J.Cell Sci. 5) 351-364(1969)Printed in Great Britain

THE ROLE OF THE NUCLEOLUS IN THE

TRANSFER OF RNA FROM NUCLEUS

TO CYTOPLASM

E. SIDEBOTTOM AND H. HARRISSir William Dunn School of Pathology, University of Oxford, England

SUMMARYThe role of the nucleolus in the transfer of RNA from nucleus to cytoplasm was examined by

means of experiments in which inactivation of the whole nucleus, or of the nucleolus, wasachieved by a microbeam of ultraviolet light. In heterokaryons in which a chick erythrocytenucleus had been reactivated, no detectable amount of RNA was transferred from the reactivatednucleus to the cytoplasm of the cell until this nucleus had developed a nucleolus. In HeLa cells,inactivation of the nucleolus alone inhibited the transfer to the cytoplasm, not only of the RNAsynthesized at the nucleolar site, but also of the RNA made elsewhere in.the nucleus. It thusappears that the nucleolus governs the transport not only of its own RNA, but also of the RNAwhich is made on other parts of the chromosomes.

INTRODUCTION

When a chick erythrocyte nucleus, which is normally metabolically inactive, isincorporated into the cytoplasm of a tissue culture cell from the same or a differentanimal species, the dormant nucleus undergoes reactivation (Harris, 1965). Thechanges detectable in the reactivated nucleus include gross enlargement (Harris, 1967),structural alterations in the chromatin (Bolund, Ringertz & Harris, 1969), and syn-thesis of DNA and RNA (Harris, 1965). The RNA synthesized by the erythrocytenucleus during the first 2-3 days after reactivation is of high molecular weight andshows polydisperse sedimentation on centrifugation in conventional sucrose gradients;but the erythrocyte nucleus fails, during this period, to determine the synthesis ofchick-specific surface antigens or of a soluble enzyme lacking ia the recipient cell.When, however, from about the third day onward, the erythrocyte nuclei developnucleoli, the synthesis of the antigens and the enzyme begins (Harris, Sidebottom,Grace & Bramwell, 1969; Harris & Cook, 1969).

Analysis of the patterns of RNA synthesis in the heterokaryons, by autoradiographictechniques involving the use of a microbeam of ultraviolet light, revealed that theerythrocyte nuclei, prior to the development of nucleoli, failed to transfer the RNAwhich they synthesized to the cytoplasm of the cell. But when these nuclei developednucleoli and began to synthesize RNA of ribosomal type, passage of radioactivityfrom the nuclear to the cytoplasmic RNA could be detected (Harris et al. 1969). Thepresent paper presents in more detail the experiments carried out with the ultravioletmicrobeam on the heterokaryons, and extends the observations to normal mononucleatecells.

352 E. Sidebottom and H. Harris

MATERIALS AND METHODS

Cells

HeLa cells were maintained in both suspension and monolayer culture essentially as de-scribed by Harris & Watts (1962). A9 cells, a mutant strain of the mouse L cell line, wereobtained from Dr. J. Littlefield of the Massachusetts General Hospital, Boston, U.S.A., andgrown as monolayer cultures in medium 199 (Glaxo Ltd., Greenford, Middlesex), containing10% foetal calf serum (Flow Laboratories, Irvine, Scotland). Chick embryo erythrocytes wereobtained from 15-day-old fertile eggs as described by Bolund et al. (1969).

Irradiation of cells

The A 9 cells were subjected to a flux of 6000 rad of gamma radiation emanating from acobalt-60 source. The cells were irradiated as a monolayer in the culture vessel. As explained inHarris et al. (1969), irradiation of the cells was necessary to suppress mitosis, so that the erythro-cyte nuclei in the heterokaryons could remain discrete long enough to develop nucleoli.

Cell fusion

The technique of cell fusion was as described in Harris, Watkins, Ford & Schoefl (1966):5 x io'irradiated A9 cells and 4 x io7 erythrocytes were treated with 250—750 haemagglutinatingunits of Sendai virus inactivated by ultraviolet light. The preparations were freed of unfusederythrocyte ghosts and debris by centrifugation as described by Harris et al. (1969).

Radioactive precursors

Uridine-5[H'] with a specific activity of 20-3 Ci/mM was obtained from the RadiochemicalCentre, Amersham, Bucks. The tracer was used at a final concentration of 10/tCi/ml.

Autoradiography

The cells, growing on quartz coverslips in specially constructed chambers, were exposed tothe radioactive precursor for 6 h. The coverslips were then removed, rinsed in phosphate-buffered saline at pH 7-3 and fixed in 1 :j acetic acid:methanol. The fixed preparations wereextracted and subjected to autoradiography as previously described (Harris et al. 1966). Thefilms were developed after 24-72 h, as required.

Culture chambers

Simple culture chambers were made by a modification of the chamber described by Munro(1963). A circle 16 mm in diameter and a connecting channel, as shown in Fig. 6, were cut outof a rectangle of glass 4 x 2-5 cm and 3 mm in thickness. This was then fixed on to a 3 x ii in.microscope slide with Araldite A.T.I. (Ciba, Duxford, Cambs). The resulting chamber issimple to clean, can be repeatedly sterilized by dry heat at 160 °C and is able to maintain thecells used in this work in a healthy state for several days. Coverslips are secured on the chamberseither with molten sterile paraffin wax or with silicone grease. When the cell suspension ormedium has been introduced into the chamber, the channel is closed with paraffin wax. Themedium can easily be changed, through the paraffin plug, by means of a needle and syringe.

Microbeam apparatus

The microbeam was constructed essentially to the design of Uretz, Bloom & Zirkle (1954).The light source was a Philips 500 W water-cooled high-pressure mercury discharge lamp. Aliquid filter consisting of a 5-cm path of a solution of NiSO4 (240 g/1.) and CoSO4 (50 g/1.) inwater served to remove heat and most of the visible light from the beam. This light source andfilter gave a heterochromatic ultraviolet beam with wavelengths from about 225 to 325 nm.A quartz lens, focal length 2J in., focused the light on to a primary aperture which was, in

Role of the nucleolus 353

effect, a field diaphragm which controlled the size and shape of the microbeam. A series ofinterchangeable disks with apertures from 0-2 to 1 mm allowed the diameter of the microbeamto be varied from about 2$ to 11 fim. Cells were selected for irradiation and mapped on anappropriate reference system with a Watson x 40 phase-contrast objective. The cells wereirradiated by means of a Beck x 72 reflecting objective, numerical aperture 0-65. At the tubelength used, the true magnification of the objective was about x 90. No attempt was made toincorporate phase-contrast optics into this system, since there was already enough contrast toidentify unequivocally those cells which had previously been located by the phase-contrastobjective. The microbeam apparatus is seen in Fig. 5.

Microbeam dosage

No measurement of incident light flux or absorbed dose of irradiation was attempted. Asuitable dose was determined empirically by varying the time of irradiation. This was usuallybetween 2 and 6 s. The output of the light source was monitored with a photocell, and anygross change in the intensity was corrected. It is likely that the intensity of irradiation was of thesame order as that measured by Dendy for a similar type of apparatus. (Dendy & Smith, 1964).Dendy calculated that the incident light flux was 2 x io~s ergs/fim'/s, and that the dose absorbedby a fibroblast nucleus was 2-5 x io~* ergs//*m'/s. Since the object of the microbeam irradiationwas to suppress RNA synthesis in the irradiated nucleus or nucleolus, the lowest dose whichcould achieve this was the optimal dose. Some preliminary experiments on binucleate A 9 cellsshowed that irradiation of one nucleus with an ultraviolet beam 10 /tm in diameter, for 3-5 s,reduced the incorporation of tritiated uridine into that nucleus by more than 90 % comparedwith the unirradiated nucleus in the same cell. When a binucleate cell with one irradiatednucleus was exposed to tritiated uridine, labelling of cytoplasmic RNA took place in theusual way. In heterokaryons containing an A9 nucleus and a reactivated erythrocyte nucleus,irradiation of the A 9 nucleus did not suppress incorporation of tritiated uridine into the erythro-cyte nucleus. At the irradiation doses used, no visible damage was seen in the irradiated nuclei.

OBSERVATIONS

Heterokaryons from 1 to 2 days after cell fusion

Heterokaryons were selected containing one A 9 nucleus and up to 4 erythrocytenuclei, which, at this stage, had not yet developed visible nucleoli. The aim of theexperiment was to compare cytoplasmic RNA labelling in these heterokaryons withcytoplasmic labelling in single A9 cells in the same cultures, after the A9 nucleus ineach case had been irradiated with the microbeam. A low level of cytoplasmic RNAlabelling takes place in single A9 cells after irradiation of the nucleus. If the reactivatederythrocyte nuclei in the heterokaryon contributed to the cytoplasmic RNA labelling,one would expect this to be greater in the heterokaryons, in which only the A9 nucleuswas irradiated, than in single A9 cells with irradiated nuclei. If, however, there was nodifference in the cytoplasmic labelling of the 2 groups, this would indicate that theerythrocyte nuclei were not contributing radioactive RNA to the cell cytoplasm. Thecultures were incubated with the radioactive precursor for 6 h after irradiation withthe microbeam, and the exposure of the autoradiographs was adjusted to give suitablegrain densities over irradiated cells.

The ratios of the total number of nuclear grains to the total number of cytoplasmicgrains for the two groups of irradiated cells are shown in Table 1. It will be seen that,in spite of considerable variation within each group of cells, these ratios were the same.The means of the cytoplasmic grain counts in the irradiated heterokaryons and in the

23 Cell Sci. 5

354 E. Sidebottom and H. Harris

Table i. Nuclear and cytoplasmic grain counts in Ag-erythrocyte heterokaryons inwhich the A 9 nucleus has been irradiated and in A 9 cells in which the nucleus has beenirradiated (1-3 days after cell fusion)

A 9 cellsHeterokaryons

No. cells

2458

Total no.

Nucleus

7991303

grains

Cytoplasm

6171023

Rationucleus/

cytoplasm

1-291-27

Table 2. Comparison of cytoplasmic labelling in irradiated heterokaryons with that inirradiated A 9 cells (1-3 days after cell fusion)

Expt

7990

919 2

no.

100

90

80

70

60

V

0 SOI

40

30

20

10

°(

Mean cytoplasmicr

Heterokaryons

19315-610-329-4

0

0

0

0

0

-

80 c

• 00

• °o

0 °01 1 1

grain counts

A 9 cells

20-518-2I I - O

35-4

0

1 1 1

) 10 20 30 40 50 60Cytoplasm

P value

o-88o-6o0-830-44

1 1

70 80

Fig. 1. Relationship of nuclear grain counts to cytoplasmic grain counts in hetero-karyons in which the A 9 nucleus has been irradiated, and in A 9 cells in which the nucleushas been irradiated (1-3 days after cell fusion). • , A9 cells; O, heterokaryons.

Role of the nucleolus 355

irradiated A 9 cells were also similar in each of the four separate experiments. Theprobability (P) values indicate that the differences between the two groups were notsignificant (Table 2). The results of two individual experiments are shown graphicallyin Fig. 1. Figure 8 shows an autoradiograph of a heterokaryon in which the A9 nucleushas been irradiated but the erythrocyte nucleus is still active. There is very littlecytoplasmic labelling compared with that seen in an unirradiated heterokaryon fromthe same culture (Fig. 7). It is clear from these experiments that the erythrocyte nucleusin the heterokaryon can continue to synthesize large amounts of RNA after the A 9nucleus has been irradiated, but this RNA is not transported in detectable amounts tothe cytoplasm of the cell.

Heterokaryons 4 days after cell fusion

Preparations at this stage have a mixed population of heterokaryons: in some, theerythrocyte nuclei show distinct nucleoli; in others, no nucleoli are yet visible. Whenheterokaryons in which the A9 nuclei have been irradiated are now compared with

90

80

70

60

5 SO

2 4 0

30

20

10

oo

0 10 20 30 40 SO 60 70 80 90 100 110 120Cytoplasm

Fig. 2. Relationship of nuclear grain counts to cytoplasmic grain counts in hetero-karyons in which the A 9 nucleus has been irradiated, and in A 9 cells in which the nucleushas been irradiated (4 days after cell fusion). • , A9 cells; O, heterokaryons.

A 9 cells in which the nuclei have been irradiated, the cytoplasmic grain counts reflectthe change which has taken place in the heterokaryons. Figure 2 shows that the cyto-plasmic grain counts in some of the heterokaryons are indistinguishable from those inthe irradiated A 9 cells, but other heterokaryons have cytoplasmic counts well abovethis range. These results give the first indication that the reactivated erythrocyte

23-2

356 E. Sidebottom and H. Harris

nuclei are able to contribute radioactivity to the cytoplasm of the heterokaryon. Thiscontribution becomes clearer at later stages, as shown in the following groups ofexperiments.

Heterokaryons 7 or more days after cell fusion

In these cells, most of the erythrocyte nuclei in the heterokaryons contained quitelarge nucleoli. Table 3 shows the ratios of the total number of nuclear grains to thetotal number of cytoplasmic grains for heterokaryons in which the A 9 nuclei have beenirradiated, and for A9 cells in which the nuclei have been irradiated. The ratio for the

Table 3. Nuclear and cytoplasmic grain counts in Ag-erythrocyte heterokaryons in whichthe A 9 nucleus has been irradiated and in A 9 cells in which the nucleus has been irradiated{7 or more days after cell fusion)

A 9 cellsHeterokaryons

No. cells

2787

Total

Nucleus

12784884

no. grains

Cytoplasm

IO456739

Rationucleus/cytoplasm

1-220-725

Table 4. Comparison of cytoplasmic labelling in irradiated heterokaryons with that inirradiated A 9 cells (7 or more days after cell fusion)

Expt no.

87939478

Mean cytoplasmic

Heterokaryons

68334870

grain counts

A 9 cells

36192030

P value

O-O20-038001009

latter is quite similar to that seen in these cells during the early stages of the experiment,thus indicating that there has been no fundamental change in the cultural conditions.But the ratio in the irradiated heterokaryons is now quite different from that in theirradiated A9 cells. The means of the cytoplasmic grain counts in the two groups of cellsare quite different in each of the four separate experiments shown in Table 4, and theP values in three of these experiments indicate that this difference is significant atthe 5 % level. The results of two individual experiments are shown graphically inFig. 3. Figure 9 shows an autoradiograph of a heterokaryon in which the erythrocytenucleus has developed a nucleolus, and in which the A 9 nucleus has been irradiated.The obvious cytoplasmic labelling is to be compared with the relative absence of suchlabelling in the heterokaryon shown in Fig. 8, where the erythrocyte nucleus has notyet developed a nucleolus.

Role of the nucleolus 357

These experiments indicate clearly that the reactivated erythrocyte nuclei cancontribute to cytoplasmic RNA labelling in the heterokaryon, but they do not do sountil they develop nucleoli.

HeLa cells

The object of the following experiments was to see whether the conclusions con-cerning the function of the nucleolus, which had previously been drawn from experi-ments on the behaviour of erythrocyte nuclei in heterokaryons, were applicable also

100 1-

90

80

70

601/1

•S 50

z

40

30

20

10

10 20 30 40 50Cytoplasm

60 70 80 90

Fig. 3. Relationship of nuclear grain counts to cytoplasmic grain counts in hetero-karyons in which the A 9 nucleus has been irradiated and in A 9 cells in which thenucleus has been irradiated (7 or more days after cell fusion). •, A9 cells; O, hetero-karyons.

to normal mononucleate cells, and to provide more direct evidence for these con-clusions by irradiation of the nucleolus alone. Nuclear and cytoplasmic labelling werecompared in 4 groups of HeLa cells from the one culture: (1) unirradiated cells; (2)cells with a single nucleolus which was irradiated with an ultraviolet microbeam3^fim in diameter; (3) cells in which a non-nucleolar part of the nucleus (nucleo-plasm) was irradiated with the same beam; (4) cells in which the whole nucleus wasirradiated with a beam 10 fim in diameter. The preparations were again exposed tothe radioactive precursor for 6 h after irradiation, and grain counts in the resultingautoradiographs were made over the nucleolus, nucleoplasm and cytoplasm in each ofthe four groups of cells.

358 E. Sidebottom and H. Harris

The total nuclear and cytoplasmic grain counts, and the means of each of thesecounts, in each group of cells, are shown in Table 5. Although the intensity of labellingvaried from experiment to experiment, the results showed the same pattern in allcases (Table 6). From these two Tables it can be seen that irradiation of the nucleolusreduced cytoplasmic labelling to about 13% of the level found in unirradiated cells.In these cells with irradiated nucleoli, nucleoplasmic labelling was just below 50% ofthat found in unirradiated cells. Irradiation of the whole nucleus reduced cytoplasmic

Table 5. Nucleoplasmic and cytoplasmic grain counts in HeLa cells in which either thewhole nucleus or the nucleolus has been irradiated

Unirradiated cellsNucleolus irradiatedWhole nucleus irradiatedNucleoplasm irradiated

No.cells

37504324

Total no.

Nucleoplasm

20161284207888

grains

Cytoplasm

1885347217806

Meannucleoplasmic

counts

54-52574-8

370

Meancytoplasmic

counts

69

33-6

Table 6. Effect of irradiation of the whole nucleus and of the nucleolusalone on cytoplasmic labelling

Expt no.

100

102

104

1 OS

Mean

Unirradiatedcells

69643432

cytoplasmic

Nucleolusirradiated

107

5-86256

counts

Whole nucleusirradiated

9-3

47

labelling to about 10% of the level found in unirradiated cells. It is thus clear that irra-diation of the nucleolus alone is just about as effective as irradiation of the whole nucleusin reducing cytoplasmic labelling, despite the fact that irradiation of the wholenucleus subjects the cell to a much higher dose of ultraviolet light. In the absenceof a functional nucleolus, the RNA synthesized in the rest of the nucleus cannot betransported, in detectable amounts, to the cytoplasm of the cell. Figure 4, whichcompares the cytoplasmic grain counts in unirradiated cells and cells in which thenucleolus has been irradiated, makes it clear that the nucleoplasm, after irradiation ofthe nucleolus, makes little or no contribution to cytoplasmic labelling. That this is nota non-specific effect of irradiation is shown in Table 5. Irradiation of the non-nucleolarparts of the nucleus with the same microbeam does not eliminate cytoplasmic labelling.Figure 10 shows an auto radiograph of an unirradiated HeLa cell, a cell with thenucleolus only irradiated and a cell with the whole nucleus irradiated.

Role of the nucleolus 359

100

90

80

70

Ea 60O-o

_«)

S 50

z

40

30

20

10I

10 20 60 70 80 9030 40 50Cytoplasm

Fig. 4. Comparison of the number of grains over the cytoplasm with the number ofgrains over the non-nucleolar parts of the nucleus (nucleoplasm) in unirradiated HeLacells and in HeLa cells in which the nucleolus has been irradiated. • , unirradiatedcells; O, cells in which the nucleolus only was irradiated.

Table 7. Effect of irradiation of the nucleolus on cytoplasmic labelling in HeLa cells:a comparison of three different studies

Authors

Takeda et al.Perry et al.Sidebottom & Harris

Percentage inhibition

Nucleolus

8085-90S1 95

of labelling

Cytoplasm

4367

> 90

DISCUSSIONThere have been two previous investigations of the effects of irradiation of the

nucleolus on the pattern of labelling of RNA in the cell. Perry, Hell & Errera (1961)concluded that the nucleolus was involved in the transport of about two-thirds of theRNA passing from nucleus to cytoplasm. The results of Takeda, Naruse & Yatani(1967) yield a figure of about two-fifths for this fraction. However, a comparison of theresults of these two studies with those obtained in the present experiments (Table 7)reveals that neither Perry et al. (1961) nor Takeda et al. (1967) inactivated the nucleoliin their preparations at all completely; and neither group irradiated whole nuclei as a

360 E. Sidebottom and H. Harris

control. It is clear from Table 7 that, in the present experiments, nucleolar activity wassuppressed much more effectively and, as a consequence, cytoplasmic RNA labellingwas much more drastically reduced.

The most important conclusion to be drawn from the present experiments is thatthe nucleolus is involved in the transfer, not only of the RNA made at the nucleolarsite, but also of the RNA made elsewhere in the nucleus. Experimental error does not,of course, permit the statement that all the RNA made in the nucleus is dependent onnucleolar activity for its transport to the cytoplasm; but it does appear that the greatbulk of the RNA made on the chromosomes, as well as that made in the nucleolus,fails to be transferred to the cytoplasm when the nucleolus is inactivated. It thereforeseems very improbable that the RNA made in the nucleolus and that made elsewherein the nucleus can be transported to the cytoplasm independently. All the resultsobtained in the present study indicate that the transport of both these classes of RNA isco-ordinated by some single process which is located at or near the nucleolus. Previousexperiments on the synthesis of specific proteins determined by the erythrocytenucleus in the heterokaryon (Harris et al. 1969; Harris & Cook, 1969) indicate that thetransport of the RNA which carries the information for protein synthesis is similarlydependent on the activity of the nucleolus. This suggests that the RNA which carriesthe information for protein synthesis and any 'structural' RNA components whichmay be made in the nucleolus leave the nucleus together.

REFERENCES

BOLUND, L., RINGERTZ, N. R. & HARRIS, H. (1969). Changes in the cytochemical properties oferythrocyte nuclei reactivated by cell fusion. J. Cell Sci. 4, 71-87.

DENDY, P. P. & SMITH, C. L. (1964). Effects of DNA synthesis of localized irradiation of cellsin tissue culture by (i) a u.v. microbeam and (ii) an a particle microbeam. Proc. R. Soc. B160, 328-344.

HARRIS, H. (1965). Behaviour of differentiated nuclei in heterokaryons of animal cells fromdifferent species. Nature, Lond, 206, 583-588.

HARRIS, H. (1967). The reactivation of the red cell nucleus, J. Cell Sci. 2, 23-32.HARRIS, H. & COOK, P. R. (1969). Synthesis of an enzyme determined by an erythrocyte

nucleus in a hybrid cell. J. Cell Sci. 5, 121-133.HARRIS, H., SIDEBOTTOM, E., GRACE, D. M. & BRAMWELL, M. E. (1969). The expression of

genetic information. A study with hybrid animal cells. J. Cell Sci. 4, 499—525.HARRIS, H., WATKINS, J. F., FORD, C. E. & SCHOEFL, G. I. (1966). Artificial heterokaryons of

animal cells from different species. J. Cell Sci. 1, 1—30.HARRIS, H. & WATTS, J. W. (1962). The relationship between nuclear and cytoplasmic ribo-

nucleic acid. Proc. R. Soc. B 156, 109-121.MUNRO, T. R. (1963). An improved chamber for micromanipulation work with cell cultures, and

a chamber that facilitates comparison of living and stained cells. Expl Cell Res. 32, 408-410.PERRY, R. P., HELL, A. & ERRERA, M. (1961). The role of the nucleolus in ribonucleic acid and

protein synthesis. 1. Incorporation of cytidine into normal and nucleolar inactivated HeLacells. Biochim. biophys. Acta 49, 47-58.

TAKEDA, S., NARUSE, S. & YATANI, R. (1967). Effects of ultraviolet microbeam irradiation ofvarious sites in HeLa cells, on the synthesis of RNA, DNA, and protein. Nature, Lond. 213,696-697.

URETZ, R. B., BLOOM, W. & ZIRKLE, R. E. (1954). Irradiation of parts of individual cells. II.Effects of an ultraviolet microbeam focused on parts of chromosomes. Science, N. Y. 120,197-199.

(Received 1 February 1969)

Role of the nucleolus 361

Fig. 5. The ultraviolet microbeam apparatus.Fig. 6. The culture chamber.

362 E. Sidebottom and H. Harris

Fig. 7. Autoradiograph of a heterokaryon exposed for 6 h to tritiated uridine. Boththe A 9 and the erythrocyte nucleus are very heavily labelled and there is also sub-stantial cytoplasmic labelling, x 2000.Fig. 8. Autoradiograph of a 3-day-old heterokaryon from the same preparation as thecell shown in Fig. 7. The A9 nucleus has been irradiated. The erythrocyte nucleus,which has not yet developed a nucleolus, is heavily labelled, but the cytoplasm containsvery little radioactivity, x 2000.Fig. 9. Autoradiograph of a io-day-old heterokaryon in which the A 9 nucleus has beenirradiated. The erythrocyte nucleus has developed a nucleolus and the cytoplasm isnow labelled, x 2000.

Role of tlie nucleolus 363

364 E. Sidebottom and H. Harris

'<•*

10Fig. 10. Autoradiograph showing a normal HeLa cell (top left), a HeLa cell in whichthe whole nucleus has been irradiated (top right), and a HeLa cell in which the nucleo-lus only has been irradiated (bottom). The preparation was exposed to tritiated uridinefor 6 h. Irradiation of the nucleolus alone, despite continued synthesis of RNA else-where in the nucleus, reduces cytoplasmic labelling to a level comparable with thatseen in the cell in which the whole nucleus has been irradiated, x 2600.