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Behaviour of nucleoli in isolated nuclei
Eqerimenfal
Cell
Research
BEHAVIOUR
34, 23S94.2
(1964)
239
OF NUCLEOLI
IN ISOLATED
NUCLEI
L. F. LACOIJR Department
of Cell
Biology,
John Innes Institute, Hem., England
Bayfordbury,
Hertford,
Received May 2, 1963
FISHER and Harris [2] have developed a method which permits the isolation of nuclei from HeLa cells without appreciable loss of nucleic acid. These authors observed that during the isolation procedure, which involved controlled stirring of the cells in an aqueous solution of “Tween 80”, the nuclei became swollen, and in the most swollen nuclei the nucleoli were no longer visible. When the swollen nuclei were suspended in phosphate-buffered physiological saline, they shrank to their normal size and the nucleoli again became visible. This effect was confirmed by electron microscopy [I]. In the present study the fate of the rapidly labelled nucleolar RNA (ribose nucleic acid) during swelling and contraction of the isolated nuclei was studied by means of autoradiography. MATERIALS
AND
METHODS
The HeLa cells growing in a suspension culture were exposed to [‘HI cytidine (specific activity 1 C/mM, supplied by Schwartz Bio Research Inc., New York) at a concentration of 2 PC/ml for 45 min at 37°C. At the end of this period most of the precursor is present in the nuclear RNA. The cells were spun out of the radioactive medium and washed twice in phosphate-buffered saline. A sample of the cells was then fixed. The nuclei were isolated from the remainder by the “Tween” technique, and a sample of the swollen isolated nuclei fixed. The remaining nuclei were transferred to phosphate-buffered saline and then fixed. All samples were fixed overnight as loose pellets in 10 per cent (v/v) neutral formalin. After fixation the pellets were rinsed in water and extracted for 30 min in 5 per cent (v/v) ice-cold trichloroacetic acid. The material was then washed, dehydrated in ethanol and embedded in methacrylate. Sections were cut at 1 p and mounted on “subbed” slides. The methacrylate was removed by steeping the slides overnight in xylol. Kodak stripping film (V1042) was used for autoradiography. After the autoradiographs had been processed the preparations were stained for 20 min in 0.02 per cent (w/v) citrate-buffered azure B bromide at pH 4. RESULTS
The preparations of the two classes of isolated nuclei confirmed that the nucleoli could not be seen in the most distended nuclei and reappeared Experimental
Cell
Research
34
L. F. LaCour
240
Fig. l.-Autoradiographs of the HeLa nuclei after exposure of the cells to [aHj cytidine for 45 min: (a) from a section of an intact cell (b) and (c) from sections of isolated nuclei. In (a) and (c) the label is mainly in the nucleoli. In (b) the nucleoli are invisible and the label is dispersed. x 3200.
such nuclei were allowed to shrink to their normal size in phosphatebuffered saline (Figs. 1 b, c). No obvious distortion of the nucleoli was observed in isolated nuclei which had been allowed to shrink to their normal size. The distribution of the numbers of nucleoli per nucleus in the isolated nuclei after these had been returned to phosphate-buffered saline was not significantly different from the distribution in intact cells (Fig. 2). This suggests that during the shrinkage of the swollen nuclei and the reappearance of the nucleoli, no fusion of nucleoli occurred. In intact cells azure B at pH 4 stains nucleoli intensely and chromatin relatively lightly. In the sections of
when
Experimental
Cell
Research
34
Nucleoli in isolated nuclei
241
swollen isolated nuclei the staining was more or less uniform. The nuclei after return to phosphate-buffered saline stained in t.he same way as nuclei in intact cells. Grain counts in autoradiographs over 50 nuclei chosen at random from each of the three classes (Table I) show that in the intact cells the nucleolar
Fig. 2.-Histograms showing distribution of the numbers of nucleoli per nucleus in sections of intact cells and of isolated nuclei which have returned to their normal size after swelling.
10 ’
I 01YUCLCOL, 3 uyy”
RNA was more heavily labelled than the RNA in the chromatin (Fig. 1 a). The mean number of grains over the whole nucleus in the preparations of swollen nuclei in which nucleoli were not visible was not different from the mean number over the nucleus in intact cells. The autoradiographs of the nuclei after return to phosphate-buffered saline show heavy labelling of the nucleoli. The mean number of grains over the whole nucleus in these preparations was not different from that in intact cells. These observations confirm that no appreciable amount of the rapidly labelled nuclear RNA is TABLE
1. Persistence of labelled RNA in the nucleoli of isolated HeLa cell nuclei after swelling
and shrinking.
Mean no. of grains
Intact cells
Isolated nuclei after swelling
Over whole nucleus Over chromatin Over nucleoli
12.06 + 0.64 3.12kO.36 8.94 + 0.48
12.80 310.52 -
l~zperimenlal
Isolated nuclei after shrinking (normal size) 11.72kO.49 3.82 k 0.34 7.90 + 0.47 Cell Research
34
242
L.F.LaCour
lost during the isolation procedure or during swelling and shrinking of the isolated nuclei. Since the nucleoli reappear undistorted and with a similar distribution when the isolated nuclei return to their normal size it seems likely that their disappearance in swollen isolated nuclei is due to expansion and consequent loss of optical density. The staining characteristics of the isolated swollen nuclei also supports this view. The present observations make it very difficult to avoid the conclusion that the nucleolar RNA is firmly bound to some stable structural component. SUMMARY
Nuclei isolated from HeLa cells by the “Tween” technique swell during the isolation procedure and the nucleoli become invisible. The nucleoli reappear when the nuclei return to their normal size in buffered saline. This effect was investigated by autoradiography after the nucleolar RNA had been labelled by exposure of the cells to [SH] cytidine. Grain counts showed that the disappearance and reappearance of the nucleoli during this procedure was not associated with any loss of labelled nucleolar RNA. The results indicate that the nucleolar RNA is tightly bound to some stable structural component. I thank
Dr H. Harris for providing
the isolated nuclei for this investigation.
REFERENCES 1. CRAWLEY, J. C. W. 2. FISHER, H. W. and
Esperimenlal
and HARRIS, HARRIS, H.,
Ceil Research
34
H., ET@ Proc. Roy.
Cell Res. 31, 70 (1963). Sot. B 156, 521 (1962).
Cell
Research
BEHAVIOUR
34, 23S94.2
(1964)
239
OF NUCLEOLI
IN ISOLATED
NUCLEI
L. F. LACOIJR Department
of Cell
Biology,
John Innes Institute, Hem., England
Bayfordbury,
Hertford,
Received May 2, 1963
FISHER and Harris [2] have developed a method which permits the isolation of nuclei from HeLa cells without appreciable loss of nucleic acid. These authors observed that during the isolation procedure, which involved controlled stirring of the cells in an aqueous solution of “Tween 80”, the nuclei became swollen, and in the most swollen nuclei the nucleoli were no longer visible. When the swollen nuclei were suspended in phosphate-buffered physiological saline, they shrank to their normal size and the nucleoli again became visible. This effect was confirmed by electron microscopy [I]. In the present study the fate of the rapidly labelled nucleolar RNA (ribose nucleic acid) during swelling and contraction of the isolated nuclei was studied by means of autoradiography. MATERIALS
AND
METHODS
The HeLa cells growing in a suspension culture were exposed to [‘HI cytidine (specific activity 1 C/mM, supplied by Schwartz Bio Research Inc., New York) at a concentration of 2 PC/ml for 45 min at 37°C. At the end of this period most of the precursor is present in the nuclear RNA. The cells were spun out of the radioactive medium and washed twice in phosphate-buffered saline. A sample of the cells was then fixed. The nuclei were isolated from the remainder by the “Tween” technique, and a sample of the swollen isolated nuclei fixed. The remaining nuclei were transferred to phosphate-buffered saline and then fixed. All samples were fixed overnight as loose pellets in 10 per cent (v/v) neutral formalin. After fixation the pellets were rinsed in water and extracted for 30 min in 5 per cent (v/v) ice-cold trichloroacetic acid. The material was then washed, dehydrated in ethanol and embedded in methacrylate. Sections were cut at 1 p and mounted on “subbed” slides. The methacrylate was removed by steeping the slides overnight in xylol. Kodak stripping film (V1042) was used for autoradiography. After the autoradiographs had been processed the preparations were stained for 20 min in 0.02 per cent (w/v) citrate-buffered azure B bromide at pH 4. RESULTS
The preparations of the two classes of isolated nuclei confirmed that the nucleoli could not be seen in the most distended nuclei and reappeared Experimental
Cell
Research
34
L. F. LaCour
240
Fig. l.-Autoradiographs of the HeLa nuclei after exposure of the cells to [aHj cytidine for 45 min: (a) from a section of an intact cell (b) and (c) from sections of isolated nuclei. In (a) and (c) the label is mainly in the nucleoli. In (b) the nucleoli are invisible and the label is dispersed. x 3200.
such nuclei were allowed to shrink to their normal size in phosphatebuffered saline (Figs. 1 b, c). No obvious distortion of the nucleoli was observed in isolated nuclei which had been allowed to shrink to their normal size. The distribution of the numbers of nucleoli per nucleus in the isolated nuclei after these had been returned to phosphate-buffered saline was not significantly different from the distribution in intact cells (Fig. 2). This suggests that during the shrinkage of the swollen nuclei and the reappearance of the nucleoli, no fusion of nucleoli occurred. In intact cells azure B at pH 4 stains nucleoli intensely and chromatin relatively lightly. In the sections of
when
Experimental
Cell
Research
34
Nucleoli in isolated nuclei
241
swollen isolated nuclei the staining was more or less uniform. The nuclei after return to phosphate-buffered saline stained in t.he same way as nuclei in intact cells. Grain counts in autoradiographs over 50 nuclei chosen at random from each of the three classes (Table I) show that in the intact cells the nucleolar
Fig. 2.-Histograms showing distribution of the numbers of nucleoli per nucleus in sections of intact cells and of isolated nuclei which have returned to their normal size after swelling.
10 ’
I 01YUCLCOL, 3 uyy”
RNA was more heavily labelled than the RNA in the chromatin (Fig. 1 a). The mean number of grains over the whole nucleus in the preparations of swollen nuclei in which nucleoli were not visible was not different from the mean number over the nucleus in intact cells. The autoradiographs of the nuclei after return to phosphate-buffered saline show heavy labelling of the nucleoli. The mean number of grains over the whole nucleus in these preparations was not different from that in intact cells. These observations confirm that no appreciable amount of the rapidly labelled nuclear RNA is TABLE
1. Persistence of labelled RNA in the nucleoli of isolated HeLa cell nuclei after swelling
and shrinking.
Mean no. of grains
Intact cells
Isolated nuclei after swelling
Over whole nucleus Over chromatin Over nucleoli
12.06 + 0.64 3.12kO.36 8.94 + 0.48
12.80 310.52 -
l~zperimenlal
Isolated nuclei after shrinking (normal size) 11.72kO.49 3.82 k 0.34 7.90 + 0.47 Cell Research
34
242
L.F.LaCour
lost during the isolation procedure or during swelling and shrinking of the isolated nuclei. Since the nucleoli reappear undistorted and with a similar distribution when the isolated nuclei return to their normal size it seems likely that their disappearance in swollen isolated nuclei is due to expansion and consequent loss of optical density. The staining characteristics of the isolated swollen nuclei also supports this view. The present observations make it very difficult to avoid the conclusion that the nucleolar RNA is firmly bound to some stable structural component. SUMMARY
Nuclei isolated from HeLa cells by the “Tween” technique swell during the isolation procedure and the nucleoli become invisible. The nucleoli reappear when the nuclei return to their normal size in buffered saline. This effect was investigated by autoradiography after the nucleolar RNA had been labelled by exposure of the cells to [SH] cytidine. Grain counts showed that the disappearance and reappearance of the nucleoli during this procedure was not associated with any loss of labelled nucleolar RNA. The results indicate that the nucleolar RNA is tightly bound to some stable structural component. I thank
Dr H. Harris for providing
the isolated nuclei for this investigation.
REFERENCES 1. CRAWLEY, J. C. W. 2. FISHER, H. W. and
Esperimenlal
and HARRIS, HARRIS, H.,
Ceil Research
34
H., ET@ Proc. Roy.
Cell Res. 31, 70 (1963). Sot. B 156, 521 (1962).