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A contractile ring and cortical changes found in the dividing Tetrahymena pyriformis
Copyright@ 1980 by Academic Press, Inc. 411 rights of reproduction m any form reserve-; 0414-48271801080407-i If02,W/O
Experimental
co
ACTILE
RING
DIVIDING TOMOYOSNI
YASUDA,
OSAMU
Cell Research 128 (1980) 4
AND
C
~E~~A~Y~~~~ NUMATA,l
KAZUQ
OHNISHI,’
and YOS
Laboratory of Technology, National Institute of Health ofJapun, Shinagawa-ku, Xamioosaki, and ‘Institute ofBiological Sciences, The University ofTsukuba, Niihari-gun, Sakura-mura, Ibaraki 355, Japan
Tokyo 141,
SUMMARY A contractile ring consisting mainly of microfilaments was found in the fission zone of dividing Tetrahymena pyriformis. Diameters of the microfilaments were widely distributed from 2.5 to 15 nm. Ring-associated structures such as lateral stripes, linkers and beads with slender tails were recognized. Lateral stripes arranged at regular intervals of about 84 nm on some parts of rnicrofilament bundles were found in both tangential and transverse sections, suggesting that they correspond to bands fastening the contractile ring microfilaments. Linkers that connect individual lateral stripes to the epiplasmic layer were present. Beads or beads with slender tails were found to be arranged on some microfilaments. The results of the present paper also indicate that drastic morphological changes occur in the cortex of the fission zone, especially in the epiplasmic layer, accompanying contraction of the division furrow. The epiplasmic layer which was proved to be a compact filamentous network in this study has been known to exist at the periphery of cytoplasm in immediate contact with one of the cell surface membranes, the inner alveolar membrane; however, in the fission zone of the dividing cell, it was frequently separated from the membrane and subsided into the cytoplasm. The subsided epiplasmic layer was then loosened and dispersed. The subsidence of the epiplasmic Iayer appears to be caused by the force generated by the contraction of the contractile ring and transmitted with the linkers to the epiplasmic layer. The changes observed in the epiplasmic layer are presumably indispensable for the rigid cortical layer contraction involved ic cytokinesis of Tetrahymena.
first found microfilaarallel to the cleavage furthe cell membrane of a egg [I]. Similar microfilaments present at the margin of the cleavage furrow were observed in many kinds of ~~~rna~ cells; e.g. 9 Loligo eggs [2], Arbacia eggs [3--j], Armandia and Aequorea 61, newt eggs [7,8], mouse mammary [9], and He%a cells [lo]. Hence, it follows that icrofilament bundles localize at the margin of division furrow correspond to a ‘contractile ring’ of a dividing cell. In some of these cells, the microfila27-801804
ment has been filament from the evi thickness of micrQf~~a that of actin filame micrQ~ame~ts were
non-muscle
antibody
* To whom
offprint
requests
should
actin
specific
be
addressed.
53r
408
Yasuda et al.
In ciliate protozoa, there have been few reports concerning division-associated microfilaments or a contractile ring. In dividing Nassula, Tucker found the contractile ring microfilaments in relatively deep division furrows and described a possible role of microfilaments in the constriction of the division furrow [Ill. However, detailed study on division-associated microfilaments in ciliates other than Nassula has not yet been made. A ciliate, Tetrahymena, used in the present experiments is known to be one of the best organisms for studying cell division from the angles of morphology, physiology, biochemistry and genetics. The present paper describes the evidence that a contractile ring consisting of microfilaments and some ring-associated structures is formed in the fission zone of dividing Tetrahymena pyriformis and that, in the fission zone, cortical structures show drastic morphological changes accompanying the contraction of the division furrow. MATERIALS
AND
METHODS
Tetrahymena pyriformis strain W was used in the present experiments. Cultivation of the cells and the induction of synchronous division were performed as described previously r121. For the electron-microscopic observation, the synchronized cells were fixed with 2% glutaraldehyde in 0.03 M phosphate buffer (pH 7.4) at room temperature for 30 mm and postfixed with 1% 0~0, in 0.1 M phosphate buffer (pH 7.4) at 0°C for 1 h. Dehydration was performed in a graded series of ethanol followed by two changes of propylene oxide. The cells were then transferred to Epon 812 [13] and embedded in a flat boat according to the procedure of Allen [14]. A cell of an appropriate dividing stage embedded in the resin plate was selected and marked under a light microscope and then a small block including the cell was cut out with a razor’s edge. The block was held at a selected orientation with a specimen holder or fixed with a larger block by epoxy resin. The block was then trimmed and sectioned with an LKB-8800 microtome. Thin sections were stained with uranyl acetate and lead citrate by the method of Venable & Coggeshall [15]. Electron micrographs were taken with a Hitachi H-500. In some experiments, cells were glycerinated according to the method of Ishikawa et al. [16], exExp CellRes
128(1980)
cept that the glycerination solution and the washing solution contained 10m4 M TLCK (Na-tosyl-L-lysylchloromethane hydrochloride, a potent protease inhibitor) and that deglycerination steps were performed more quickly. After deglycerination, the cells were fixed for electron-microscopic observations.
RESULTS Fine structure of the contractile ring in dividing Tetrahymena Microfilaments running parallel to a fission zone were detected in a tangential section cutting along the dividing surface. The width of the zone including parallel tilaments was estimated to be 1.5 pm at the maximum (fig. 1 a). In a more magnified photograph (fig. lb), unique lateral stripes were seen on some part of parallel-running microfilaments. Usually, 5-8 stripes were arranged at regular intervals of about 84 nm (83.6k4.4 nm in the estimations of 60 stripes) and the dimension of each stripe was 20-25 nm wide and 40-50 nm long. In fig. 1 c, other structures like beads or beads with slender tails arranged on a microfilament were observed. The beads, each having the diameter of 16 nm, were spacing at the distance of about 35 nm. The microfilaments running parallel to the fission line were also observed in transverse sections (figs 24). Fig. 2 shows a transverse section of the furrow region of a dividing cell having shallow constriction, the diameter of which is 12 pm. In the cell, microfilaments were present close to the epiplasmic layer and the distance from the cell surface to the inner margin of microfilament zone was estimated to be about 0.1 pm. On the other hand, figs 3 and 4 show the transverse sections of dividing cells having deep constrictions, the diameters of which are about 3 pm. In these cells, contractile ring microfilaments run deep below the cell surface, and not closer to the epiplasmic layer. The inner periphery of the
Fig. 1. Microfilaments along the fission zone of dividing Teetruhymena (tangential section). (a) Parallel-running microfilaments are seen in the fission zone (a, bracket). CIA, Oral apparatus; EpL, epiplasmic layer; FZ, fission zone. (6) Enlargement of the rectangular
contractile ring microfilaments was very distinct as compared with the outer periphery and was clearly distinguished from the endoplasm. The distance from the cell
region ‘a’ of (a). Lateral stripes are seen on micro(c) Enlargement of the rectangular filaments (arrows). region ‘b’ of (a). Beads or beads with slender tails are seen (arrows).
microfi~ame~ts
were ranged from
2,5 to
ExpCrOR?_\128him?)
410
Yasuda et al.
Fig. 2. Microfilaments in the furrow region of dividing cell with shallow constriction (transverse section). Note that contractile ring microfilaments (CRM) are closely associated with the epiplasmic layer (EpL).
Fig. 3. Contractile ring in the furrow region of dividing cell with deep constriction (transverse section). EpL, Epiplasmic layer; CR, contractile ring. As for arrows, see the text.
Fig. 4. Contractile ring and its associated structures in the furrow region of dividing cell with deep constriction (transverse section cutting with a little oblique angle). (a) A wide view of the contractile ring and its associated structures and of subsidence of epiplasmic layer. CM, Cell membrane; OAM, outer alveolar membrane; I&W, inner alveolar membrane; EpE, epi-
plasmic layer; s.EpL, subsided epiplasmic layer; CR, contractile ring; L, linkers; LS, later& stripes. (b, c) Higher magnifications of the rectangular regions ‘a’ and ‘b’ of (a), respectiveiy. Lateral stripes and hnkers (arrows) are more clearly seen. Note that microfilamerits having the diameters of l&t5 nip are prominent in (c).
412
Yasuda et al.
15 nm. A histogram of the diameters of 165 sharply outlined filaments from 21 enlarged micrographs suggested that microfilaments may roughly be classified into three groups;
2.5, 5 and lo-15 nm in diameter (fig. 5). These microfilaments except the thinnest one give an impression that they were not so solid and straight as actin filaments in appearance. The lateral stripes on the contractile ring microfilaments were also observed in a transverse section (fig. 4). Since the features of the lateral stripes are the same as those observed in the tangential section shown before (fig. 1 b), it is likely that the lateral stripes are regularly arranged rings (or disks) which fasten the microfilaments. From the respective lateral stripes, filamentous linkers were started to join the epiplasmic layer (fig. 4b). The dimensions of linkers were 10-20 nmx0.15-0.2 pm.
Fig. 6. Fine structures of the epiplasmic layers in normal (a) and glycerinated cells (b, c, d). CM, Cell membrane; OAM, outer alveolar membrane; ZAM, inner alveolar membrane; EpL, epiplasmic layer; K,
kinetosome; Kd, kinetodesmal fiber; BM, broken mitochondrion. Bridge structures between inner alveolar membrane and epiplasmic filaments and between epiplasmic filaments are shown by arrows (c, d).
Diameters
of microfilaments
lnm)
Fig. 5. Histogram of diameters of contractile ring microfilaments. Only sharply outlined microfilaments were selected from the micrographs of x~OOOOO250 000.
Exp CrllRes
IZS(1980)
Contractile
ring in dividing
~et~~~yrn~~~
443
Figs 7-10. Subsidence of epiplasmic layer. Figs 7-9 represent the epiplasmic layer subsidences seen in the fission zones of dividing cel!s with shallow constrictier,. A part of epiplasmic !ayer is about to be tugged inside (fig. 7, arrml). Limited parts of epiplasmic layer are tugged inside (fig. 8. U~KWS). Epiplasmic layer is just removed frolm inner alveolar membrane (fig. 9, arrow.). Fig. 10 represents the epiplasmic layer
subsidence seen in the fission zone of dividkg deli with deep constriction. In the epiplasmic subsidence. contractile ring microfilaments (CRM), lateral stripes (LS), Linkers (I,) and subsided epiplasmic layer $@Li are obviously in unison. CM, Cell membra;re; OAM, outer alveolar membrane; IAM, inner alveolar membrane
Changes in cortex accompanied with contraction of division furrow
somes and their associated fibers [ 141 and 1171. Immediately before the appears, ~~frac~~~at~~e sys-
the cell morphology is by cortical structm-es such as the infraciliature system including kinetoIn
gion
and the so-called
‘fission
zone’
is
414
Yasuda et al.
formed [18]. However, solid epiplasm still in fig. 11. The three-layered membranes exists during fission. The epiplasmic layer and epiplasmic layer were recognized to be exists just below the inner alveolar meminvaginated into cytoplasm. brane as a thin, homogeneous layer showThe drastic changes in the epiplasmic ing a relatively high electron-density in or- layer were only detected in the fission zone dinary electron micrographs (fig. 6a). Howor its very vicinity of dividing cell, but ever, when the cells were glycerinated, the neither in any other region of the dividing epiplasmic layer was expanded to a con- cell nor in interphase cell. These events siderable extent and showed a fine filapresumably represent a passive contraction mentous network (reticulum) adhering process of the cortex, in contrast to an acclosely to the inner alveolar membrane with tive contraction supposed to occur in the some bridges (fig. 6b, c, d). This indicates contractile ring. that the epiplasmic layer is basically fibrous in composition. In the cortex of the fission DISCUSSION zone, drastic morphological changes appeared to be involved in the contraction of In the present paper we demonstrated the the contractile ring. The most prominent contractile ring consisting mainly of microwere the separation of the epiplasmic layer filaments in dividing Tetrahymena cells from the inner alveolar membrane, the sub- (figs 2-4). This contractile ring is seemingsidence of epiplasmic layer toward the in- ly very similar to those of other animal side, and the loosening of the subsided epi- cells, which suggests that the contractile plasm. As shown in fig. 4a, the epiplasmic ring of Tetrahymena also plays an imporlayer seemed to be tugged into the cyto- tant role in cytokinesis. However, the applasm to the large extent. The subsided pearance of microfilaments in Tetrahymena epiplasm was somewhat expanded and differed somewhat from that of actin filafluffed, suggesting that the disorganization ments in other animal cells. As far as we of the stiff layer was in progress. At an know, there is no direct evidence showing early stage of the cell division, the sub- the presence of actin in any ciliate proto. sidence of epiplasmic layer tended to occur zoon. In our other papers it is shown that to a limited extent in association mainly (1) although we failed to detect any typical with microfilaments (figs 7-9), whereas at a actin in Tetrahymena, 38 000 D protein can late stage the subsidence of epiplasmic be isolated from Tetrahymena acetone layer occurred to a larger extent in associa- powder by applying the isolation method tion with the combined structure of microfor non-muscle actin [19]; (2) that the profilaments, lateral stripes and linkers (fig. 10). The subsidence of the epiplasmic layerII. Invagination of cell membrane, outer and inner alveolar membrane complex was also Fig. inner alveolar membranes and euiplasmic layer. Four seen in the fission zone (arrows in fig. 3). serial transverse sections (ad -in the aider) are shown. (a) Middle of fission plane; (b, c) posterior The most effective way of cutback of the regions bf’the fission zone (parts of contrakite ring cortex is thought to be invagination of cell are still observable); (d) posterior part just outside the fission zone. Arrows indicate the invagination of cell membrane, outer and inner alveolar memmembrane (CM), outer alveolar membrane (OAM), branes and epiplasmic layer altogether into inner alveol& n&mbrane (ZAM) and epiplasmic layer altogether in (d), and the invaginated vesicle the cytoplasm. Occurrence of this process (EpL) being absorbed in (c). CR, Contractile ring; M, mitois assumed from the serial sections shown chondrion. Exp Ceil
Res 128 (1980)
416
Yasuda et al.
tein is localized in the fission zone of dividing Tetrahymena [20], and (3) that the protein can be assembled into 13-15 nm filaments in vitro [21]. Concerning the medium size microfilaments (about 5 nm in diameter), they did not form arrowhead complex with heavy meromyosin (unpublished data). It is, therefore, likely that the contractile ring of Tetrahymena is similar to those of other animal cells, but its protein component(s) is somewhat dissimilar. In ciliates, a contractile ring is found in Tetrahymena (the present paper) in addition to Nassula [l I], but some difference exists between both species: virtually no microtubule is present in the fission plane of Tetvahymena (fig. 3), but a large number of microtubules is present between the cell surface and the contractile ring in Nassula. This suggests that the mechanism of cytokinesis in Tetvahymena differs slightly from that in Nassula, presumably in the formation of the fission zone. In the present observation, we found several contractile ring-associated structures. The first type is the lateral stripes which seem to fasten contractile ring microfilaments. Intervals between stripes are always about 84 nm wherever the stripes are observed. Such a defined interval probably reflects a certain periodicity within the microfilaments. Recently, Sattler & Staehelin reported the dense bands oriented on the fine filaments at intervals of 82.5-105 nm in the cortex of the oral ribs of Tetrahymena pyriformis [22]. Whether the bands are the same with the lateral stripes is not known at present. The second type of the ringassociated structures is linkers that connect the contractile ring to the epiplasmic layer. Most of them appear to start from the lateral stripes (fig. 4~). The third type is minute structures on microfilaments, beads or beads with slender tails. These minute Exp Cell
Res 128 t/980)
structures might have a relation to the sliding of microfilaments. In the present paper, we first found the drastic morphological changes in the cortex of the fission zone accompanied with the deepening of the division furrow. The most prominent was the subsidence of the epiplasmic layer (figs 4, T-10). At an early dividing stage, epiplasmic layer is tugged to a limited small extent presumably by contraction of the associated microfilaments (figs 7-9). On the other hand, tugging of the epiplasmic layer occurs to a larger extent at a late dividing stage (figs 4, 10). The tugging may result from the following process: the force primarily generated by the contraction of the contractile ring is subsequently transmitted to epiplasmic layer via the linkers. The subsidence of epiplasmic layer is probably indispensable for facilitating the cutback of the rigid cortex, in another sense, for the cytokinesis of ciliate Tetrahymena. The drastic cortical changes, as seen in the furrow region of Tetrahymena, have not yet been reported in other animal cells. This might reflect the differences in property of the cortices between Tetrahymena and other animal cells, although interaction between contractile ring and cortex would occur in all cell types.
To elucidate the components of contractile ring and the morphological process of the cell division is considered to be the utmost important step for solving the molecular mechanism of cell division. Especially, in Tetrahymena, Frankel et al. have succeeded in the isolation of several tsmutants affecting the cell division [23, 241, and we also obtained similar mutants quite recently (unpublished data). Some of these mutants are prevented from completing division but form an incomplete division furrow at a restrictive temperature. We are
now able difference e cells servation. biological sion-related
to scrutinize the ultrastructural between ts-mutants and wildon the basis of the present obSuch a study will connect the function and structure of a divisubstance. EFERENCES
I. Schroeder, T E, Exp cell res 53 (1968) 272. 2. Arnold, J M, J cell biol 41 (1969) 894. 3. Tilney, L G & Marsland, D. J cell biol 42 (1969) 170, 4. Schroeder. T E. Biol bull 137 (1969) 413. 5. - J cell biol 53 (1972) 419. 6. Szollosi. D, J cell bio! 44 (1970) 192. 7. Seiman, G G & Perry, MM, J cell scih (1970)207. 8. Perry, M M. John, H A & Thomas, N S T, Exp ccl! res 65 (1971) 249. 9. Scott,DG&Daniel,CW,Jcellbiol45(l970)46l. 10. Schroeder, T E, Proc natl acad sci US 70 (1973) 1688.
Pnntcd
:,I Sweden
il. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Tucker, J B, J cell sci 8 (1971) 557. Watanabe. Y. Exo cell res 68 (1971) 431 Luft, J H, J biophys biochem cytol’9 (1961) 409, Allen, R D, J cell biol 40 (1969) 716. Venable. J H & Coggeshall, R, J ceil biol 25 (1965) 407. Ishikawa, H, Bischoff. R & Holtzer, H. : cell biol 43 (1969) 3i2. Peck. R K. J cell sci 25 (1977) 367. Franked, J, J exp zoo1 155 (1964) 403. Numata. 0, Yasuda. T. Hirabavashi. T 4% Watanabe. Y. Submitted for publication. - Submitted for publication. Numata. 0, Uasuda. T, Ohnishi, K & Watanabe. I’. Submitted for publication. Saitler. C M & Staehelin, t A. J uiirastruct res 66 (1979) 132. Frankei. 3, Jenkins. L M, Nelsen, E M & Doerder. F P, Genetics 83 (I 976) 489. Franked. J, Nelsen, E M&Jenkins, L M. Dev hiol 58 (1977) 255.
Received December 5, 1979 Accepted February 12, 1980
Experimental
co
ACTILE
RING
DIVIDING TOMOYOSNI
YASUDA,
OSAMU
Cell Research 128 (1980) 4
AND
C
~E~~A~Y~~~~ NUMATA,l
KAZUQ
OHNISHI,’
and YOS
Laboratory of Technology, National Institute of Health ofJapun, Shinagawa-ku, Xamioosaki, and ‘Institute ofBiological Sciences, The University ofTsukuba, Niihari-gun, Sakura-mura, Ibaraki 355, Japan
Tokyo 141,
SUMMARY A contractile ring consisting mainly of microfilaments was found in the fission zone of dividing Tetrahymena pyriformis. Diameters of the microfilaments were widely distributed from 2.5 to 15 nm. Ring-associated structures such as lateral stripes, linkers and beads with slender tails were recognized. Lateral stripes arranged at regular intervals of about 84 nm on some parts of rnicrofilament bundles were found in both tangential and transverse sections, suggesting that they correspond to bands fastening the contractile ring microfilaments. Linkers that connect individual lateral stripes to the epiplasmic layer were present. Beads or beads with slender tails were found to be arranged on some microfilaments. The results of the present paper also indicate that drastic morphological changes occur in the cortex of the fission zone, especially in the epiplasmic layer, accompanying contraction of the division furrow. The epiplasmic layer which was proved to be a compact filamentous network in this study has been known to exist at the periphery of cytoplasm in immediate contact with one of the cell surface membranes, the inner alveolar membrane; however, in the fission zone of the dividing cell, it was frequently separated from the membrane and subsided into the cytoplasm. The subsided epiplasmic layer was then loosened and dispersed. The subsidence of the epiplasmic Iayer appears to be caused by the force generated by the contraction of the contractile ring and transmitted with the linkers to the epiplasmic layer. The changes observed in the epiplasmic layer are presumably indispensable for the rigid cortical layer contraction involved ic cytokinesis of Tetrahymena.
first found microfilaarallel to the cleavage furthe cell membrane of a egg [I]. Similar microfilaments present at the margin of the cleavage furrow were observed in many kinds of ~~~rna~ cells; e.g. 9 Loligo eggs [2], Arbacia eggs [3--j], Armandia and Aequorea 61, newt eggs [7,8], mouse mammary [9], and He%a cells [lo]. Hence, it follows that icrofilament bundles localize at the margin of division furrow correspond to a ‘contractile ring’ of a dividing cell. In some of these cells, the microfila27-801804
ment has been filament from the evi thickness of micrQf~~a that of actin filame micrQ~ame~ts were
non-muscle
antibody
* To whom
offprint
requests
should
actin
specific
be
addressed.
53r
408
Yasuda et al.
In ciliate protozoa, there have been few reports concerning division-associated microfilaments or a contractile ring. In dividing Nassula, Tucker found the contractile ring microfilaments in relatively deep division furrows and described a possible role of microfilaments in the constriction of the division furrow [Ill. However, detailed study on division-associated microfilaments in ciliates other than Nassula has not yet been made. A ciliate, Tetrahymena, used in the present experiments is known to be one of the best organisms for studying cell division from the angles of morphology, physiology, biochemistry and genetics. The present paper describes the evidence that a contractile ring consisting of microfilaments and some ring-associated structures is formed in the fission zone of dividing Tetrahymena pyriformis and that, in the fission zone, cortical structures show drastic morphological changes accompanying the contraction of the division furrow. MATERIALS
AND
METHODS
Tetrahymena pyriformis strain W was used in the present experiments. Cultivation of the cells and the induction of synchronous division were performed as described previously r121. For the electron-microscopic observation, the synchronized cells were fixed with 2% glutaraldehyde in 0.03 M phosphate buffer (pH 7.4) at room temperature for 30 mm and postfixed with 1% 0~0, in 0.1 M phosphate buffer (pH 7.4) at 0°C for 1 h. Dehydration was performed in a graded series of ethanol followed by two changes of propylene oxide. The cells were then transferred to Epon 812 [13] and embedded in a flat boat according to the procedure of Allen [14]. A cell of an appropriate dividing stage embedded in the resin plate was selected and marked under a light microscope and then a small block including the cell was cut out with a razor’s edge. The block was held at a selected orientation with a specimen holder or fixed with a larger block by epoxy resin. The block was then trimmed and sectioned with an LKB-8800 microtome. Thin sections were stained with uranyl acetate and lead citrate by the method of Venable & Coggeshall [15]. Electron micrographs were taken with a Hitachi H-500. In some experiments, cells were glycerinated according to the method of Ishikawa et al. [16], exExp CellRes
128(1980)
cept that the glycerination solution and the washing solution contained 10m4 M TLCK (Na-tosyl-L-lysylchloromethane hydrochloride, a potent protease inhibitor) and that deglycerination steps were performed more quickly. After deglycerination, the cells were fixed for electron-microscopic observations.
RESULTS Fine structure of the contractile ring in dividing Tetrahymena Microfilaments running parallel to a fission zone were detected in a tangential section cutting along the dividing surface. The width of the zone including parallel tilaments was estimated to be 1.5 pm at the maximum (fig. 1 a). In a more magnified photograph (fig. lb), unique lateral stripes were seen on some part of parallel-running microfilaments. Usually, 5-8 stripes were arranged at regular intervals of about 84 nm (83.6k4.4 nm in the estimations of 60 stripes) and the dimension of each stripe was 20-25 nm wide and 40-50 nm long. In fig. 1 c, other structures like beads or beads with slender tails arranged on a microfilament were observed. The beads, each having the diameter of 16 nm, were spacing at the distance of about 35 nm. The microfilaments running parallel to the fission line were also observed in transverse sections (figs 24). Fig. 2 shows a transverse section of the furrow region of a dividing cell having shallow constriction, the diameter of which is 12 pm. In the cell, microfilaments were present close to the epiplasmic layer and the distance from the cell surface to the inner margin of microfilament zone was estimated to be about 0.1 pm. On the other hand, figs 3 and 4 show the transverse sections of dividing cells having deep constrictions, the diameters of which are about 3 pm. In these cells, contractile ring microfilaments run deep below the cell surface, and not closer to the epiplasmic layer. The inner periphery of the
Fig. 1. Microfilaments along the fission zone of dividing Teetruhymena (tangential section). (a) Parallel-running microfilaments are seen in the fission zone (a, bracket). CIA, Oral apparatus; EpL, epiplasmic layer; FZ, fission zone. (6) Enlargement of the rectangular
contractile ring microfilaments was very distinct as compared with the outer periphery and was clearly distinguished from the endoplasm. The distance from the cell
region ‘a’ of (a). Lateral stripes are seen on micro(c) Enlargement of the rectangular filaments (arrows). region ‘b’ of (a). Beads or beads with slender tails are seen (arrows).
microfi~ame~ts
were ranged from
2,5 to
ExpCrOR?_\128him?)
410
Yasuda et al.
Fig. 2. Microfilaments in the furrow region of dividing cell with shallow constriction (transverse section). Note that contractile ring microfilaments (CRM) are closely associated with the epiplasmic layer (EpL).
Fig. 3. Contractile ring in the furrow region of dividing cell with deep constriction (transverse section). EpL, Epiplasmic layer; CR, contractile ring. As for arrows, see the text.
Fig. 4. Contractile ring and its associated structures in the furrow region of dividing cell with deep constriction (transverse section cutting with a little oblique angle). (a) A wide view of the contractile ring and its associated structures and of subsidence of epiplasmic layer. CM, Cell membrane; OAM, outer alveolar membrane; I&W, inner alveolar membrane; EpE, epi-
plasmic layer; s.EpL, subsided epiplasmic layer; CR, contractile ring; L, linkers; LS, later& stripes. (b, c) Higher magnifications of the rectangular regions ‘a’ and ‘b’ of (a), respectiveiy. Lateral stripes and hnkers (arrows) are more clearly seen. Note that microfilamerits having the diameters of l&t5 nip are prominent in (c).
412
Yasuda et al.
15 nm. A histogram of the diameters of 165 sharply outlined filaments from 21 enlarged micrographs suggested that microfilaments may roughly be classified into three groups;
2.5, 5 and lo-15 nm in diameter (fig. 5). These microfilaments except the thinnest one give an impression that they were not so solid and straight as actin filaments in appearance. The lateral stripes on the contractile ring microfilaments were also observed in a transverse section (fig. 4). Since the features of the lateral stripes are the same as those observed in the tangential section shown before (fig. 1 b), it is likely that the lateral stripes are regularly arranged rings (or disks) which fasten the microfilaments. From the respective lateral stripes, filamentous linkers were started to join the epiplasmic layer (fig. 4b). The dimensions of linkers were 10-20 nmx0.15-0.2 pm.
Fig. 6. Fine structures of the epiplasmic layers in normal (a) and glycerinated cells (b, c, d). CM, Cell membrane; OAM, outer alveolar membrane; ZAM, inner alveolar membrane; EpL, epiplasmic layer; K,
kinetosome; Kd, kinetodesmal fiber; BM, broken mitochondrion. Bridge structures between inner alveolar membrane and epiplasmic filaments and between epiplasmic filaments are shown by arrows (c, d).
Diameters
of microfilaments
lnm)
Fig. 5. Histogram of diameters of contractile ring microfilaments. Only sharply outlined microfilaments were selected from the micrographs of x~OOOOO250 000.
Exp CrllRes
IZS(1980)
Contractile
ring in dividing
~et~~~yrn~~~
443
Figs 7-10. Subsidence of epiplasmic layer. Figs 7-9 represent the epiplasmic layer subsidences seen in the fission zones of dividing cel!s with shallow constrictier,. A part of epiplasmic !ayer is about to be tugged inside (fig. 7, arrml). Limited parts of epiplasmic layer are tugged inside (fig. 8. U~KWS). Epiplasmic layer is just removed frolm inner alveolar membrane (fig. 9, arrow.). Fig. 10 represents the epiplasmic layer
subsidence seen in the fission zone of dividkg deli with deep constriction. In the epiplasmic subsidence. contractile ring microfilaments (CRM), lateral stripes (LS), Linkers (I,) and subsided epiplasmic layer $@Li are obviously in unison. CM, Cell membra;re; OAM, outer alveolar membrane; IAM, inner alveolar membrane
Changes in cortex accompanied with contraction of division furrow
somes and their associated fibers [ 141 and 1171. Immediately before the appears, ~~frac~~~at~~e sys-
the cell morphology is by cortical structm-es such as the infraciliature system including kinetoIn
gion
and the so-called
‘fission
zone’
is
414
Yasuda et al.
formed [18]. However, solid epiplasm still in fig. 11. The three-layered membranes exists during fission. The epiplasmic layer and epiplasmic layer were recognized to be exists just below the inner alveolar meminvaginated into cytoplasm. brane as a thin, homogeneous layer showThe drastic changes in the epiplasmic ing a relatively high electron-density in or- layer were only detected in the fission zone dinary electron micrographs (fig. 6a). Howor its very vicinity of dividing cell, but ever, when the cells were glycerinated, the neither in any other region of the dividing epiplasmic layer was expanded to a con- cell nor in interphase cell. These events siderable extent and showed a fine filapresumably represent a passive contraction mentous network (reticulum) adhering process of the cortex, in contrast to an acclosely to the inner alveolar membrane with tive contraction supposed to occur in the some bridges (fig. 6b, c, d). This indicates contractile ring. that the epiplasmic layer is basically fibrous in composition. In the cortex of the fission DISCUSSION zone, drastic morphological changes appeared to be involved in the contraction of In the present paper we demonstrated the the contractile ring. The most prominent contractile ring consisting mainly of microwere the separation of the epiplasmic layer filaments in dividing Tetrahymena cells from the inner alveolar membrane, the sub- (figs 2-4). This contractile ring is seemingsidence of epiplasmic layer toward the in- ly very similar to those of other animal side, and the loosening of the subsided epi- cells, which suggests that the contractile plasm. As shown in fig. 4a, the epiplasmic ring of Tetrahymena also plays an imporlayer seemed to be tugged into the cyto- tant role in cytokinesis. However, the applasm to the large extent. The subsided pearance of microfilaments in Tetrahymena epiplasm was somewhat expanded and differed somewhat from that of actin filafluffed, suggesting that the disorganization ments in other animal cells. As far as we of the stiff layer was in progress. At an know, there is no direct evidence showing early stage of the cell division, the sub- the presence of actin in any ciliate proto. sidence of epiplasmic layer tended to occur zoon. In our other papers it is shown that to a limited extent in association mainly (1) although we failed to detect any typical with microfilaments (figs 7-9), whereas at a actin in Tetrahymena, 38 000 D protein can late stage the subsidence of epiplasmic be isolated from Tetrahymena acetone layer occurred to a larger extent in associa- powder by applying the isolation method tion with the combined structure of microfor non-muscle actin [19]; (2) that the profilaments, lateral stripes and linkers (fig. 10). The subsidence of the epiplasmic layerII. Invagination of cell membrane, outer and inner alveolar membrane complex was also Fig. inner alveolar membranes and euiplasmic layer. Four seen in the fission zone (arrows in fig. 3). serial transverse sections (ad -in the aider) are shown. (a) Middle of fission plane; (b, c) posterior The most effective way of cutback of the regions bf’the fission zone (parts of contrakite ring cortex is thought to be invagination of cell are still observable); (d) posterior part just outside the fission zone. Arrows indicate the invagination of cell membrane, outer and inner alveolar memmembrane (CM), outer alveolar membrane (OAM), branes and epiplasmic layer altogether into inner alveol& n&mbrane (ZAM) and epiplasmic layer altogether in (d), and the invaginated vesicle the cytoplasm. Occurrence of this process (EpL) being absorbed in (c). CR, Contractile ring; M, mitois assumed from the serial sections shown chondrion. Exp Ceil
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tein is localized in the fission zone of dividing Tetrahymena [20], and (3) that the protein can be assembled into 13-15 nm filaments in vitro [21]. Concerning the medium size microfilaments (about 5 nm in diameter), they did not form arrowhead complex with heavy meromyosin (unpublished data). It is, therefore, likely that the contractile ring of Tetrahymena is similar to those of other animal cells, but its protein component(s) is somewhat dissimilar. In ciliates, a contractile ring is found in Tetrahymena (the present paper) in addition to Nassula [l I], but some difference exists between both species: virtually no microtubule is present in the fission plane of Tetvahymena (fig. 3), but a large number of microtubules is present between the cell surface and the contractile ring in Nassula. This suggests that the mechanism of cytokinesis in Tetvahymena differs slightly from that in Nassula, presumably in the formation of the fission zone. In the present observation, we found several contractile ring-associated structures. The first type is the lateral stripes which seem to fasten contractile ring microfilaments. Intervals between stripes are always about 84 nm wherever the stripes are observed. Such a defined interval probably reflects a certain periodicity within the microfilaments. Recently, Sattler & Staehelin reported the dense bands oriented on the fine filaments at intervals of 82.5-105 nm in the cortex of the oral ribs of Tetrahymena pyriformis [22]. Whether the bands are the same with the lateral stripes is not known at present. The second type of the ringassociated structures is linkers that connect the contractile ring to the epiplasmic layer. Most of them appear to start from the lateral stripes (fig. 4~). The third type is minute structures on microfilaments, beads or beads with slender tails. These minute Exp Cell
Res 128 t/980)
structures might have a relation to the sliding of microfilaments. In the present paper, we first found the drastic morphological changes in the cortex of the fission zone accompanied with the deepening of the division furrow. The most prominent was the subsidence of the epiplasmic layer (figs 4, T-10). At an early dividing stage, epiplasmic layer is tugged to a limited small extent presumably by contraction of the associated microfilaments (figs 7-9). On the other hand, tugging of the epiplasmic layer occurs to a larger extent at a late dividing stage (figs 4, 10). The tugging may result from the following process: the force primarily generated by the contraction of the contractile ring is subsequently transmitted to epiplasmic layer via the linkers. The subsidence of epiplasmic layer is probably indispensable for facilitating the cutback of the rigid cortex, in another sense, for the cytokinesis of ciliate Tetrahymena. The drastic cortical changes, as seen in the furrow region of Tetrahymena, have not yet been reported in other animal cells. This might reflect the differences in property of the cortices between Tetrahymena and other animal cells, although interaction between contractile ring and cortex would occur in all cell types.
To elucidate the components of contractile ring and the morphological process of the cell division is considered to be the utmost important step for solving the molecular mechanism of cell division. Especially, in Tetrahymena, Frankel et al. have succeeded in the isolation of several tsmutants affecting the cell division [23, 241, and we also obtained similar mutants quite recently (unpublished data). Some of these mutants are prevented from completing division but form an incomplete division furrow at a restrictive temperature. We are
now able difference e cells servation. biological sion-related
to scrutinize the ultrastructural between ts-mutants and wildon the basis of the present obSuch a study will connect the function and structure of a divisubstance. EFERENCES
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Received December 5, 1979 Accepted February 12, 1980