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Cyclic nucleolar changes during the cell cycle
Q 1968 by Academic Experimenlal
Cell
Press Inc. Research
50, 599-615
CYCLIC
I.
VARIATIONS
NUCLEOLAR CHANGES THE CELL CYCLE1
IN
SISTER Department
of Biology,
599
(1968)
NUMBER,
PAULA
The Catholic
SIZE,
GONZALEZ2 University Received
MORPHOLOGY
and
AND
POSITION
R. M. NARDONE
of America, July
DURING
Washington,
D.C.
20017,
USA
26, 19663
the past decade many investigations have been made to characterize the events of the cell cycle. Through the use of autoradiography and various cytochemical techniques it has been found in many cell types that deoxyribonucleic acid (DNA) synthesis occurs only during a portion of the cell cycle [20, 28, 531. Ribonucleic acid (RNA) has been found to be synthesized continuously except for the brief period from late prophase to mid-telophase [5, 11, 441. There seem to be marked differences in rates of RNA synthesis among individual logarithmic-phase cells, as determined by grain counts of autoradiograms made following brief pulse-labeling with tritiated precursors [12, 14, 26, 56j. A threefold increase between the lowest and highest RNA synthesis rates over the cell cycle has been reported in synchronously dividing populations of HeLa cells [56]. These showed an almost constant rate of RNA synthesis during the four to five hour period immediately after mitosis, followed by a linear increase until the next mitosis. Curves included data from only two experiments, and though one curve was nearly linear, the other was suggestive of possible cyclic changes in rate of RNA synthesis and distribution of RNA. In recent studies a distinct drop in RNA synthesis during the S phase has been reported in synchronously dividing mammalian cells [26, 271 and in slime mold [38]. It has been clearly established that the nucleolus plays a key role in the synthesis and distribution of RNA (see reviews: [4, 16, 431. A striking feature of a typical monolayer of Strain L cells is the great variety we have noted in morphology, position, number and size of nucleoli, DURING
1 This work was supported in part by a USPHS from the Department of the Army. 2 Present address: College of Mount St. Joseph, 3 Revised version received July 25, 1967.
Fellowship Mount
and Contract
St. Joseph,
Ohio
Experimental
DA-49-193-MD-2127 45051,
USA.
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Sister Paula Gonzblez and R. n/I. Nardone
as observed by a number of methods. Staining patterns of nucleoli and cytoplasm vary considerably both in quality and intensity. Careful study of autoradiograms made after pulse-labeling of logarithmic phase cultures with 3H-uridine also indicates a great range of metabolic activity, particularly with respect to synthesis of RNA. Cinemicrographic studies reveal much nucleolar movement, along with the nuclear rotation and cellular migration reported by many investigators [29, 37, 401. Such observations suggested that if these differences are reflections of a cyclic pattern of nucleolar behavior, the nucleolus might serve as a useful indicator of various stages of interphase. In order to determine if nucleolar changes are of a cyclic nature the use of synchronized populations of cells was necessary. A method was developed for obtaining routinely an adequate number of strain L cells in which a high degree of division synchrony was evident. These cells were observed throughout interphase and the first division, using a combination of techniques, in an attempt to characterize nucleolar events which might serve as an index of the stage of a cell in interphase. MATERIALS
AND
METHODS
Culture procedure.-Ceils used were derived from a culture of Strain L-929 cells [47], kindly provided by Dr David Axelrod. Stock cultures were grown as monolayers in prescription bottles containing Eagle’s Minimum Essential Medium (MEM) [9] in Hanks’ balanced salt solution supplemented with fetal bovine serum at a level of 5 per cent. Fifty units each of penicillin and streptomycin/ml were added (Microbiological Assoc., Bethesda, Md). Synchronously dividing cells were harvested and planted in modified SykesMoore chambers (Bellco Glass, Inc., Vineland, N. J.) and/or on coverslips in Leighton tubes for autoradiographic and cytochemical determinations. Preparation of cells for planting included trypsinizing, resuspendingand counting them with an electronic counter (Coulter Electronics, Hialeah, Fla). Photographic procedure.-Time-lapse motion pictures were made of cells grown on No. 1 l/2 coverslips in Sykes-Moore chambers which were modified by using a flat silicone gasket (Emdeco; Houston, Tex.). Cinemicrography was carried out at 37 f0.5”C, usually at the rate of I frame/min, for periods up to 72 h; during this period growth paralleled that of Leighton tube cultures. Kodak 16 mm reversal film was used for motion pictures; analysis of these films was done on a Project0 movie editor, equipped with a frame counter (Craig). All other photomicrographs were made using a Zeissresearch microscope equipped both with bright-field and phase-contrast optics( xl00 oil immersion objectives, apochromatic and neofluar, respectively). Kodak Ektachrome B and Kodak Plus-X 35 mm films were utilized. Autoradiographic procedure.-For determination of DNA synthesis cells were Experimental
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grown for 30 min in medium which contained 0.25 ,&/ml of SH-thymidine; specific activity, 1.9 C/m&f (Schwarz BioResearch). Coverslips were rinsed with serum-free MEM, and after three saline rinses the cells were fixed for 5 min in methanol. After a quick rinse in distilled water the coverslips were air-dried and mounted, cells up, with Permount. The slides were dried thoroughly and dipped in Kodak Nuclear Track Emulsion, Type NTB-2, diluted 1:l with 0.05 per cent sodium lauryl sulfate. After 48 h of exposure in the dark at 4°C they were developed 2 l/2 min in Kodak D-19, washed 10 to 15 min in running tap water, and stained through the emulsion with Giemsa stain. The percentage of labeled cells at each 2 h interval was determined by counting approximately 1000 cells for each period. Staining procedure.-5 min staining in stock Giemsa stain, diluted 1: 50 with glass distilled water (pH 6.8-7.0) was used for all routine preparations. After rapid dehydration through acetone to xylol, coverslips were mounted with Permount. Cytological observations.-The major and minor axes of nucleoli were measuredby using an ocular micrometer; becauseof the great changesin thickness of the nucleoli during the cycle, it was decided that the absolute diameter and/or area were really not indicative of changesin overall size. Calculation of approximate areasand volumes was not done becausesuch figures could be very misleading, due to the great changes in thickness, density and morphology, as noted in motion pictures. Therefore, in order to assessat least gross differences between Gl, S and G2 cells, nucleoli were grouped into size classeson the basis of the area of rectangles formed by the multiplication of the axes, rather than on absolute area. OBSERVATIONS Attainment
AND RESULTS
of synchrony
Populations of cells in which 85 to 95 per cent completed division within 1 to 2 h were obtained by modification of a selective harvesting technique [56]. Cells tend to round up prior to mitosis, and many are detached from the glass surface and float free in the medium. By agitating the cultures gently about 1 per cent of the cells could be collected. Many cells which were obviously in division remained attached to the glass surface, but efforts to remove a larger number by more vigorous agitation resulted in the dislodging of non-mitotic cells. Though many investigators [46, 48, 611 have obtained much larger numbers of synchronously dividing cells by alterations of medium or temperature and by various mechanical separation methods, it was decided that cells which were in a condition as physiologically normal as possible were absolutely necessary for characterization of stages in the life cycle. Experimental cultures were prepared by trypsinizing cultures which were in late logarithmic phase and seeding 1 x lo6 cells in 32-0~ prescription bottles, each containing 25 ml of medium. Forty-eight hours later medium was Experimental
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Sister Paula Gonzdlez and R. M. Nardone
Fig.
l.--Synchronous
cells 1 h after
planting.
replaced, and after 12 h the bottles were agitated gently about 100 times, using a rocking motion to “wash” the surface of the monolayer. Twelve hours later cells were again harvested by the same procedure. Cultures planted from the first harvest provided samples for study of cells from 12 to 24 h; the second harvest, 12 h later, provided samples for study of cells between 0 to 12 and 24 to 30 h. Overlapping samples (12 and 24 h) from the two plantings were found to be very similar, as determined by the percentage of cells labeled with tritiated thymidine. The distance between cells in the cultures from which the mitotic cells were harvested was very important, as the removal of mitotic cells was efficient only if they had few, if any cell-to-cell attachments at the time they began mitosis. Planting of approximately 1 x lo5 cells per square inch of culture vessel (32-0~ prescription bottle) surface was found to yield optimal harvests with respect to very close synchrony (Fig. 1). To plant Leighton tubes, three to four ml of the medium containing synchronized cells were used as an inoculum for Leighton tubes. This gave a total cell count of approximately 4 x 104/ml; 85 to 95 per cent of the cells were in some stage between late prophase and late telophase, and completed division within 1 to 2 h. The inoculum used for the seeding of SykesMoore chambers was obtained by gently centrifuging the cell suspension for one minute and then removing sufficient supernatant to obtain a concentraExperimental
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Cyclic nrrcleolar
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tion of approximately 7.5 x 104/ml. A 26-gauge needle fitted to a 2 ml syringe was used to deliver the cell suspension into the growth chambers; the final number of cells per unit surface area was comparable to that used in Leighton tube cultures. 3
I+..-@ 0
, 24
, 48
y\ 72
I 96
0
12
I 24
I
1 .rcr
411 36
Fig. Z.-Growth rate of synchronous cultures as determined bv visual counter. Abscissa: h; ordinate: cell number. -, Visual count, count, expt 91; w - - - w , Coulter count, expt 95; .-.-e, non-synchronous
0
12
I 24
I 36
observation and electronic expt 62; A. . . A, Coulter cultures.
Fig. 3.-Mitotic index determined by counting fixed cells at 2 h intervals in sublines of strain L cells with (a) a peak of mitosis at about 20 b; (b) a peak of mitosis at about 24 h. Abscissae: h; ordinates: % cells dividing. (a), A Expt 51, 0, 52 (chambers); 0, 62; A, 91 (tubes); (b) A, expt 94; 0, 95 (tubes).
Growth
rate
That these cells have a growth rate similar to non-synchronous log-phase cultures is clearly seen in Fig. 2. The initial slope is very steep, showing a doubling time between 23 and 24 h, an indication that there was a high degree of synchrony in the original inoculum. The slope of the growth curve then rather quickly parallels that of non-synchronous exponentially multiplying populations. Growth rate was measured by direct visual counting of cells in masked areas of Leighton tube cultures. A total of at least 100 cells, in 10 masked areas, was counted at the beginning of the period of observation. The growth rate as measured in this manner was very similar to that of Leighton tube cultures of synchronous cells which were trypsinized at intervals and counted with a Coulter counter. At least four samples were used for each point on the graph. The growth data suggest that the overall physiological condition of these synchronous cultures was comparable to that of nonsynchronous log-phase cultures. This was further emphasized by the fact that in several motion pictures that were analyzed 99 per cent of those cells which did not migrate out of the field, and which could be followed to the Experimental
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Sister Paula Gonzcilez and R. M. Kardone
next division, completed third generation. Generation
division
within
the normal
time range,
even at the
time
The synchrony decreased considerably within a very few hours of planting. This was due largely to the great differences in individual cell generation times, which have been reported as typical in mammalian cell systems [13, 21, SO]. In three motion pictures in which pairs of daughter cells could be followed through the second division, the average difference in generation time was 112 min, with a range from 26 min to 6 h. There was considerable difference in generation times even between daughter cells. For the 41 pairs of cells which could be followed the average generation time was 19.46 h, with a range from 14.80 to 24.70 h. The overlapping of the points for experiments carried out both in Leighton tubes and chambers indicated that there was probably little difference between the two systems, at least in division time (Fig. 3a). During the period when the motion picture experiments were being performed the cells being used showed a peak mitotic index at about 20 h (Fig. 3 a). Later experiments, using cells obtained from the same source, constantly showed longer generation times, with the peak of mitosis at approx. 23 h (Fig. 3b). In all experiments represented by Fig. 3 the cells were sampled at 2-h intervals, fixed, and stained with Giemsa stain. Because too many of the mitotic cells were floating in the medium and they would have been lost in the pre-fixation rinses, actual dividing figures were not counted for determination of mitotic index. Instead, cells which were spread fully on the glass but not yet separated were counted. This configuration, designated “cytoplasmic bridge stage” (CB), which occurs about 1 h after planting, was easy to recognize (see Fig. 1). Because complete separation of the daughter cells usually occurred within 1 h, and since most counts were done at intervals of 2 h, there was little danger of recounting them. In Fig. 4 the curve obtained by counting mitotic figures is compared with that obtained by counting cells in CB stage. It can be seen that the pattern of the curves was essentially the same, but showed a higher mitotic index for the spread cells; this was much more in line with the motion picture observations than the curve from mitotic figures. Establishment of Gl, S and G2 periods In order to\ establish the duration of the pre-DNA synthesis period the DNA-synthesis period (S), and the post-DNA-synthesis period duplicate cultures of synchronously dividing cells were incubated Esperimenfal
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(Gl), (G2), with
Cyclic nucleolur
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tritiated thymidine (3HTdR) at 2-h intervals. Fig. 5 shows the percentage of cells in S phase at various times after harvesting, as well as the mitotic index The average number of dividing cells in of the ensuing burst of division. non-synchronous cultures under these conditions was always about 2 per cent. In synchronous cultures virtually no dividing cells were seen for 18 h,
0
Fig. 4.-Comparison 1 h intervals. 0 -
of mitotic index 0, Mitotic figures;
!J
lb
15
determined by counting mitotic figures n--n, “CB” cells. Abscissa: h; ordinafe:
i0
25
30
and “CB” cells at % cells dividing.
Fig. 5.-Pattern of DNA synthesis in synchronously dividing cultures of strain L cells. The mitotic index of the first synchronous burst of division is shown. The triangles and circles refer to two separate experiments. -, Uptake of 3HTdR; - - - -, mitotic index. Abscissa: h; ordinale: (left) “6 cells labeled $HTdR; (right) 5: dividing cells.
at which time a rather sharp rise in percentage of dividing cells became apparent. By the end of 28 h most of the cells had completed the cell cycle. Very little incorporation of radioactive thymidine occurred before the fifth hour after planting; in many experiments this rise did not begin until about 7 or 8 h, and the rise of the curve was steeper. From about 9 to 16 h after planting, over 90 per cent of the cells were always labeled; then the curve dropped less steeply, due to the asynchrony which had developed by this time. Though there was considerable asynchrony, due largely to generation time differences among individual cells, the system was found suitable for use in characterizing cells in the three interphase periods of the cell cycle. For all nucleolar studies, cells at 3, 14 and 19 h after planting were chosen to represent Gl, S and G2 periods, respectively, and they are referred to in this manner throughout this paper. Observations
on cellulnr
morphology
and mouement
Motion picture analysis provided information on gross changes in cellular and nucleolar shape and movement during interphase. When planted, most cells were in telophase, though cells in metaphase were also present. Frameby-frame analysis of several cells indicated that the time from a recognizable Experimental
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Sister Paula Gonzcilez and R. M. Nardone
metaphase to the beginning of constriction was only about 6 min. Il’ithin about 3 min the chromosomes had reached the poles and soon formed an amorphous mass. Recognizable nucleoli were seen within 10 to 20 min. Nucleoli were probably evident much sooner, judging from the stained preparations; however, this was difficult to determine in the live cells because their spherical shape made observations on the nucleoli very difficult. The doublets remained rounded until the division furrow was almost complete and then they spread out on the glass very rapidly, showing a great movement of vacuoles into the cytoplasm and often exhibiting large undulating membranes. The vacuoles were quite large, and their entrance into the cell appeared to be associated with very rapid movement of the cptoplasmic projections within which the vacuoles first appeared. From the projections or undulating membranes the vacuoles moved through the cytoplasm tobvard the nucleus and soon disappeared. During the first few hours after planting, the cells maintained a somelvhat stellate appearance, with several cytoplasmic processes furnishing attachment to the glass. By about 8 to 10 h many of the cells were spindle-shaped, usually having only two main cytoplasmic processes. They she\\-ed little migratory activity, though wave-like movement back and forth along the length of the cell was observed. This more quiescent period coincided with that of DNA synthesis. The onset of prophase was recognizable by a return to the more stellate configuration, which was characterized by a great deal deal of movement, followed by a rounding up which occurred rather suddenly and was accomplished by the rapid retraction of the cytoplasmic processes which had characterized the early prophase condition. Movement of the cellular inclusions in the perinuclear region of the cytoplasm was particularly marked, especially in the area of a nuclear imagination \vhich was often seen in cells during this period. Observations
on nucleolar
morphology,
position,
Analysis of nucleolar changes from motion of the poor focus often occasioned by cellular
size and number pictures was difficult because and nucleolar movement and
Fig. 6.-Strain L cells at various times after planting of synchronous cultures. (a) 30 min; Most of the forming nucleoli can be clearly seen to be in direct or indirect contact with the nuclear membrane ( x 1500); (b) 3 h: The irregular morphology of nucleoli and their association with the nuclear membrane are evident ( x 1250); (c) 6 h: Most nucleoli are nearly oval and centrally located. The nucleolus-associated chromatin and the chromocenters appear quite reddishpurple ( x 1250); (d) 14 h: The density of the nucleoli is striking, and the deep blue-black color contrasts strongly with the reddish-purple nucleoplasm which can be observed at this time ( x 1250); (e) 19 h: In G2 cells nucleoli and chromocenters exhibit a diffuse appearance, as though they were losing material at the periphery ( x 1250); (I) Early prophase: The diminished size of the diffuse, irregular-shaped nucleoli is apparent ( x 1250). Experimental
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lack of flatness. In order to obtain a clearer picture of these changes experiments were performed in which synchronous cultures were fixed and stained with neutral Giemsa stain. It was thus possible to study the characteristic changes in nucleolar features which had been suggested by observation of motion picture films and to note the variations in nucleoprotein distribution using Giemsa stain. Using dilute Giemsa stain, Jacobson and Webb [24, 251 found that the structures in the interphase nucleus known to contain deoxyribonucleoprotein (DNP) stained reddish-purple; the ribonucleoprotein-rich (RNP) nucleoli and cytoplasm stained blue. In mitotic cells prophase and telophase chromosomes looked reddish-purple, but metaphase and anaphase chromosomes had a dark, blue-black appearance. That this material surrounding metaphase and anaphase chromosomes is RNP has been established by these two workers using Giemsa stain with selective enzymatic digestion to differentiate the nucleoproteins. In the present study of Giemsa-stained Strain L cells nucleoprotein distribution similar to that reported [24, 251 was obtained. Since the accuracy of azure-eosinate stains has been questioned we have studied the same sequence of events using toluidine blue-molybdate staining to differentiate the nucleoproteins; results of these cytochemical studies will be reported later. Using this staining method Love and his associates [31, 33, 341 reported RNP distribution very similar to that seen in Giemsa-stained preparations. Nine types of RNP have been said to be demonstrable by this technique [33]. The advantage of this method is that it includes use of a formaldehydecontaining fixative which inactivates protein-bound amino groups. This eliminates staining of surrounding protein such as occurs with azure-eosinate stains; used in conjunction with DNase digestion it provides excellent differentiation of the nucleoproteins [32]. In the present investigation subtle differences in staining qualities were noted among cells in non-synchronous cultures. That these variations might be related to cyclic differences in nucleic acid distribution during interphase is suggested by Giemsa staining of synchronous cultures. From the time they could be recognized most nucleoli exhibited irregular morphology, many having pointed projections which gave them a somewhat stellate appearance (Fig. 6a and b); these projections were nearly always seen to be in contact with the nuclear membrane. As interphase proceeded, the nucleoli became quite discrete, and the various other RNP-containing sites diminished in staining intensity. Ry 6 h after planting most nucleoli had assumed a fairly oval shape; use of very careful focusing suggested that few, if any, were in contact with the nuclear membrane (Fig. SC). Similar conclusions resulted Experimental
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from careful study of living cells during motion picture studies. This was similar for S-phase cells at 14 h (Fig. 6d). The nucleolus-associated chromatin stained quite reddish-purple in the late Gl cells; but it was not visible in S-phase cells. Both the deep blue-black color of the nucleoli and of the chromocenters during S-phase and the increase in size of these structures were
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40-
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20 -
20GIS Gz
GIS Gz
GGGz
GtSGz
Fig. 7.-Changes in nucleolar number ordinate: y0 of cells. (a) Gl, 3 h, aver. Fig. 8.-Changes units): 1, O-15; cells.
e
f
GiSGz
1 GIS GI
in Gl, S and G2 cells. Abscissa: Number of nucleoli per cell; 5.66; (b) S, 14 h, aver. 4.02; (c) G2, 19 h, aver. 3.71.
in nucleolar size in Gl (3 h); S (14 h) and G2 (19 h). Size classes (in arbitrary 2, 16-30; 3, 31-45; 4, 46-60; 5, 61-75; 6, > 75. Abscissa: Size class; ordinate: % of
Fig. 9.-Changes in nucleolar shape (left) and nucleolar G2 (19 h) cells. (a) Irregular; (b) oval; (c)round; (d) nuclear Stage of cycle; ordinates: y0 of cells.
position membrane;
(right) in Gl (3 h); S (14 h); (e) edge; (f) center. Abscissae:
noteworthy. Whether these changes in color and size were due to the great density of the S-phase nucleoli and chromocenters which may obscure the heterochromatin, or reflect a real change in position or chemical nature of this material was not clear. By G2 (19 h after planting), the asynchrony was considerable, but this made it possible to attempt to reconstruct the sequence of pre-prophase events, for a considerable range of pre-division cells was present in every field studied. In Fig. 6e is shown a “typical” G2 cell, with large, irregular nucleoli, many in contact with the nuclear membrane. The density of nucleolar staining was much less intense, especially around the edges, which appeared quite diffuse. This was in strong contrast with the condition of Sphase nucleoli, which were intensely stained throughout. This same apExperimental
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Sister Paula Gonzcilez and R. M. Nardone
pearance also characterized the chromocenters, many of which were now closely associated with the nuclear membrane. By the time prophase was well along, the nucleoli presented a highly diffuse, rather “empty” appearance among the thickening chromosomes; they were also considerably smaller than they had been just prior to the onset of prophase (Fig. 6f). Study of the cells stained by this method revealed that the average number of nucleoli in cells at the three interphase periods varied considerably (Fig. 7). It was interesting to note that by G2 there were scarcely any cells with more than five nucleoli, though in Gl the range extended up to ten nucleoli per cell and the average number was 5.66. Correlated with this change in number was a shift in the size pattern (Fig. 8). The drop in number of very small nucleoli as the cell passed from Gl to S to G2 was due largely to incorporation of these smaller nucleoli into the larger ones by fusion. Had the shift been due only to growth a shift of the percentage of Gl cells to an intermediate size class would be expected; such a peak did not appear, either in S or G2. Nucleolar fusion was noted in several motion picture sequences and has been reported by many investigators [45, 551. The decrease in nucleolar number due to fusion apparently occurred throughout the cycle, though it was probably most rapid in early interphase. The areas of the histograms in Fig. 8 were determined, and the results suggested that most of the increase in size due to growth must have occurred after 14 h. The total areas (in arbitrary units), for nucleoli of the 50 cells representing each stage were: Gl (3 h), 295; S (14 h), 280; and G2 (19 h), 410. Two other very interesting aspects of nucleolar change have been noted above. Changes in shape and nucleolar position were seen to occur in a cyclic manner also. Because of the importance of these phenomena in helping to clarify the mode of formation and possible function of the nucleolus, an attempt was made to quantify the observations (Fig. 9). The proportion of nucleoli of irregular shape decreased as the cells moved from Gl to S, and then increased greatly in G2. Oval-shaped nucleoli, on the other hand, were very prevalent in S-phase cells. The number of round nucleoli (these were usually very small), did not change much until G2, where the decrease in number was probably due to their fusion with larger nucleoli. Changes in position paralleled changes in shape, as can be seen clearly in Fig. 9 b. Irregularly-shaped nucleoli were often associated with the nuclear membrane or were, at least, peripherally located within the nucleus. Analyses of motion pictures had shown that the irregular morphology of the nucleoli was at least partly due to movement of the nucleoli themselves; this did not necessarily appear to be correlated with nuclear rotation. Experimental
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DISCUSSION
The observations reported here have shown that there is a definite cyclic pattern to the morphological changes seen in nucleoli during the cell cycle. Changes in number appear to be due largely to nucleolar fusion; size changes are due both to nucleolar growth and fusion, the latter entailing nucleolar movement. A marked increase in the percentage of large nucleoli occurred late in the cycle; this was apparently the result of actual nucleolar growth, as most of the fusion of smaller nucleoli had occurred by the end of 14 h. Swift [55] reported a similar drop in nucleolar number after early interphase and attributed it to fusion. An increased rate of RNA synthesis late in the cycle has been reported [7, 26, 56, 581, and preliminary autoradiographic experiments by the present authors strongly suggest that a pre-prophase spurt of RNA synthesis occurs in strain L cells. Movement of nucleoli was very evident immediately after their formation in telophase, and was largely responsible both for their irregular morphology and for the fusion seen, much of which was observed early in the Gl period, especially in motion picture studies. In Giemsa-stained preparations just after telophase the characteristic blue RNP color was arranged in irregular masses along the newly formed nuclear membrane. This arrangement has been observed by many investigators [13, 24, 25, 52, 551. It is this RNP material which appears to be organized into the discrete bodies which are usually thought of as characteristic interphase nucleoli. Similar observations were made using toluidine blue-molybdate staining, as will be reported later. In pre-prophase cells, late in G2, the reverse of this process was observed, with RNP material appearing to move from the nucleoli toward the nuclear membrane, presumably along some type of structural component. The fact that irregular morphology, diffuse appearance, active movement and close association of nucleoli with the nuclear membrane are always associated with very early and very late interphase argues for a close interdependence of these occurrences. Several studies which described a correlation between diffuse, irregularly-shaped nucleoli and the nuclear membrane have suggested that these changes reflect varying stages in the preparation of the cell for division [lo, 13, 22, 45, 51, 571. The changes in cellular and nucleolar morphology also suggest that nucleocytoplasmic exchange during the Gl and G2 periods is more active than in the S period. In motion pictures perinuclear and nucleolar activity seemed especially marked during the first two or three hours of interphase and just prior to prophase. The percentage of nucleoli which exhibited 39
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Sister Paula Gonzcilez and R. M. Nardone
attachments to the nuclear membrane was very high at both of these times; a recent study of nucleolar positions in HeLa cells indicated that only about 11 per cent of nucleoli were in direct or indirect contact with the nuclear membrane in non-synchronous cultures [2 j. The fact that nucleoli of cells in early Gl and late G2 exhibited considerable similarity of shape, position, movement, staining characteristics and association with the nuclear membrane suggested that there may be a very close relationship between formation and disappearance of nucleoli and that of the nuclear membrane. It seems probable that the “organization” of nucleoli may be accomplished through cyclic changes undergone by the nucleoli, the nuclear membrane, and what might be called the “heterochromatic which comprises the nucleolus-associated chromatin and intrasystem”, nucleolar chromatin. Recently Woolam et al. suggested that observed cytological phenomena of both meiosis and mitosis might be explained on the basis of three processes only: (a) replication of DNA; (b) the degree of coiling and uncoiling of the chromosomal elements, and (c) sol-gel relationships [59]. If the present results are viewed in the light of recent electron microscope studies it seems feasible to consider the possibility of accounting for the “appearance” and “disappearance” of nucleoli as a cyclic process of condensation and decondensation of nucleoproteins similar to that which accounts for these phenomena in chromosomes. Nucleoli may perhaps be formed by the interaction of several chromosomes [ 19, 49, 601. Many accounts of a meshwork joining heterochromatin and nucleoli have been reported. Fell and Hughes [13] observed that the surfaces of developing nucleoli were studded with dark heterochromatic granules, from each of which line filaments were seen to emerge in such a way as to “knit the nucleolar components together by an intricate mesh of threads”. They noted that other heterochromatic granules adhered to the nuclear membrane, a phenomenon always observed in the strain L cells used in this study. Irregularly-shaped lumps of heterochromatin which continue into thinner threads of chromatin, presumably the chromonemata, have been observed [S, 13, 30, 41, 431. Reitalu also noted that by destaining gradually after the Feulgen technique, Feulgen-positive threads within the nucleolus could be seen [45!. Possible involvement of this interphase chromatinic network in nucleolar formation and movement is suggested by several electron microscope studies [22, 23, 36, 601. Observation of an interphase RNP network with strands radiating from the nucleoli has been made by Smetana and his associates [Sl]. Recently intraExperimental
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Cyclic nucleolar
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613
nucleolar chromatin Iibrils have been shown to be in close relationship to this network, as well as being attached to the nucleolus-associated chromatin [41, 421. A number of electron microscope investigations have shown that intranucleolar chromatin and nucleolus-associated chromatin form a single system [17, 18, 22, 411. In such studies the nucleolus is seen as “an active genetic site on a chromosome “[22], an observation which gains considerable support from two recent findings: (1) that DNA replication in heterochromatin and euchromatin is asynchronous [3, 61; and (2) that DNA synthesis in nucleoli begins later than in chromatin, and occurs in two distinct phases
[261. Evidence is increasing that there is an intimate association between chromosomes and the nuclear membrane. A “synaptinemal complex” has been described in a variety of meiotic prophase chromosomes [lo, 39, 59]; in such studies the ends of all the chromosomes were found to be polarized and anchored at the nuclear membrane. Most significant of all was the fact that at the stage when chromosomal cores were present in the nucleus but distinct chromosomes had not yet formed, the nucleoli were found at the periphery of the nucleus and vacuoles appeared to be discharging their contents into the karyoplasm along the nuclear membrane. Many investigators have mentioned discharge of material from the nucleoli [l, 15, 30, 35, ,571; cyclic variations of the discharge of various granules in pre-prophase strain L cells will be reported later. That chromosomes of non-meiotic cells may also be attached to the nuclear membrane throughout interphase has been shown in Ehrlich ascites cells [BO] and in meristematic cells of Plantago ovata [22]. Further clarification of these intimate associations of chromatin, nucleoli and nuclear membrane may aid in explaining the cyclic nucleolar movements and morphological changes, if the pre-nucleolar materials are considered to be chromosomal products.
SUMMARY
1. Development of a technique for selective harvesting of synchronous strain L cells has made possible the study of nucleolar changes during the cell cycle. The pattern of DNA synthesis was determined autoradiographically, and cultures which had been planted for 3, 14 and 19 h were chosen as representative of the Gl, S and G2 periods, respectively. Changes in morphology, position, number and size of nucleoli were assessed, both cinematographically and using fixed preparations. 2. The significant reduction in nucleolar number between Gl and mitosis Experimental
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was found to be due largely to fusion. Increase in size resulted from both fusion and synthetic activity within the nucleolus. 3. Irregular morphology of nucleoli, marked association with the nuclear membrane and amoeboid movement were characteristic of nucleoli both in Gl and G2. During S phase nucleoli were oval-shaped and centrallylocated; they exhibited much less movement. 4. The possible significance of these cyclic nucleolar phenomena as related to other nuclear changes during the cell cycle has been discussed.
REFERENCES 1. ALTMANN, 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.
H. W., Z. Krebsforsch. 58, 632 (1952). ALVAREZ-LOUELI, B., Bol. Inst. Eslud. Med. Biol. 21, 15 (1963). BAER, D., Genetics 52, 275 (1965). BUSCH. H., BYVOET, P. and SMETANA, K., Cancer Res. 23. 313 (1963). CASPE&SO~, T. O., Cell Growth and Cell Function. W. W: No&on i Co., New York, 1950. CHURCH, K., Genetics 52, 843 (1965). CRIPPA, M., Exptl Cell Res. 42, 371 (1966). DAVIS, J. M. G., in J. S. MITCHELL (ed.), The Cell Nucleus. Butterworths, London, 1965. EAGLE, H., J. Biol. Chem. 214, 839 (1955). FAWCETT, D. W., J. Biophys. Biochem. Cytol. 2, 403 (1956). FEINENDEGEN, L. E. and BOND, V. P., Expptl Cell Res. 30, 393 (1963). FEINENDEGEN, L. E., BOND, V. P., SHREEVE, W. W. and PAINTER, R. B., Exptl Cell Res. 19, 443 (1960). FELL, H. B. and HUGHES, A. F., Quart. J. Microscop. Sot. 90, 355 (1949). GOLDSTEIN, L. and MICOU, J., J. Biophys. Biochem. Cytol. 6, 301 (1959). GONZALEZ-RAMIREZ, J., ZUEIGA, M. and NUREZ, B. A., Bol. Inst. Estud. Med. Biol. 17, 51 (1959). GRAHAM, A. F. and RAKE, A. V., Ann. Reu. Microbial. 17, 139 (1963). GRANBOULAN, N. and GRANBOULAN, P., Exptl Cell Res. 34, 71 (1964). ibid. 38, 604 (1965). HERICH, R.. Caryologia 16, 521 (1963). HOWARD, A. and PELC, S. F., Exptl Cell Res. 2, 178 (1951). Hsu, T. C., Tex. Repts BioI. Med. 18, 31 (1960). HYDE, B. B., J. UItrastruct. Res. 18, 25 (1967). JACOB, J., and SIRLIN, J. L., J. Cell Biol. 17, 153 (1963). JACOBSO?;, W. and WEBB, M., Endeauour 11, 200 (1952).
__
Exptl
Cell Res. 3, 163 (1952).
KASTEN, F. H. and STRASSER, F. F., Nature 211, 135 (1966). KIM, J. H. and PEREZ, A. G., Nature 207, 974 (1965). LAJTHA, L. G., OLIVER, R. and ELLIS, F., Brit. J. Cancer 8, 367 (1954). LEONE, V., Hsu, T. C. and POMERAT, C. M., Z. Zellforsch. 41, 481 (1955). LETTRE, R. and SIEBS, W., Chemotherapia 2, 223 (1961). LOVE, R. and LILES, R. H., J. Histochk Cytochem. 7, 164 (1959). LOVE, R. and RABOTTI, G., ibid. 11, 603 (1963). LOVE, R. and SUSKIND, R. G., Ann. Hisfochim. 6, 437 (1962). LOVE, R. and WALSH, R. J., J. Histochem. Cytochem. 11, 188 (1963). LUDFORD. R. J.. Proc. Rou. Sot. 98. 457 (1925). MARISOZ& V., >. Ulfrast~uet. Res. i0, 433 (1964). MCOUILKIN, W. T. and EARLE, W. R., J. Natl Cancer Inst. 28, 763 (1962). MIT~ERMAY&R, C., BRAUN, R. and R&H, H. P., Biochim. Biophys. ‘Acta MOSES, M. J., J. Biophys. Biochem. Cytol. 4, 633 (1958). NAKAI, J., Am. .7. Anal. 99, 81 (1956).
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91, 399 (1964)
Cyclic nucleolar 41. 42. 43. 44. 45. 46. 47. 48. 49.
50. 51. 52. 53. 54. 55. 56. 57. 58. 59.
60. 61.
changes
during
cell cycle. I
615
NARAYAN, K. S., MURAMATSU, M., SMETANA, K. and BUSCH, H., Exptl Cell Res. 41, 81 (1966). NARAYAN, K. S., STEELE, W. J., SMETANA, K. and BUSCH, H., Exptl Cell Res. 46, 65 (1967). PRESCOTT, D. M., Progr. Nucleic Acid Res. Molec. Biol. 3, 33 (1964). PRESCOTT, D. M. and BENDER, M. A., Exptl Cell Res. 26, 260 (1962). RF.ITALU,.J., Acta Pathol. Microbial. &and. 41, 257 (1957). ROBBINS, E. and MARCUS, P. I., Science 144, 1152 (1964). SANFORD, K. K., EARLE, W. R. and LIKELY, G. D., J. Nat1 Cancer Inst. 9, 229 (1948). SCHERBAUM. 0. H., Ann. Rev. Microbial. 14, 283 (1960). SIRLIN, J. L., Endkaoour 20, 146 (1961). ~ ’ SISKEN, J. E. and KINOSITA, R., Exptl Cell Res. 22, 521 (1961). SMET?LNA, K., STEELE, W. J. and BUSCH, H., Exptl Cell Res. 31, 198 (1963). SOSA, J. M., An. Fat. Med. (Montevideo) 30, 319 (1945). STANNERS, C. P. and TILL. J. E., Biochim. Biophys. Acta 37, 406 (1960). SWIFT, H., Intern. Reu. Cytol. 2, 1 (1953). -in R. E. ZIRKLE, (ed.), A symposium on Molecular Biology. Univ. of Chicago Press, Chicago, 1959. TERASIMA, T. and TOLMACH, L. J., Exptl Cell Res. 30, 344 (1963). WEISSENFELS, N., Z. Zellforsch. 62, 667 (1964). WOODARD, J., RASCH, E. and SWIFT, H., J. Biophus. Biochem. Cutol. 9, 445 (1961). WOOLLAM; D.. H. M., FORD, E. H. R. and MILLEN~J. W., Exptl Cell R&. 42,‘657 (1966). YASUZUMI, G. and SUGIHARA, R., Exptl Cell Res. 37, 207 (1965). ZEUTHEN,.E., Ado. Riol. Med. Phys. b, 37 (1958).
Experimental
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Press Inc. Research
50, 599-615
CYCLIC
I.
VARIATIONS
NUCLEOLAR CHANGES THE CELL CYCLE1
IN
SISTER Department
of Biology,
599
(1968)
NUMBER,
PAULA
The Catholic
SIZE,
GONZALEZ2 University Received
MORPHOLOGY
and
AND
POSITION
R. M. NARDONE
of America, July
DURING
Washington,
D.C.
20017,
USA
26, 19663
the past decade many investigations have been made to characterize the events of the cell cycle. Through the use of autoradiography and various cytochemical techniques it has been found in many cell types that deoxyribonucleic acid (DNA) synthesis occurs only during a portion of the cell cycle [20, 28, 531. Ribonucleic acid (RNA) has been found to be synthesized continuously except for the brief period from late prophase to mid-telophase [5, 11, 441. There seem to be marked differences in rates of RNA synthesis among individual logarithmic-phase cells, as determined by grain counts of autoradiograms made following brief pulse-labeling with tritiated precursors [12, 14, 26, 56j. A threefold increase between the lowest and highest RNA synthesis rates over the cell cycle has been reported in synchronously dividing populations of HeLa cells [56]. These showed an almost constant rate of RNA synthesis during the four to five hour period immediately after mitosis, followed by a linear increase until the next mitosis. Curves included data from only two experiments, and though one curve was nearly linear, the other was suggestive of possible cyclic changes in rate of RNA synthesis and distribution of RNA. In recent studies a distinct drop in RNA synthesis during the S phase has been reported in synchronously dividing mammalian cells [26, 271 and in slime mold [38]. It has been clearly established that the nucleolus plays a key role in the synthesis and distribution of RNA (see reviews: [4, 16, 431. A striking feature of a typical monolayer of Strain L cells is the great variety we have noted in morphology, position, number and size of nucleoli, DURING
1 This work was supported in part by a USPHS from the Department of the Army. 2 Present address: College of Mount St. Joseph, 3 Revised version received July 25, 1967.
Fellowship Mount
and Contract
St. Joseph,
Ohio
Experimental
DA-49-193-MD-2127 45051,
USA.
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Sister Paula Gonzblez and R. n/I. Nardone
as observed by a number of methods. Staining patterns of nucleoli and cytoplasm vary considerably both in quality and intensity. Careful study of autoradiograms made after pulse-labeling of logarithmic phase cultures with 3H-uridine also indicates a great range of metabolic activity, particularly with respect to synthesis of RNA. Cinemicrographic studies reveal much nucleolar movement, along with the nuclear rotation and cellular migration reported by many investigators [29, 37, 401. Such observations suggested that if these differences are reflections of a cyclic pattern of nucleolar behavior, the nucleolus might serve as a useful indicator of various stages of interphase. In order to determine if nucleolar changes are of a cyclic nature the use of synchronized populations of cells was necessary. A method was developed for obtaining routinely an adequate number of strain L cells in which a high degree of division synchrony was evident. These cells were observed throughout interphase and the first division, using a combination of techniques, in an attempt to characterize nucleolar events which might serve as an index of the stage of a cell in interphase. MATERIALS
AND
METHODS
Culture procedure.-Ceils used were derived from a culture of Strain L-929 cells [47], kindly provided by Dr David Axelrod. Stock cultures were grown as monolayers in prescription bottles containing Eagle’s Minimum Essential Medium (MEM) [9] in Hanks’ balanced salt solution supplemented with fetal bovine serum at a level of 5 per cent. Fifty units each of penicillin and streptomycin/ml were added (Microbiological Assoc., Bethesda, Md). Synchronously dividing cells were harvested and planted in modified SykesMoore chambers (Bellco Glass, Inc., Vineland, N. J.) and/or on coverslips in Leighton tubes for autoradiographic and cytochemical determinations. Preparation of cells for planting included trypsinizing, resuspendingand counting them with an electronic counter (Coulter Electronics, Hialeah, Fla). Photographic procedure.-Time-lapse motion pictures were made of cells grown on No. 1 l/2 coverslips in Sykes-Moore chambers which were modified by using a flat silicone gasket (Emdeco; Houston, Tex.). Cinemicrography was carried out at 37 f0.5”C, usually at the rate of I frame/min, for periods up to 72 h; during this period growth paralleled that of Leighton tube cultures. Kodak 16 mm reversal film was used for motion pictures; analysis of these films was done on a Project0 movie editor, equipped with a frame counter (Craig). All other photomicrographs were made using a Zeissresearch microscope equipped both with bright-field and phase-contrast optics( xl00 oil immersion objectives, apochromatic and neofluar, respectively). Kodak Ektachrome B and Kodak Plus-X 35 mm films were utilized. Autoradiographic procedure.-For determination of DNA synthesis cells were Experimental
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Cyclic nucleolar
changes
during
601
cell cycle. I
grown for 30 min in medium which contained 0.25 ,&/ml of SH-thymidine; specific activity, 1.9 C/m&f (Schwarz BioResearch). Coverslips were rinsed with serum-free MEM, and after three saline rinses the cells were fixed for 5 min in methanol. After a quick rinse in distilled water the coverslips were air-dried and mounted, cells up, with Permount. The slides were dried thoroughly and dipped in Kodak Nuclear Track Emulsion, Type NTB-2, diluted 1:l with 0.05 per cent sodium lauryl sulfate. After 48 h of exposure in the dark at 4°C they were developed 2 l/2 min in Kodak D-19, washed 10 to 15 min in running tap water, and stained through the emulsion with Giemsa stain. The percentage of labeled cells at each 2 h interval was determined by counting approximately 1000 cells for each period. Staining procedure.-5 min staining in stock Giemsa stain, diluted 1: 50 with glass distilled water (pH 6.8-7.0) was used for all routine preparations. After rapid dehydration through acetone to xylol, coverslips were mounted with Permount. Cytological observations.-The major and minor axes of nucleoli were measuredby using an ocular micrometer; becauseof the great changesin thickness of the nucleoli during the cycle, it was decided that the absolute diameter and/or area were really not indicative of changesin overall size. Calculation of approximate areasand volumes was not done becausesuch figures could be very misleading, due to the great changes in thickness, density and morphology, as noted in motion pictures. Therefore, in order to assessat least gross differences between Gl, S and G2 cells, nucleoli were grouped into size classeson the basis of the area of rectangles formed by the multiplication of the axes, rather than on absolute area. OBSERVATIONS Attainment
AND RESULTS
of synchrony
Populations of cells in which 85 to 95 per cent completed division within 1 to 2 h were obtained by modification of a selective harvesting technique [56]. Cells tend to round up prior to mitosis, and many are detached from the glass surface and float free in the medium. By agitating the cultures gently about 1 per cent of the cells could be collected. Many cells which were obviously in division remained attached to the glass surface, but efforts to remove a larger number by more vigorous agitation resulted in the dislodging of non-mitotic cells. Though many investigators [46, 48, 611 have obtained much larger numbers of synchronously dividing cells by alterations of medium or temperature and by various mechanical separation methods, it was decided that cells which were in a condition as physiologically normal as possible were absolutely necessary for characterization of stages in the life cycle. Experimental cultures were prepared by trypsinizing cultures which were in late logarithmic phase and seeding 1 x lo6 cells in 32-0~ prescription bottles, each containing 25 ml of medium. Forty-eight hours later medium was Experimental
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Sister Paula Gonzdlez and R. M. Nardone
Fig.
l.--Synchronous
cells 1 h after
planting.
replaced, and after 12 h the bottles were agitated gently about 100 times, using a rocking motion to “wash” the surface of the monolayer. Twelve hours later cells were again harvested by the same procedure. Cultures planted from the first harvest provided samples for study of cells from 12 to 24 h; the second harvest, 12 h later, provided samples for study of cells between 0 to 12 and 24 to 30 h. Overlapping samples (12 and 24 h) from the two plantings were found to be very similar, as determined by the percentage of cells labeled with tritiated thymidine. The distance between cells in the cultures from which the mitotic cells were harvested was very important, as the removal of mitotic cells was efficient only if they had few, if any cell-to-cell attachments at the time they began mitosis. Planting of approximately 1 x lo5 cells per square inch of culture vessel (32-0~ prescription bottle) surface was found to yield optimal harvests with respect to very close synchrony (Fig. 1). To plant Leighton tubes, three to four ml of the medium containing synchronized cells were used as an inoculum for Leighton tubes. This gave a total cell count of approximately 4 x 104/ml; 85 to 95 per cent of the cells were in some stage between late prophase and late telophase, and completed division within 1 to 2 h. The inoculum used for the seeding of SykesMoore chambers was obtained by gently centrifuging the cell suspension for one minute and then removing sufficient supernatant to obtain a concentraExperimental
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Cyclic nrrcleolar
changes
during
cell cycle. I
603
tion of approximately 7.5 x 104/ml. A 26-gauge needle fitted to a 2 ml syringe was used to deliver the cell suspension into the growth chambers; the final number of cells per unit surface area was comparable to that used in Leighton tube cultures. 3
I+..-@ 0
, 24
, 48
y\ 72
I 96
0
12
I 24
I
1 .rcr
411 36
Fig. Z.-Growth rate of synchronous cultures as determined bv visual counter. Abscissa: h; ordinate: cell number. -, Visual count, count, expt 91; w - - - w , Coulter count, expt 95; .-.-e, non-synchronous
0
12
I 24
I 36
observation and electronic expt 62; A. . . A, Coulter cultures.
Fig. 3.-Mitotic index determined by counting fixed cells at 2 h intervals in sublines of strain L cells with (a) a peak of mitosis at about 20 b; (b) a peak of mitosis at about 24 h. Abscissae: h; ordinates: % cells dividing. (a), A Expt 51, 0, 52 (chambers); 0, 62; A, 91 (tubes); (b) A, expt 94; 0, 95 (tubes).
Growth
rate
That these cells have a growth rate similar to non-synchronous log-phase cultures is clearly seen in Fig. 2. The initial slope is very steep, showing a doubling time between 23 and 24 h, an indication that there was a high degree of synchrony in the original inoculum. The slope of the growth curve then rather quickly parallels that of non-synchronous exponentially multiplying populations. Growth rate was measured by direct visual counting of cells in masked areas of Leighton tube cultures. A total of at least 100 cells, in 10 masked areas, was counted at the beginning of the period of observation. The growth rate as measured in this manner was very similar to that of Leighton tube cultures of synchronous cells which were trypsinized at intervals and counted with a Coulter counter. At least four samples were used for each point on the graph. The growth data suggest that the overall physiological condition of these synchronous cultures was comparable to that of nonsynchronous log-phase cultures. This was further emphasized by the fact that in several motion pictures that were analyzed 99 per cent of those cells which did not migrate out of the field, and which could be followed to the Experimental
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Sister Paula Gonzcilez and R. M. Kardone
next division, completed third generation. Generation
division
within
the normal
time range,
even at the
time
The synchrony decreased considerably within a very few hours of planting. This was due largely to the great differences in individual cell generation times, which have been reported as typical in mammalian cell systems [13, 21, SO]. In three motion pictures in which pairs of daughter cells could be followed through the second division, the average difference in generation time was 112 min, with a range from 26 min to 6 h. There was considerable difference in generation times even between daughter cells. For the 41 pairs of cells which could be followed the average generation time was 19.46 h, with a range from 14.80 to 24.70 h. The overlapping of the points for experiments carried out both in Leighton tubes and chambers indicated that there was probably little difference between the two systems, at least in division time (Fig. 3a). During the period when the motion picture experiments were being performed the cells being used showed a peak mitotic index at about 20 h (Fig. 3 a). Later experiments, using cells obtained from the same source, constantly showed longer generation times, with the peak of mitosis at approx. 23 h (Fig. 3b). In all experiments represented by Fig. 3 the cells were sampled at 2-h intervals, fixed, and stained with Giemsa stain. Because too many of the mitotic cells were floating in the medium and they would have been lost in the pre-fixation rinses, actual dividing figures were not counted for determination of mitotic index. Instead, cells which were spread fully on the glass but not yet separated were counted. This configuration, designated “cytoplasmic bridge stage” (CB), which occurs about 1 h after planting, was easy to recognize (see Fig. 1). Because complete separation of the daughter cells usually occurred within 1 h, and since most counts were done at intervals of 2 h, there was little danger of recounting them. In Fig. 4 the curve obtained by counting mitotic figures is compared with that obtained by counting cells in CB stage. It can be seen that the pattern of the curves was essentially the same, but showed a higher mitotic index for the spread cells; this was much more in line with the motion picture observations than the curve from mitotic figures. Establishment of Gl, S and G2 periods In order to\ establish the duration of the pre-DNA synthesis period the DNA-synthesis period (S), and the post-DNA-synthesis period duplicate cultures of synchronously dividing cells were incubated Esperimenfal
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(Gl), (G2), with
Cyclic nucleolur
chctnges during
605
cell cycle. I
tritiated thymidine (3HTdR) at 2-h intervals. Fig. 5 shows the percentage of cells in S phase at various times after harvesting, as well as the mitotic index The average number of dividing cells in of the ensuing burst of division. non-synchronous cultures under these conditions was always about 2 per cent. In synchronous cultures virtually no dividing cells were seen for 18 h,
0
Fig. 4.-Comparison 1 h intervals. 0 -
of mitotic index 0, Mitotic figures;
!J
lb
15
determined by counting mitotic figures n--n, “CB” cells. Abscissa: h; ordinafe:
i0
25
30
and “CB” cells at % cells dividing.
Fig. 5.-Pattern of DNA synthesis in synchronously dividing cultures of strain L cells. The mitotic index of the first synchronous burst of division is shown. The triangles and circles refer to two separate experiments. -, Uptake of 3HTdR; - - - -, mitotic index. Abscissa: h; ordinale: (left) “6 cells labeled $HTdR; (right) 5: dividing cells.
at which time a rather sharp rise in percentage of dividing cells became apparent. By the end of 28 h most of the cells had completed the cell cycle. Very little incorporation of radioactive thymidine occurred before the fifth hour after planting; in many experiments this rise did not begin until about 7 or 8 h, and the rise of the curve was steeper. From about 9 to 16 h after planting, over 90 per cent of the cells were always labeled; then the curve dropped less steeply, due to the asynchrony which had developed by this time. Though there was considerable asynchrony, due largely to generation time differences among individual cells, the system was found suitable for use in characterizing cells in the three interphase periods of the cell cycle. For all nucleolar studies, cells at 3, 14 and 19 h after planting were chosen to represent Gl, S and G2 periods, respectively, and they are referred to in this manner throughout this paper. Observations
on cellulnr
morphology
and mouement
Motion picture analysis provided information on gross changes in cellular and nucleolar shape and movement during interphase. When planted, most cells were in telophase, though cells in metaphase were also present. Frameby-frame analysis of several cells indicated that the time from a recognizable Experimental
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Sister Paula Gonzcilez and R. M. Nardone
metaphase to the beginning of constriction was only about 6 min. Il’ithin about 3 min the chromosomes had reached the poles and soon formed an amorphous mass. Recognizable nucleoli were seen within 10 to 20 min. Nucleoli were probably evident much sooner, judging from the stained preparations; however, this was difficult to determine in the live cells because their spherical shape made observations on the nucleoli very difficult. The doublets remained rounded until the division furrow was almost complete and then they spread out on the glass very rapidly, showing a great movement of vacuoles into the cytoplasm and often exhibiting large undulating membranes. The vacuoles were quite large, and their entrance into the cell appeared to be associated with very rapid movement of the cptoplasmic projections within which the vacuoles first appeared. From the projections or undulating membranes the vacuoles moved through the cytoplasm tobvard the nucleus and soon disappeared. During the first few hours after planting, the cells maintained a somelvhat stellate appearance, with several cytoplasmic processes furnishing attachment to the glass. By about 8 to 10 h many of the cells were spindle-shaped, usually having only two main cytoplasmic processes. They she\\-ed little migratory activity, though wave-like movement back and forth along the length of the cell was observed. This more quiescent period coincided with that of DNA synthesis. The onset of prophase was recognizable by a return to the more stellate configuration, which was characterized by a great deal deal of movement, followed by a rounding up which occurred rather suddenly and was accomplished by the rapid retraction of the cytoplasmic processes which had characterized the early prophase condition. Movement of the cellular inclusions in the perinuclear region of the cytoplasm was particularly marked, especially in the area of a nuclear imagination \vhich was often seen in cells during this period. Observations
on nucleolar
morphology,
position,
Analysis of nucleolar changes from motion of the poor focus often occasioned by cellular
size and number pictures was difficult because and nucleolar movement and
Fig. 6.-Strain L cells at various times after planting of synchronous cultures. (a) 30 min; Most of the forming nucleoli can be clearly seen to be in direct or indirect contact with the nuclear membrane ( x 1500); (b) 3 h: The irregular morphology of nucleoli and their association with the nuclear membrane are evident ( x 1250); (c) 6 h: Most nucleoli are nearly oval and centrally located. The nucleolus-associated chromatin and the chromocenters appear quite reddishpurple ( x 1250); (d) 14 h: The density of the nucleoli is striking, and the deep blue-black color contrasts strongly with the reddish-purple nucleoplasm which can be observed at this time ( x 1250); (e) 19 h: In G2 cells nucleoli and chromocenters exhibit a diffuse appearance, as though they were losing material at the periphery ( x 1250); (I) Early prophase: The diminished size of the diffuse, irregular-shaped nucleoli is apparent ( x 1250). Experimental
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Cyclic nucleolar
changes
during
cell cycle. I
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Sister Paula
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lack of flatness. In order to obtain a clearer picture of these changes experiments were performed in which synchronous cultures were fixed and stained with neutral Giemsa stain. It was thus possible to study the characteristic changes in nucleolar features which had been suggested by observation of motion picture films and to note the variations in nucleoprotein distribution using Giemsa stain. Using dilute Giemsa stain, Jacobson and Webb [24, 251 found that the structures in the interphase nucleus known to contain deoxyribonucleoprotein (DNP) stained reddish-purple; the ribonucleoprotein-rich (RNP) nucleoli and cytoplasm stained blue. In mitotic cells prophase and telophase chromosomes looked reddish-purple, but metaphase and anaphase chromosomes had a dark, blue-black appearance. That this material surrounding metaphase and anaphase chromosomes is RNP has been established by these two workers using Giemsa stain with selective enzymatic digestion to differentiate the nucleoproteins. In the present study of Giemsa-stained Strain L cells nucleoprotein distribution similar to that reported [24, 251 was obtained. Since the accuracy of azure-eosinate stains has been questioned we have studied the same sequence of events using toluidine blue-molybdate staining to differentiate the nucleoproteins; results of these cytochemical studies will be reported later. Using this staining method Love and his associates [31, 33, 341 reported RNP distribution very similar to that seen in Giemsa-stained preparations. Nine types of RNP have been said to be demonstrable by this technique [33]. The advantage of this method is that it includes use of a formaldehydecontaining fixative which inactivates protein-bound amino groups. This eliminates staining of surrounding protein such as occurs with azure-eosinate stains; used in conjunction with DNase digestion it provides excellent differentiation of the nucleoproteins [32]. In the present investigation subtle differences in staining qualities were noted among cells in non-synchronous cultures. That these variations might be related to cyclic differences in nucleic acid distribution during interphase is suggested by Giemsa staining of synchronous cultures. From the time they could be recognized most nucleoli exhibited irregular morphology, many having pointed projections which gave them a somewhat stellate appearance (Fig. 6a and b); these projections were nearly always seen to be in contact with the nuclear membrane. As interphase proceeded, the nucleoli became quite discrete, and the various other RNP-containing sites diminished in staining intensity. Ry 6 h after planting most nucleoli had assumed a fairly oval shape; use of very careful focusing suggested that few, if any, were in contact with the nuclear membrane (Fig. SC). Similar conclusions resulted Experimental
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Cyclic nucleolar
changes during
609
cell cycle. I
from careful study of living cells during motion picture studies. This was similar for S-phase cells at 14 h (Fig. 6d). The nucleolus-associated chromatin stained quite reddish-purple in the late Gl cells; but it was not visible in S-phase cells. Both the deep blue-black color of the nucleoli and of the chromocenters during S-phase and the increase in size of these structures were
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78910
9
m- a
b
c
80-d
60-
60.
40-
40-
20 -
20GIS Gz
GIS Gz
GGGz
GtSGz
Fig. 7.-Changes in nucleolar number ordinate: y0 of cells. (a) Gl, 3 h, aver. Fig. 8.-Changes units): 1, O-15; cells.
e
f
GiSGz
1 GIS GI
in Gl, S and G2 cells. Abscissa: Number of nucleoli per cell; 5.66; (b) S, 14 h, aver. 4.02; (c) G2, 19 h, aver. 3.71.
in nucleolar size in Gl (3 h); S (14 h) and G2 (19 h). Size classes (in arbitrary 2, 16-30; 3, 31-45; 4, 46-60; 5, 61-75; 6, > 75. Abscissa: Size class; ordinate: % of
Fig. 9.-Changes in nucleolar shape (left) and nucleolar G2 (19 h) cells. (a) Irregular; (b) oval; (c)round; (d) nuclear Stage of cycle; ordinates: y0 of cells.
position membrane;
(right) in Gl (3 h); S (14 h); (e) edge; (f) center. Abscissae:
noteworthy. Whether these changes in color and size were due to the great density of the S-phase nucleoli and chromocenters which may obscure the heterochromatin, or reflect a real change in position or chemical nature of this material was not clear. By G2 (19 h after planting), the asynchrony was considerable, but this made it possible to attempt to reconstruct the sequence of pre-prophase events, for a considerable range of pre-division cells was present in every field studied. In Fig. 6e is shown a “typical” G2 cell, with large, irregular nucleoli, many in contact with the nuclear membrane. The density of nucleolar staining was much less intense, especially around the edges, which appeared quite diffuse. This was in strong contrast with the condition of Sphase nucleoli, which were intensely stained throughout. This same apExperimental
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pearance also characterized the chromocenters, many of which were now closely associated with the nuclear membrane. By the time prophase was well along, the nucleoli presented a highly diffuse, rather “empty” appearance among the thickening chromosomes; they were also considerably smaller than they had been just prior to the onset of prophase (Fig. 6f). Study of the cells stained by this method revealed that the average number of nucleoli in cells at the three interphase periods varied considerably (Fig. 7). It was interesting to note that by G2 there were scarcely any cells with more than five nucleoli, though in Gl the range extended up to ten nucleoli per cell and the average number was 5.66. Correlated with this change in number was a shift in the size pattern (Fig. 8). The drop in number of very small nucleoli as the cell passed from Gl to S to G2 was due largely to incorporation of these smaller nucleoli into the larger ones by fusion. Had the shift been due only to growth a shift of the percentage of Gl cells to an intermediate size class would be expected; such a peak did not appear, either in S or G2. Nucleolar fusion was noted in several motion picture sequences and has been reported by many investigators [45, 551. The decrease in nucleolar number due to fusion apparently occurred throughout the cycle, though it was probably most rapid in early interphase. The areas of the histograms in Fig. 8 were determined, and the results suggested that most of the increase in size due to growth must have occurred after 14 h. The total areas (in arbitrary units), for nucleoli of the 50 cells representing each stage were: Gl (3 h), 295; S (14 h), 280; and G2 (19 h), 410. Two other very interesting aspects of nucleolar change have been noted above. Changes in shape and nucleolar position were seen to occur in a cyclic manner also. Because of the importance of these phenomena in helping to clarify the mode of formation and possible function of the nucleolus, an attempt was made to quantify the observations (Fig. 9). The proportion of nucleoli of irregular shape decreased as the cells moved from Gl to S, and then increased greatly in G2. Oval-shaped nucleoli, on the other hand, were very prevalent in S-phase cells. The number of round nucleoli (these were usually very small), did not change much until G2, where the decrease in number was probably due to their fusion with larger nucleoli. Changes in position paralleled changes in shape, as can be seen clearly in Fig. 9 b. Irregularly-shaped nucleoli were often associated with the nuclear membrane or were, at least, peripherally located within the nucleus. Analyses of motion pictures had shown that the irregular morphology of the nucleoli was at least partly due to movement of the nucleoli themselves; this did not necessarily appear to be correlated with nuclear rotation. Experimental
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DISCUSSION
The observations reported here have shown that there is a definite cyclic pattern to the morphological changes seen in nucleoli during the cell cycle. Changes in number appear to be due largely to nucleolar fusion; size changes are due both to nucleolar growth and fusion, the latter entailing nucleolar movement. A marked increase in the percentage of large nucleoli occurred late in the cycle; this was apparently the result of actual nucleolar growth, as most of the fusion of smaller nucleoli had occurred by the end of 14 h. Swift [55] reported a similar drop in nucleolar number after early interphase and attributed it to fusion. An increased rate of RNA synthesis late in the cycle has been reported [7, 26, 56, 581, and preliminary autoradiographic experiments by the present authors strongly suggest that a pre-prophase spurt of RNA synthesis occurs in strain L cells. Movement of nucleoli was very evident immediately after their formation in telophase, and was largely responsible both for their irregular morphology and for the fusion seen, much of which was observed early in the Gl period, especially in motion picture studies. In Giemsa-stained preparations just after telophase the characteristic blue RNP color was arranged in irregular masses along the newly formed nuclear membrane. This arrangement has been observed by many investigators [13, 24, 25, 52, 551. It is this RNP material which appears to be organized into the discrete bodies which are usually thought of as characteristic interphase nucleoli. Similar observations were made using toluidine blue-molybdate staining, as will be reported later. In pre-prophase cells, late in G2, the reverse of this process was observed, with RNP material appearing to move from the nucleoli toward the nuclear membrane, presumably along some type of structural component. The fact that irregular morphology, diffuse appearance, active movement and close association of nucleoli with the nuclear membrane are always associated with very early and very late interphase argues for a close interdependence of these occurrences. Several studies which described a correlation between diffuse, irregularly-shaped nucleoli and the nuclear membrane have suggested that these changes reflect varying stages in the preparation of the cell for division [lo, 13, 22, 45, 51, 571. The changes in cellular and nucleolar morphology also suggest that nucleocytoplasmic exchange during the Gl and G2 periods is more active than in the S period. In motion pictures perinuclear and nucleolar activity seemed especially marked during the first two or three hours of interphase and just prior to prophase. The percentage of nucleoli which exhibited 39
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attachments to the nuclear membrane was very high at both of these times; a recent study of nucleolar positions in HeLa cells indicated that only about 11 per cent of nucleoli were in direct or indirect contact with the nuclear membrane in non-synchronous cultures [2 j. The fact that nucleoli of cells in early Gl and late G2 exhibited considerable similarity of shape, position, movement, staining characteristics and association with the nuclear membrane suggested that there may be a very close relationship between formation and disappearance of nucleoli and that of the nuclear membrane. It seems probable that the “organization” of nucleoli may be accomplished through cyclic changes undergone by the nucleoli, the nuclear membrane, and what might be called the “heterochromatic which comprises the nucleolus-associated chromatin and intrasystem”, nucleolar chromatin. Recently Woolam et al. suggested that observed cytological phenomena of both meiosis and mitosis might be explained on the basis of three processes only: (a) replication of DNA; (b) the degree of coiling and uncoiling of the chromosomal elements, and (c) sol-gel relationships [59]. If the present results are viewed in the light of recent electron microscope studies it seems feasible to consider the possibility of accounting for the “appearance” and “disappearance” of nucleoli as a cyclic process of condensation and decondensation of nucleoproteins similar to that which accounts for these phenomena in chromosomes. Nucleoli may perhaps be formed by the interaction of several chromosomes [ 19, 49, 601. Many accounts of a meshwork joining heterochromatin and nucleoli have been reported. Fell and Hughes [13] observed that the surfaces of developing nucleoli were studded with dark heterochromatic granules, from each of which line filaments were seen to emerge in such a way as to “knit the nucleolar components together by an intricate mesh of threads”. They noted that other heterochromatic granules adhered to the nuclear membrane, a phenomenon always observed in the strain L cells used in this study. Irregularly-shaped lumps of heterochromatin which continue into thinner threads of chromatin, presumably the chromonemata, have been observed [S, 13, 30, 41, 431. Reitalu also noted that by destaining gradually after the Feulgen technique, Feulgen-positive threads within the nucleolus could be seen [45!. Possible involvement of this interphase chromatinic network in nucleolar formation and movement is suggested by several electron microscope studies [22, 23, 36, 601. Observation of an interphase RNP network with strands radiating from the nucleoli has been made by Smetana and his associates [Sl]. Recently intraExperimental
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nucleolar chromatin Iibrils have been shown to be in close relationship to this network, as well as being attached to the nucleolus-associated chromatin [41, 421. A number of electron microscope investigations have shown that intranucleolar chromatin and nucleolus-associated chromatin form a single system [17, 18, 22, 411. In such studies the nucleolus is seen as “an active genetic site on a chromosome “[22], an observation which gains considerable support from two recent findings: (1) that DNA replication in heterochromatin and euchromatin is asynchronous [3, 61; and (2) that DNA synthesis in nucleoli begins later than in chromatin, and occurs in two distinct phases
[261. Evidence is increasing that there is an intimate association between chromosomes and the nuclear membrane. A “synaptinemal complex” has been described in a variety of meiotic prophase chromosomes [lo, 39, 59]; in such studies the ends of all the chromosomes were found to be polarized and anchored at the nuclear membrane. Most significant of all was the fact that at the stage when chromosomal cores were present in the nucleus but distinct chromosomes had not yet formed, the nucleoli were found at the periphery of the nucleus and vacuoles appeared to be discharging their contents into the karyoplasm along the nuclear membrane. Many investigators have mentioned discharge of material from the nucleoli [l, 15, 30, 35, ,571; cyclic variations of the discharge of various granules in pre-prophase strain L cells will be reported later. That chromosomes of non-meiotic cells may also be attached to the nuclear membrane throughout interphase has been shown in Ehrlich ascites cells [BO] and in meristematic cells of Plantago ovata [22]. Further clarification of these intimate associations of chromatin, nucleoli and nuclear membrane may aid in explaining the cyclic nucleolar movements and morphological changes, if the pre-nucleolar materials are considered to be chromosomal products.
SUMMARY
1. Development of a technique for selective harvesting of synchronous strain L cells has made possible the study of nucleolar changes during the cell cycle. The pattern of DNA synthesis was determined autoradiographically, and cultures which had been planted for 3, 14 and 19 h were chosen as representative of the Gl, S and G2 periods, respectively. Changes in morphology, position, number and size of nucleoli were assessed, both cinematographically and using fixed preparations. 2. The significant reduction in nucleolar number between Gl and mitosis Experimental
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was found to be due largely to fusion. Increase in size resulted from both fusion and synthetic activity within the nucleolus. 3. Irregular morphology of nucleoli, marked association with the nuclear membrane and amoeboid movement were characteristic of nucleoli both in Gl and G2. During S phase nucleoli were oval-shaped and centrallylocated; they exhibited much less movement. 4. The possible significance of these cyclic nucleolar phenomena as related to other nuclear changes during the cell cycle has been discussed.
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