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Identification of early S phase nuclei by observation of centriole replication in cultured human lymphocytes
Copyright All rights
Q 1972 by Academic Press, Inc. reproduction in my form resewed
of
Experimental
Cell Research 73 (1972) 17-24
IDENTIFICATION OF EARLY S PHASE NUCLEI BY OBSERVATION OF CENTRIOLE REPLICATION IN CULTURED HUMAN LYMPHOCYTES K. T. TOKUYASU’ Anita M. University
Baldwin Electron Microscope Laboratory, Department of California School qf Medicine, Los Angeles, Cal&
of Pathology, 90024, USA
SUMMARY Early S phase nuclei of cultured human lymphocytes are identified by the observation of the early stage of centriole replication. In such nuclei, condensed chromatin is seen to cover the entire inner surface of nuclear envelope, except the sites of nuclear pores. When the centriole replication is more advanced, chromatin aggregates are found to be in a state of reduced condensation? the loosened state. The association of inactive condensed chromatin with the nuclear envelope m the early S phase makes it quite unlikely, at least in cultured human lymphocytes, that DNA replication is initiated exclusively at the nuclear membrane. The restriction of DNA synthesis to the nuclear periphery is considered to be characteristic of the terminal S phase where chromatin is almost completely dispersed. The effect of amethopterin, a synchrony inducing agent, upon the nuclear transformation is discussed. It is proposed that the transformation of inactive chromatin from the condensed to loosened and further to dispersed state during S phase is a slow and gradual process.
In our previous autoradiographic study of cultured lymphocytes [l], the early S phase nuclei were identified as those which resembled the G 1 phase nuclei in that condensed chromatin aggregates were still found in the nuclei, particularly along the nuclear envelope. The mid S and late S phase nuclei were similarly identified by the principle that these nuclei, together with the early S phase nuclei, should form a morphological continuum with the Gl and G2 phase nuclei. The majority of the label appeared to be associated with chromatin of a reduced condensation, loosened chromatin, throughout S phase or to be located near the interface between condensed and diffuse chromatin in early S phase. Both the DNA synthetic 1 Present address: Department of Biology, University of California, La Jolla, Calif. 92037, USA. a - 721807
activity and the quantity of loosened chromatin were highest in the mid S phase nuclei. In the late S phase nuclei, loosened chromatin was recognized in a much lesser quantity as a very thin layer along the nuclear envelope and a few small aggregates inside. Otherwise, these nuclei showed almost completely dispersed chromatin. Peripheral chromatin was observed to be similarly thin in the very early prophase and absent in the typical early prophase nuclei, except for the attachment of chromosomes to discrete regions of the nuclear envelope. On the other hand, Comings & Kakefuda [2] studied the DNA replication by means of autoradiography in cultured human amnion cells which were synchronized first with excess thymidine and then with amethopterin in the absence of thymidine. The nuclei at the onset Exptl
Cell Res 73 (1972)
18 K. T. Tokuyasu of the synchronized DNA synthesis showed an almost complete dispersion of chromatin and, in many of them, the silver grains were found to be mostly restricted to the nuclear periphery. On this basis, it was concluded that DNA replication may be initiated at the nuclear membrane. Thus, the nuclei with an almost complete dispersion of chromatin and the restriction of grains mainly to the nuclear periphery were identified to be in the earliest S phase in their study and in the terminal S phase in ours. Grains in the early S phase nuclei of our study were found mostly inside the nuclei and only occasionally in association with the regions of the periphery where chromatin was thin. If chromatin in the nuclei of unsynchronized human amnion cells is dispersed throughout S phase, as observed in those of synchronized cells at the onset of S phase, the disparity between the two studies would be simply explained as originating from the difference in the cell species used, and the problem would be reduced to the question of which one would be more generally applicable to different cell types. In the nuclei of some unsynchronized cells in their study, however, numerous chromatin aggregates were distributed inside the nucleus as well as along the envelope, in a manner similar to the pattern of chromatin aggregates in the mid S phase nuclei of our study. The association of most of the silver grains with such aggregates was also similar in the nuclei of both cell types. It is very unlikely that the identical fixation and preparation procedures causean artificial dispersion of chromatin only in nuclei at the onset of S phase but not in those in S phase. The possibility is, therefore, that a difference in culture conditions is the major cause of the disparity. The rather drastic method of synchronization in their study could disturb Exptl
Cd
Res 73 (1972)
the normal nuclear transformation during the S phase. Robbins and co-workers [3] achieved the synchronization of HeLa cell culture much more mildly by utilizing the property of the mitotic cells that they are easily detached from the substrate [4], and clearly demonstrated that the replication of centriole begins at/or near the initiation of DNA synthesis. This knowledge provides an independent means to identify the early S phase nuclei. In the present study of cultured human lymphocytes, it will be shown that nuclei of the cells in the early stage of the centriole replication are of the type identified as the early S phase nuclei in our previous study and are not of the late S phase type. MATERIALS
AND
METHODS
Culture, preparation, and examination procedure of normal human peripheral blood lymphocytes were described in detail in the previous paper [l]. Therefore, only a summary of essential information is given here. Lymphocytes cultured with 0.01 ml of phytohemagglutinin M (Difco Laboratories, Detroit, Mich.) per ml of culture medium for 4, 24, 48, and 72 h are doubly fixed with 5 % glutaraldehyde and 1 % OSO, in 0.1. M phosphate buffer, pH 7.4, and embedded in Epon 812 after the dehydration with ethanol. All sections, 200-500 A thick; are doubly stained with uranyl acetate and lead citrate. For convenience, cells cultured for n h are referred to as 12h cells and their nuclei as n h nuclei in this paper.
RESULTS The examination of about 5 000 cells of 24 h cultures, including those in a large number of serial sections, has failed to reveal replicating centrioles or more than one pair of centrioles in a cell. On the other hand, the separation of centrioles, about 0.7-0.8 pm, as well as displacement from the orthogonal arrangement is rather often found in cells of this stage (figs 1, 2). Since no or very few cells are in the S phase during the first 24 h of culture [5], this result is in accordance with
E!-uly S phase nuclei of lymphocytes
I9
All figures are of thin sections of human peripheral blood lymphocytes cultured for the time period indicated in individual captions. Directions of cellular and nuclear profiles are referred to the axes passing through nuclei and centrosotnes. Key to symbols: C, centriole; G, Golgi apparatus; A4, mitochondria; IWT, microtubules; N, nucleus; NL, nucleolus. Figs I, 2. Parts of 24 h cells. Centrioles, C, and C1, are separated by a distance of 0.7-0.8 porn and are displaced from the orthogonal arrangement. Microtubules (MT) radiate from a dense mass lying between centrioles and also from a satellite (arrow, fig. 1). Prominent Golgi apparatus (G) is found around centrioles. Thick aggregates of condensed chromatin are seen to cover the inner surface of the nuclear envelope except the sites of nuclear pores. Both, ‘< 35 000.
the observation [4] in HeLa
of Robbins and co-workers cells in late G 1 phase. In the
lymphocytes of 24 h cultures, however, centrospherical or centrosomal regions are found to be more enlarged than in the previous periods and prominent microtubules and Golgi complex are frequently observed in at their vicinity (figs 1, 2). This is somewhat variance with their report that in Gl phase is not clearly of HeLa cells, the centrosome
defined and only occasional spindle tubule is visible. In fig. 3 of a part of a 48 h cell, an oblique profile of a centriole, is accompanied by a procentriole, C,‘. A cross sectioned centriole, C,, found at a position about 1 pm apart from the above pair is, therefore, considered to belong to another pair. In fig. 5 of a part of another 48 h cell, one pair of centrioles, long C, and short C,‘, Exptl
Cell Res 73 (1972)
20
K. T. Tokuyasu
Figs 3,4. Parts of a 48 h ceil. In fig. 3, an obliquely sectioned centriole, C,, is accompanied by a longitudinally sectioned procentriole, C,‘. A cross-sectioned centriole, Cz, is separated from the above pair by a distance of about 1 pm. The nucleus in fig. 4 shows the persistence of condensed chromatin along the nuclear envelope. The overall pattern of chromatin aggregates is similar to that in a typical 24 h nucleus such as shown in fig. 8. Fig. 3, x 35 000; fig. 4, x 10 000.
show an orthogonal arrangement, while barely skimmed surfaces of centrioles, C, and C2’, form another pair. They are mutually separated by a distance of about 1 ,um. A whole oblique profile of one of the latter pair, C,, is found in fig. 6, or in higher magnification in fig. 7, of an adjacent section. Nuclei of the above cells, shown in figs 4 and 6, show condensed chromatin aggregates along the nuclear envelope as well as inside the inner areas. It will be evident that these nuclei are in interphase when compared with a typical 24 h nucleus in fig. 8 (see also figs 1, 2). The average width of the peripheral chromatin is smaller in the 48 h nuclei than in the 24 h nucleus and this appears to be in correspondence
with
the much
enlarged
sur-
face areas of the 48 h nuclei. Interruptions of the peripheral chromatin represent the sites of nuclear pores and the greatly increased number of the pores in the 48 h nuclei is considered to indicate an enhanced nucleocytoplasmic interaction in these cells. Procentrioles at the earliest stage of repliExptl
Cell
Res 73 (1972)
cation are known to be seen as poorly defined, ring-like structures [6, 71. The fact that more or less well defined cylinders of procentrioles in figs 3 and 5 are already about half of parent centrioles in length indicates that these procentrioles do not represent the very beginning of the centriole replication but a somewhat advanced stage. Therefore, the nucleus at the initial replication stage is probably even closer to the 24 h nucleus in the pattern of chromatin aggregates than the nucleus in figs 4 or 6. In fig. 9 of a 72 h cell, two centrioles are found to be separated by a distance of about 3 pm, This wide separation suggests that they belong individually to two different pairs and also that the cell is more advanced in the stage of centriole replication, and therefore of S phase, than those of figs 4 and 6. In fact, the nucleus of this cell has been shown in the previous paper as a probable mid S phase nucleus (fig. 7 (6) or 15 in [l]). A part of the nucleus is shown in the insert of fig. 9, at low magnification, to allow the
Eady
S phase nuclei of lymphocytes
21
Figs 5-7. Parts of a 48 h cell. In fig. 5, parent centriole, C,, and daughter centriole, Cl’, are orthogonally paired. Barley skimmed surfaces of centrioles, C, and C,’ form another pair. A whole oblique profile of C, is seen in fig. 6 or, at higher magnification, in fig. 7 of an adjacent section. Some of enlarged lymphocytes in 48 or 72 h culture are found to be bell- or cup-shaped. The nuclear profile in fig. 6 represents an oblique section of such a nucleus. Condensed chromatin persists along the nuclear periphery. This nucleus is quite different from that of fig. 4. in morphology but similar with respect to size and distribution of chromatin aggregates. Figs 5, 7, y 35 000; fig. 6, x 10 000.
direct comparison with the nucleus of the earlier stage in fig. 4 or 6. Chromatin aggregates in the insert may be seento be lower in density than those in figs 4 or 6 and uniformly reduced to be about 0.1 ,um in width.
S-IO] persists in association with the nuclear envelope except at the sites of nuclear pores. One of the finest demonstrations of the early phase of centriole replication was pre-
The above observations confirm the conclusion of our previous study that in the early S phase nuclei of cultured lymphocytes, condensed chromatin which is known to be inactive in both DNA and RNA synthesis [I,
Fig. 8. A typical 24 h nucleus. The 48 h nuclei in figs. 4 and 6 are similar to this nucleus with respect to the distribution of condensed chromatin aggregates, although perioheral condensed chromatin in this nucleus is thicker on average than in the larger 48 h nuclei and the number of nuclear pores is smaller.
x 10000. Exptl Cell Res 73 (1972)
22
K. T. Tokuyasu
Fig. 9. Part of a 72 h cell. Two centriolar profiles, cross-sectioned C, and obliquely cut Ce, are separated by a distance of about 3 ,nm, which is considered to indicate that these centrioles belong to two different pairs. The smaller diameter of C, than C, suggests that C, may be a procentriole. Many satellites (arrows) are observed around C,. Three profiles of Golgi complex are recognized; two, G, and Gz, in the vicinity of each centriole and the third, Ga, in the central region. A part of the nucleus is shown in the inset at the same magnification as that of figs 4, 6 or 8. Peripheral and central chromatin aggregates are approximately uniformly reduced in density as well as in width to about 0.1 pm. x 20 000. Inset, x 10 000.
sented by Murray and co-workers in rat thymic lymphocytes ([l 11, their fig 9). The phase of the cell was then considered to be an early prophase. Data as to the structure of the prophase nucleus have since then accumulated [l, 3, 12-141 and it will now be justified to identify the nucleus as that of the interphase. The association of condensed chromatin with the nuclear envelope is clearly seen in this demonstration. Interphase nuclei of mammalian cells vary greatly in the amount, width, or distribution of condensed chromatin aggregates. Chromatin aggregates are hardly recognized in the interphase nuclei of mammalian oogonia [ 16, 171, whereas the nucleus of the peripheral blood lymphocyte is known as one of the richest in the amount of condensed chromatin. In this aspect, nuclei of other cell types distribute between these two extremes. The human peripheral blood lymphocytes and rat thymic lymphocytes, therefore, could be two of the exceptional cases. Nonetheless, any exceptions Exptl
CeN Res 73 (1972)
will cast doubt as to the range of the generalization of a theory. The results of Comings & Kakefuda [2] cannot be generalized without qualification. The fact that abundant chromatin aggregates are recognized in the S phase nuclei of some unsynchronized human amnion cells in their study leaves the possibility that chromatin aggregates may also be abundant at the onset of S phase if the amnion cells are not synchronized with excess thymidine and amethopterin. It is very difficult to consider that chromatin aggregates which commonly occur in G 1 phase almost completely disperse at the onset of S phase only to reaggregate during S phase. A drastic effect of amethopterin upon the nuclear function is well known. According to Rueckert & Mueller [15], when HeLa cells were grown in thymidine-deficient amethopterin medium for a period of about two-thirds of a generation time, they began to undergo an irreversible change resulting in a rapid ‘killing’ of the cells. They stated
Early S phase nuclei of lymphocytes
that addition of thymidine at the brink of imminent death rescued them and resulted in a burst of mitosis. In the study of Comings & Kakefuda, human amnion cells were incubated for 14 h, about three quarters of the generation time of 19 h, in the amethopterin medium. It is possible or rather likely that the nuclear transformation proceeds even without DNA synthesis and the inability of nuclei to enter into G2 phase due to the lack of the additional set of chromosomes forces the nuclei of originally different phases to come to the same phase, that is, the end of G 1 phase with respect to DNA synthesis but the terminal S phase with respect to transformation. The irreversibility of the transformation will cause the death of the cells when such a state is prolonged without thymidine. The generalization of findings on drastically synchronized cells to normal cellular or nuclear processes may need to be critically evaluated. The amount of peripheral chromatin at the onset of S phase will be variable among different cell types, as described above, and there will be a certain variation in the pattern of the initial DNA synthesis. In extreme cases of certain cells such as oogonia, nuclear chromatin could be normally dispersed throughout the S phase. Nonetheless, in most mammalian cell types, the restriction of the majority of label to the nuclear periphery may be the feature of the late S phase nuclei but not that of the early S and mid S phase nuclei. In fact, recently, Huberman and coworkers (personal communication) labeled unsynchronized HeLa cells and Chinese hamster ovary cells using pulse times as short as 30 set and found silver grains distributed over the entire nucleus even at the very beginning of the S phase; only in late S were sites of synthesis confined to the nuclear and nucleolar peripheries. There is a basic difficulty in autoradiography
23
for relating the DNA synthetic activity to specific nuclear locations such as the nuclear envelope. The highest attainable resolution by commonly used autoradiographic technique is in the order of 1 000 A, whereas the width of a chromatin fiber is about 200 A. When a large number of silver grains are mutually superposed or form clumps, the resolution falls to the order of 0.5-l pm. The autoradiographic data alone, therefore, will be inadequate for clarifying whether the DNA synthesis occurs in association with the chromatin fiber immediately adjacent to the nuclear envelope or several fiber layers apart from the envelope. The overall information as to the nuclear structure and its transformation may be indispensable for the critical interpretation of autoradiographic data. This difficulty of autoradiography also applies to our autoradiographic data: It cannot be undisputably stated that silver grains lying on loosened chromatin aggregates of average width of 0.1 ,um really represent label in loosened chromatin but not that in neighboring diffuse chromatin. On the other hand, it is known that the major portion of chromatin, 80-90 % in many cases, is inactive [18, 191. Since loosened chromatin appears to be derived from inactive condensed chromatin, it is not strange even if the number of grains associated with loosened chromatin is much larger than that associated with active diffuse chromatin. The labeling period in our previous study was 2 h, about one sixth of the S phase duration of cultured lymphocytes [5]. Despite such a relatively long labeling period, the majority of silver grains were observed to be localized on loosened chromatin aggregates. The autoradiographs of some unsynchronized cells labeled for only 10 min in the study of Comings & Kakefuda [2] showed a great resemblance to our autoradiographs in the distribution of silver grains. The loosening and further dispersion of conExptl
Cell Res 73 (1972)
24
K. T. Tokuyasu
densed chromatin may be a slow and gradual process and the autoradiographic result will be essentially the same whether the labeling period is in the order of hours or shorter. The rate of DNA replication is estimated to be 0.5 to 2.5 ,um/min [20, 211. Such a fast rate is not necessarily in conflict with the prolonged confinement of label within the loosened chromatin networks, since all or most of the replicated DNA of inactive chromatin may remain inactive in loosened chromatin. The differentiation of inactive and active chromatin to condensed and diffuse states of chromatin in the G 1 phase is probably modified to loosened and diffuse states in S phase until such a differentiation becomes undetectable in the dispersed state in the terminal S phase. Chromatin of different cell types of a mammalian species, with variable proportions of inactive chromatin in Gl or S phase, may attain the morphologically uniform state of dispersion at the end of S phase before transforming to a set of chromosomes which is uniform for all cell types. The author wishes to express his appreciation to Dr S. C. Madden and Dr L. J. Zeldis for their constant encouragement. and Dr T. Fukushima for assistance in the preparation of lymphocyte cultures. The skillful technical assistance of Miss M. D. Coffman is also gratefully acknowledged.
Exptl
Cell Res 73 (1972)
This work was supported in part by research grants Nos. CA-07409 and HD-02562 from NIH, USPHS.
REFERENCES 1.
2. 3. 4. 5. 6. 7. ;: 10. 11. 12.
Tokuyasu, K, Madden, S C & Zeldis, L J, J cell biol 39 (1968) 630. Comings, G E & Kakefuda, T, J mol biol 33 (1968) 225. Robbins, E, Jentzsch, G & Michali, A, J cell biol 36 (1968) 329. Terashima, T & Tolmach, L J, Exptl cell res 30 (1963) 344. Sasaki, M S & Norman, A, Nature 210 (1966) 913. Gall, J G, J biophys biochem cytol 10 (1961) 163. Sorokin, S P, J cell sci 3 (1968) 207. Hay, E D & Revel, J P, J cell biol 16 (1963) 29. Littau, V C, Allfrey, V G, Frenster, J H & Mirsky, A E, Proc natl acad sci US 52 (1964) 93. Karasaki, S, J cell biol 26 (1965) 937. Murray. R C. Murray. A S & Pizzo, A, J cell -.biol 28 (1965)‘601. FT9bbins, E & Gonatas, N K, J cell biol21 (1964)
13. Daiis, H G & Tooze, J, J cell sci 1 (1966) 331. 14. Comings, D E & Okada, T A, Exptl cell res 63 (1970) 471. 15. Rueckert, R R & Mueller, C C, Cancer res 20 (1960) 1584. 16. Weakley, B S, J anat 101 (1967) 435. 17. Baker, T G & Franchi, L L, J cell sci 2 (1967) 213. 18. Bonner, J, Dahmus, M E, Fambrough, D, Huang, R C C, Marushige, K & Tuan, D Y H, Science 159 (1968) 47. 19. Shih, T Y & Bonner, J, J mol biol 50 (1970) 333. 20. Cairns, J, J mol biol 15 (1966) 372. 21. Huberman, J A & Riggs, A D, J mol biol 32 (1968) 327. Received November 22, 1971 Revised version received February 1, 1972
Q 1972 by Academic Press, Inc. reproduction in my form resewed
of
Experimental
Cell Research 73 (1972) 17-24
IDENTIFICATION OF EARLY S PHASE NUCLEI BY OBSERVATION OF CENTRIOLE REPLICATION IN CULTURED HUMAN LYMPHOCYTES K. T. TOKUYASU’ Anita M. University
Baldwin Electron Microscope Laboratory, Department of California School qf Medicine, Los Angeles, Cal&
of Pathology, 90024, USA
SUMMARY Early S phase nuclei of cultured human lymphocytes are identified by the observation of the early stage of centriole replication. In such nuclei, condensed chromatin is seen to cover the entire inner surface of nuclear envelope, except the sites of nuclear pores. When the centriole replication is more advanced, chromatin aggregates are found to be in a state of reduced condensation? the loosened state. The association of inactive condensed chromatin with the nuclear envelope m the early S phase makes it quite unlikely, at least in cultured human lymphocytes, that DNA replication is initiated exclusively at the nuclear membrane. The restriction of DNA synthesis to the nuclear periphery is considered to be characteristic of the terminal S phase where chromatin is almost completely dispersed. The effect of amethopterin, a synchrony inducing agent, upon the nuclear transformation is discussed. It is proposed that the transformation of inactive chromatin from the condensed to loosened and further to dispersed state during S phase is a slow and gradual process.
In our previous autoradiographic study of cultured lymphocytes [l], the early S phase nuclei were identified as those which resembled the G 1 phase nuclei in that condensed chromatin aggregates were still found in the nuclei, particularly along the nuclear envelope. The mid S and late S phase nuclei were similarly identified by the principle that these nuclei, together with the early S phase nuclei, should form a morphological continuum with the Gl and G2 phase nuclei. The majority of the label appeared to be associated with chromatin of a reduced condensation, loosened chromatin, throughout S phase or to be located near the interface between condensed and diffuse chromatin in early S phase. Both the DNA synthetic 1 Present address: Department of Biology, University of California, La Jolla, Calif. 92037, USA. a - 721807
activity and the quantity of loosened chromatin were highest in the mid S phase nuclei. In the late S phase nuclei, loosened chromatin was recognized in a much lesser quantity as a very thin layer along the nuclear envelope and a few small aggregates inside. Otherwise, these nuclei showed almost completely dispersed chromatin. Peripheral chromatin was observed to be similarly thin in the very early prophase and absent in the typical early prophase nuclei, except for the attachment of chromosomes to discrete regions of the nuclear envelope. On the other hand, Comings & Kakefuda [2] studied the DNA replication by means of autoradiography in cultured human amnion cells which were synchronized first with excess thymidine and then with amethopterin in the absence of thymidine. The nuclei at the onset Exptl
Cell Res 73 (1972)
18 K. T. Tokuyasu of the synchronized DNA synthesis showed an almost complete dispersion of chromatin and, in many of them, the silver grains were found to be mostly restricted to the nuclear periphery. On this basis, it was concluded that DNA replication may be initiated at the nuclear membrane. Thus, the nuclei with an almost complete dispersion of chromatin and the restriction of grains mainly to the nuclear periphery were identified to be in the earliest S phase in their study and in the terminal S phase in ours. Grains in the early S phase nuclei of our study were found mostly inside the nuclei and only occasionally in association with the regions of the periphery where chromatin was thin. If chromatin in the nuclei of unsynchronized human amnion cells is dispersed throughout S phase, as observed in those of synchronized cells at the onset of S phase, the disparity between the two studies would be simply explained as originating from the difference in the cell species used, and the problem would be reduced to the question of which one would be more generally applicable to different cell types. In the nuclei of some unsynchronized cells in their study, however, numerous chromatin aggregates were distributed inside the nucleus as well as along the envelope, in a manner similar to the pattern of chromatin aggregates in the mid S phase nuclei of our study. The association of most of the silver grains with such aggregates was also similar in the nuclei of both cell types. It is very unlikely that the identical fixation and preparation procedures causean artificial dispersion of chromatin only in nuclei at the onset of S phase but not in those in S phase. The possibility is, therefore, that a difference in culture conditions is the major cause of the disparity. The rather drastic method of synchronization in their study could disturb Exptl
Cd
Res 73 (1972)
the normal nuclear transformation during the S phase. Robbins and co-workers [3] achieved the synchronization of HeLa cell culture much more mildly by utilizing the property of the mitotic cells that they are easily detached from the substrate [4], and clearly demonstrated that the replication of centriole begins at/or near the initiation of DNA synthesis. This knowledge provides an independent means to identify the early S phase nuclei. In the present study of cultured human lymphocytes, it will be shown that nuclei of the cells in the early stage of the centriole replication are of the type identified as the early S phase nuclei in our previous study and are not of the late S phase type. MATERIALS
AND
METHODS
Culture, preparation, and examination procedure of normal human peripheral blood lymphocytes were described in detail in the previous paper [l]. Therefore, only a summary of essential information is given here. Lymphocytes cultured with 0.01 ml of phytohemagglutinin M (Difco Laboratories, Detroit, Mich.) per ml of culture medium for 4, 24, 48, and 72 h are doubly fixed with 5 % glutaraldehyde and 1 % OSO, in 0.1. M phosphate buffer, pH 7.4, and embedded in Epon 812 after the dehydration with ethanol. All sections, 200-500 A thick; are doubly stained with uranyl acetate and lead citrate. For convenience, cells cultured for n h are referred to as 12h cells and their nuclei as n h nuclei in this paper.
RESULTS The examination of about 5 000 cells of 24 h cultures, including those in a large number of serial sections, has failed to reveal replicating centrioles or more than one pair of centrioles in a cell. On the other hand, the separation of centrioles, about 0.7-0.8 pm, as well as displacement from the orthogonal arrangement is rather often found in cells of this stage (figs 1, 2). Since no or very few cells are in the S phase during the first 24 h of culture [5], this result is in accordance with
E!-uly S phase nuclei of lymphocytes
I9
All figures are of thin sections of human peripheral blood lymphocytes cultured for the time period indicated in individual captions. Directions of cellular and nuclear profiles are referred to the axes passing through nuclei and centrosotnes. Key to symbols: C, centriole; G, Golgi apparatus; A4, mitochondria; IWT, microtubules; N, nucleus; NL, nucleolus. Figs I, 2. Parts of 24 h cells. Centrioles, C, and C1, are separated by a distance of 0.7-0.8 porn and are displaced from the orthogonal arrangement. Microtubules (MT) radiate from a dense mass lying between centrioles and also from a satellite (arrow, fig. 1). Prominent Golgi apparatus (G) is found around centrioles. Thick aggregates of condensed chromatin are seen to cover the inner surface of the nuclear envelope except the sites of nuclear pores. Both, ‘< 35 000.
the observation [4] in HeLa
of Robbins and co-workers cells in late G 1 phase. In the
lymphocytes of 24 h cultures, however, centrospherical or centrosomal regions are found to be more enlarged than in the previous periods and prominent microtubules and Golgi complex are frequently observed in at their vicinity (figs 1, 2). This is somewhat variance with their report that in Gl phase is not clearly of HeLa cells, the centrosome
defined and only occasional spindle tubule is visible. In fig. 3 of a part of a 48 h cell, an oblique profile of a centriole, is accompanied by a procentriole, C,‘. A cross sectioned centriole, C,, found at a position about 1 pm apart from the above pair is, therefore, considered to belong to another pair. In fig. 5 of a part of another 48 h cell, one pair of centrioles, long C, and short C,‘, Exptl
Cell Res 73 (1972)
20
K. T. Tokuyasu
Figs 3,4. Parts of a 48 h ceil. In fig. 3, an obliquely sectioned centriole, C,, is accompanied by a longitudinally sectioned procentriole, C,‘. A cross-sectioned centriole, Cz, is separated from the above pair by a distance of about 1 pm. The nucleus in fig. 4 shows the persistence of condensed chromatin along the nuclear envelope. The overall pattern of chromatin aggregates is similar to that in a typical 24 h nucleus such as shown in fig. 8. Fig. 3, x 35 000; fig. 4, x 10 000.
show an orthogonal arrangement, while barely skimmed surfaces of centrioles, C, and C2’, form another pair. They are mutually separated by a distance of about 1 ,um. A whole oblique profile of one of the latter pair, C,, is found in fig. 6, or in higher magnification in fig. 7, of an adjacent section. Nuclei of the above cells, shown in figs 4 and 6, show condensed chromatin aggregates along the nuclear envelope as well as inside the inner areas. It will be evident that these nuclei are in interphase when compared with a typical 24 h nucleus in fig. 8 (see also figs 1, 2). The average width of the peripheral chromatin is smaller in the 48 h nuclei than in the 24 h nucleus and this appears to be in correspondence
with
the much
enlarged
sur-
face areas of the 48 h nuclei. Interruptions of the peripheral chromatin represent the sites of nuclear pores and the greatly increased number of the pores in the 48 h nuclei is considered to indicate an enhanced nucleocytoplasmic interaction in these cells. Procentrioles at the earliest stage of repliExptl
Cell
Res 73 (1972)
cation are known to be seen as poorly defined, ring-like structures [6, 71. The fact that more or less well defined cylinders of procentrioles in figs 3 and 5 are already about half of parent centrioles in length indicates that these procentrioles do not represent the very beginning of the centriole replication but a somewhat advanced stage. Therefore, the nucleus at the initial replication stage is probably even closer to the 24 h nucleus in the pattern of chromatin aggregates than the nucleus in figs 4 or 6. In fig. 9 of a 72 h cell, two centrioles are found to be separated by a distance of about 3 pm, This wide separation suggests that they belong individually to two different pairs and also that the cell is more advanced in the stage of centriole replication, and therefore of S phase, than those of figs 4 and 6. In fact, the nucleus of this cell has been shown in the previous paper as a probable mid S phase nucleus (fig. 7 (6) or 15 in [l]). A part of the nucleus is shown in the insert of fig. 9, at low magnification, to allow the
Eady
S phase nuclei of lymphocytes
21
Figs 5-7. Parts of a 48 h cell. In fig. 5, parent centriole, C,, and daughter centriole, Cl’, are orthogonally paired. Barley skimmed surfaces of centrioles, C, and C,’ form another pair. A whole oblique profile of C, is seen in fig. 6 or, at higher magnification, in fig. 7 of an adjacent section. Some of enlarged lymphocytes in 48 or 72 h culture are found to be bell- or cup-shaped. The nuclear profile in fig. 6 represents an oblique section of such a nucleus. Condensed chromatin persists along the nuclear periphery. This nucleus is quite different from that of fig. 4. in morphology but similar with respect to size and distribution of chromatin aggregates. Figs 5, 7, y 35 000; fig. 6, x 10 000.
direct comparison with the nucleus of the earlier stage in fig. 4 or 6. Chromatin aggregates in the insert may be seento be lower in density than those in figs 4 or 6 and uniformly reduced to be about 0.1 ,um in width.
S-IO] persists in association with the nuclear envelope except at the sites of nuclear pores. One of the finest demonstrations of the early phase of centriole replication was pre-
The above observations confirm the conclusion of our previous study that in the early S phase nuclei of cultured lymphocytes, condensed chromatin which is known to be inactive in both DNA and RNA synthesis [I,
Fig. 8. A typical 24 h nucleus. The 48 h nuclei in figs. 4 and 6 are similar to this nucleus with respect to the distribution of condensed chromatin aggregates, although perioheral condensed chromatin in this nucleus is thicker on average than in the larger 48 h nuclei and the number of nuclear pores is smaller.
x 10000. Exptl Cell Res 73 (1972)
22
K. T. Tokuyasu
Fig. 9. Part of a 72 h cell. Two centriolar profiles, cross-sectioned C, and obliquely cut Ce, are separated by a distance of about 3 ,nm, which is considered to indicate that these centrioles belong to two different pairs. The smaller diameter of C, than C, suggests that C, may be a procentriole. Many satellites (arrows) are observed around C,. Three profiles of Golgi complex are recognized; two, G, and Gz, in the vicinity of each centriole and the third, Ga, in the central region. A part of the nucleus is shown in the inset at the same magnification as that of figs 4, 6 or 8. Peripheral and central chromatin aggregates are approximately uniformly reduced in density as well as in width to about 0.1 pm. x 20 000. Inset, x 10 000.
sented by Murray and co-workers in rat thymic lymphocytes ([l 11, their fig 9). The phase of the cell was then considered to be an early prophase. Data as to the structure of the prophase nucleus have since then accumulated [l, 3, 12-141 and it will now be justified to identify the nucleus as that of the interphase. The association of condensed chromatin with the nuclear envelope is clearly seen in this demonstration. Interphase nuclei of mammalian cells vary greatly in the amount, width, or distribution of condensed chromatin aggregates. Chromatin aggregates are hardly recognized in the interphase nuclei of mammalian oogonia [ 16, 171, whereas the nucleus of the peripheral blood lymphocyte is known as one of the richest in the amount of condensed chromatin. In this aspect, nuclei of other cell types distribute between these two extremes. The human peripheral blood lymphocytes and rat thymic lymphocytes, therefore, could be two of the exceptional cases. Nonetheless, any exceptions Exptl
CeN Res 73 (1972)
will cast doubt as to the range of the generalization of a theory. The results of Comings & Kakefuda [2] cannot be generalized without qualification. The fact that abundant chromatin aggregates are recognized in the S phase nuclei of some unsynchronized human amnion cells in their study leaves the possibility that chromatin aggregates may also be abundant at the onset of S phase if the amnion cells are not synchronized with excess thymidine and amethopterin. It is very difficult to consider that chromatin aggregates which commonly occur in G 1 phase almost completely disperse at the onset of S phase only to reaggregate during S phase. A drastic effect of amethopterin upon the nuclear function is well known. According to Rueckert & Mueller [15], when HeLa cells were grown in thymidine-deficient amethopterin medium for a period of about two-thirds of a generation time, they began to undergo an irreversible change resulting in a rapid ‘killing’ of the cells. They stated
Early S phase nuclei of lymphocytes
that addition of thymidine at the brink of imminent death rescued them and resulted in a burst of mitosis. In the study of Comings & Kakefuda, human amnion cells were incubated for 14 h, about three quarters of the generation time of 19 h, in the amethopterin medium. It is possible or rather likely that the nuclear transformation proceeds even without DNA synthesis and the inability of nuclei to enter into G2 phase due to the lack of the additional set of chromosomes forces the nuclei of originally different phases to come to the same phase, that is, the end of G 1 phase with respect to DNA synthesis but the terminal S phase with respect to transformation. The irreversibility of the transformation will cause the death of the cells when such a state is prolonged without thymidine. The generalization of findings on drastically synchronized cells to normal cellular or nuclear processes may need to be critically evaluated. The amount of peripheral chromatin at the onset of S phase will be variable among different cell types, as described above, and there will be a certain variation in the pattern of the initial DNA synthesis. In extreme cases of certain cells such as oogonia, nuclear chromatin could be normally dispersed throughout the S phase. Nonetheless, in most mammalian cell types, the restriction of the majority of label to the nuclear periphery may be the feature of the late S phase nuclei but not that of the early S and mid S phase nuclei. In fact, recently, Huberman and coworkers (personal communication) labeled unsynchronized HeLa cells and Chinese hamster ovary cells using pulse times as short as 30 set and found silver grains distributed over the entire nucleus even at the very beginning of the S phase; only in late S were sites of synthesis confined to the nuclear and nucleolar peripheries. There is a basic difficulty in autoradiography
23
for relating the DNA synthetic activity to specific nuclear locations such as the nuclear envelope. The highest attainable resolution by commonly used autoradiographic technique is in the order of 1 000 A, whereas the width of a chromatin fiber is about 200 A. When a large number of silver grains are mutually superposed or form clumps, the resolution falls to the order of 0.5-l pm. The autoradiographic data alone, therefore, will be inadequate for clarifying whether the DNA synthesis occurs in association with the chromatin fiber immediately adjacent to the nuclear envelope or several fiber layers apart from the envelope. The overall information as to the nuclear structure and its transformation may be indispensable for the critical interpretation of autoradiographic data. This difficulty of autoradiography also applies to our autoradiographic data: It cannot be undisputably stated that silver grains lying on loosened chromatin aggregates of average width of 0.1 ,um really represent label in loosened chromatin but not that in neighboring diffuse chromatin. On the other hand, it is known that the major portion of chromatin, 80-90 % in many cases, is inactive [18, 191. Since loosened chromatin appears to be derived from inactive condensed chromatin, it is not strange even if the number of grains associated with loosened chromatin is much larger than that associated with active diffuse chromatin. The labeling period in our previous study was 2 h, about one sixth of the S phase duration of cultured lymphocytes [5]. Despite such a relatively long labeling period, the majority of silver grains were observed to be localized on loosened chromatin aggregates. The autoradiographs of some unsynchronized cells labeled for only 10 min in the study of Comings & Kakefuda [2] showed a great resemblance to our autoradiographs in the distribution of silver grains. The loosening and further dispersion of conExptl
Cell Res 73 (1972)
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K. T. Tokuyasu
densed chromatin may be a slow and gradual process and the autoradiographic result will be essentially the same whether the labeling period is in the order of hours or shorter. The rate of DNA replication is estimated to be 0.5 to 2.5 ,um/min [20, 211. Such a fast rate is not necessarily in conflict with the prolonged confinement of label within the loosened chromatin networks, since all or most of the replicated DNA of inactive chromatin may remain inactive in loosened chromatin. The differentiation of inactive and active chromatin to condensed and diffuse states of chromatin in the G 1 phase is probably modified to loosened and diffuse states in S phase until such a differentiation becomes undetectable in the dispersed state in the terminal S phase. Chromatin of different cell types of a mammalian species, with variable proportions of inactive chromatin in Gl or S phase, may attain the morphologically uniform state of dispersion at the end of S phase before transforming to a set of chromosomes which is uniform for all cell types. The author wishes to express his appreciation to Dr S. C. Madden and Dr L. J. Zeldis for their constant encouragement. and Dr T. Fukushima for assistance in the preparation of lymphocyte cultures. The skillful technical assistance of Miss M. D. Coffman is also gratefully acknowledged.
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This work was supported in part by research grants Nos. CA-07409 and HD-02562 from NIH, USPHS.
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Tokuyasu, K, Madden, S C & Zeldis, L J, J cell biol 39 (1968) 630. Comings, G E & Kakefuda, T, J mol biol 33 (1968) 225. Robbins, E, Jentzsch, G & Michali, A, J cell biol 36 (1968) 329. Terashima, T & Tolmach, L J, Exptl cell res 30 (1963) 344. Sasaki, M S & Norman, A, Nature 210 (1966) 913. Gall, J G, J biophys biochem cytol 10 (1961) 163. Sorokin, S P, J cell sci 3 (1968) 207. Hay, E D & Revel, J P, J cell biol 16 (1963) 29. Littau, V C, Allfrey, V G, Frenster, J H & Mirsky, A E, Proc natl acad sci US 52 (1964) 93. Karasaki, S, J cell biol 26 (1965) 937. Murray. R C. Murray. A S & Pizzo, A, J cell -.biol 28 (1965)‘601. FT9bbins, E & Gonatas, N K, J cell biol21 (1964)
13. Daiis, H G & Tooze, J, J cell sci 1 (1966) 331. 14. Comings, D E & Okada, T A, Exptl cell res 63 (1970) 471. 15. Rueckert, R R & Mueller, C C, Cancer res 20 (1960) 1584. 16. Weakley, B S, J anat 101 (1967) 435. 17. Baker, T G & Franchi, L L, J cell sci 2 (1967) 213. 18. Bonner, J, Dahmus, M E, Fambrough, D, Huang, R C C, Marushige, K & Tuan, D Y H, Science 159 (1968) 47. 19. Shih, T Y & Bonner, J, J mol biol 50 (1970) 333. 20. Cairns, J, J mol biol 15 (1966) 372. 21. Huberman, J A & Riggs, A D, J mol biol 32 (1968) 327. Received November 22, 1971 Revised version received February 1, 1972