after incubation with 3H-uridine at 5°C. b 15 mln after incubation with SH-uridine at 21“C; at the moment of adding “cold” uridine and actinomycin D (the total time of incubation is 135 min). c 60 min after adding “cold” uridine and actinomycin D at 21°C. Stages 0 and I represent cells before mitosis (mother cells); stage II after mitosis (generation of daughter cells).

of the original content of the labelled RNA in a mother cell. This result has been obtained from the following calculations. Since the ratio of volumes of a mother and a daughter cell is equal to MmlMd = 2/l, the ratio of the radii of the 3

cells will be 1/2 (assuming the cells are spherical). The amount of labelled RNA per cell area is proportional to the grain number per cell area (m) while the amount of labelled RNA per cell volume (M) is proportional to M=m(4/3)r. Hence it follows that the ratio of the amount of labelled RNA per daughter and mother cells should be equal to Md/Mm i.e. the = md rdlm, r, = 64185 h=O.59, amount of RNA per volume makes in a daughter cell 59 % of the original amount of RNA. This value is in a good agreement with that expected (50 “/o). The small difference, however, can be explained in one of three ways: (1) RNA synthesis was not blocked by actinomycin D immediately; (2) some proportion of cells had not passed mitosis during the time of the experiment; (3) possible miscounts cannot be neglected. However, the above figures show that in our experiments RNA synthesis was virtually blocked by actinoExptl Cell Res 65

mycin D and the bulk of the cells have undergone mitosis. Distribution of labeled RNA between nucleus and cytoplasm Fig. 1a and table 1 show that after incubation with 3H-UdR the label is localized mainly over the nuclei in the interphase cells. 88 % of the label was counted over nucleus and 12 % over cytoplasm. In metaphase cells, newly synthesized RNA is mainly distributed throughout the cytoplasm (fig. 1 b-c). In metaphase cells only 18 % of the label was found over metaphase chromosomes (if the latter were taken for “nucleus” (table 1). After mitosis, the amount of label again accounted for 76 % in nuclei of interphase cells of the next generation (fig. 1d-e; table 1). Thus, the data allow the conclusion, with a high degree of confidence, that newly synthesized RNA (DNA-like RNA) which enters cytoplasm during mitosis, returns after mitosis to the nuclei of the daughter cells. According to preliminary observation, this occurs in late telophase. The results of the present work do not allow any conclusions about the mechanism determining the return of RNA to nucleus. From the data suggesting

Migration of RNA during mitosis. I

343

Fig. 1. Distribution of labelled RNA over the nuclear structures and cytoplasm (a) Interphase before the onset of mitosis. The label is localized over the nucleus; fixation at the moment of adding actinomycin D. x 1 250; (b, c) Metaphases; the label is localized mainly over the cytoplasm; fixation 45 min after adding actinomycin D; (b) x 1 250, (c) x 450; (d, e) Interphases after completion of mitosis; the label is again localized over the nuclei; fixation 60 min after adding actinomycin D; x 1 250.

of labelled RNA molecules from donor nucleus to host nucleus in amoeba [3], it can be assumed that newly synthesized RNA is reversibly bound to nuclear structures and that the equilibrium is shifted to the bound form of RNA. In interphase cells, and after time-lapse, some RNA molecules are released from the association and enter cytoplasm, irreversibly in this case. During migration

mitosis, simultaneously with chromosome spiralization and dispersion of the nuclear membrane, RNA molecules enter the cytoplasm and return to the daughter cell nuclei when the interphase structure of the nuclei is restored. Recent experiments with cultured Chinese hamster fibroblasts [l l] showed some new features of the behavior of newly synthesized Exptt CeIi Res65

344

A. A. Neyfakh & A. A. Kostomarova

RNA and may be useful for the elucidation of the mechanisms of RNA transfer from nucleus to cytoplasm. The authors wish to thank Professor A, P. Dyban for his helpful suggestions and criticism during the course of this investigation. The authors wish to express their gratitude to Mrs N. S. Korobtsova for her skilled technical assistance.

Note added in proof: After the preliminary communication had been published (Neyfakh, Priroda, N 7, p. 44, July 1970) and the present paper submitted for publication, an article appeared (Rao & Prescott, Exptl cell res 62 (1970) 286) in which migration of a large proportion of RNA to the nuclei of the daughter cells was demonstrated in amoeba. Although in this work RNA was not shown to leave the nucleus during mitosis, one may suggest that the phenomenon of newly synthesized RNA returning to the nuclei of the daughter cells occurs in unicellular eucariots as well.

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ExptI Cell Res 65

2. Georgiev, G P & Samarina, 0 P, Advances in biological chemistry, vol. 10, p. 5. Nauka, Moscow (1969). 3. Goldstein. L. Rao. M V N & Prescott. D M. Ann embryo1 ‘mdrphoi, suppl. 1. Trav&x d; VI congr&s international d’embryologie, p. 189 (1969). 4. Ignatieva, G M & Kostomarova, A A, Dokl akad nauk, SSSR 154 (1966) 1221. In Russian. 5. Kafiani, C A, Timofeeva, M J, Neyfakh, A A, Melnikova, N L & Rachkus. J A. J embrvol exptl morph01 21 (1969) 295. ’ 6. King, D W & Barnhisel, N L, J cell biol 33 (1967) 265. 7. Konrad, C G, J cell biol 19 (1963) 267. 8. Kostomarova, A A & Neyfakh, A A, Zh obshch biol 25 (1964) 386. In Russian. 9. Kostomarova, A A & Korobtsova, N S, Dokl akad nauk SSST 191 (1970) 493. In Russian. 10. Neyfakh, A A & Rott, N N, Dokl akad nauk SSSR 125 (1959) 432. In Russian. 11. Neyfakh, A A, Abramova, N B & Bagrova, A M, Exptl cell res 65 (1971) 000. 12. Prescott, D M & Bender, M A, Exptl cell res 26 (1962) 260. 13. - J cell camp nhvsiol62. sunnl. 1 (1963) 175. 14. Rao, M V N &Prescott, b h;r; J ceil bioi 35, part 2 (1967) 109A. Received October 16, 1970