Unorthodox Mitosis in Trichonympha Agilis: Kinetochore Differentiation and Chromosome Movement

J. Cell Set. 13, 511-552 (i973) 511 Printed in Great Britain

UNORTHODOX MITOSIS IN TRICHONYMPHA AGILIS: KINETOCHORE DIFFERENTIATION AND CHROMOSOME MOVEMENT

DONNA F. KUBAI Department of Zoology, University of Wisconsin, Madison, Wisconsin 53706, U.S.A.

SUMMARY Changes in rostral structures and the nuclear events which occur in dividing cells of Tricho- nympha agilis (obtained from experimentally refaunated termites) were studied by means of electron microscopy of serial sections. It is possible to characterize 5 stages of division: Stage I. During this earliest recognizable division stage, the bilaterally symmetrical hemirostra have begun to separate and spindle microtubules appear in the intervening space. As in interphase, the kinetochore regions of chromosomes are distinguishable as fibrillar masses underlying the intact nuclear envelope; and, in individual sections, they are often seen to occur in pairs. These pairs are taken to be sister kinetochores. Stage II. The extranuclear spindle has become established between the posterior ends of well separated hemirostral tubes. Elaboration of daughter rostral structures begins and will continue through the subsequent stages of division. Kinetochores differentiate, becoming bipartite structures consisting of a fibrillar element underlain by a dense disk. The fibrillar kinetochore element is associated with the still-intact nuclear envelope which lies between kinetochores and cytoplasmic spindle microtubules. Reconstruction from serial sections shows all kinetochores to be disposed in pairs which are distributed randomly over the nuclear surface. Stage III. The fibrillar elements of kinetochores are enclosed in evaginations of the nuclear envelope, while the disk elements have come to lie in the plane of the nuclear surface. Kineto- chores remain separated from the extranuclear spindle microtubules by the intact nuclear envelope. The distribution of kinetochores has changed relative to that seen in stage II: kinetochores no longer appear to be paired, and they are confined to that hemisphere of the nuclear surface closest to the spindle. Stage IV. The nuclear envelope opens at the sites of kinetochores, leaving the dense disk kinetochore element inserted in pore-like discontinuities of the nuclear envelope and the fibrillar element in the cytoplasm. Direct interaction between fibrillar kinetochore element and extranuclear spindle microtubules is, however, not yet established. Stage V. The cytoplasmically situated fibrillar elements of 'inserted' kinetochores are now in direct contact with spindle microtubules. As seen in reconstructions of the nucleus from serial sections, kinetochores have become segregated in 2 groups on the nuclear surface, one near each spindle pole. It is during this stage that final elaboration of rostral structures takes place. On the basis of the observed changes in kinetochore distribution which occur between stages II and III while the intact nuclear envelope prevents any direct interaction between intranuclear kinetochores and extranuclear spindle microtubules, it is suggested that kinetochore- membrane interaction is involved in early chromosome movement in Trichonympha agilis. Only during stage V, when direct contact between kinetochores and spindle microtubules is established, may the microtubules assume their usual role in chromosome movement. 512 D.F.Kubai

INTRODUCTION In typical eukaryotes, chromosome segregation is under the control of the complex mitotic spindle apparatus. Though the detailed structural organization of the spindle may vary, especially among lower forms such as algae and fungi (Leedale, 1970), the direct involvement of spindle microtubules in chromosome movement is considered characteristic of eukaryotes (Stanier, 1970). In prokaryotes, on the other hand, microtubules are non-existent, and genophore segregation is thought to be a membrane- mediated phenomenon (Ryter, 1968). The nuclear division of at least one group of organisms, the dinoflagellates, appears to combine features of both prokaryotes and eukaryotes (Leadbeater & Dodge, 1967; Kubai & Ris, 1969; Soyer, 1969, 1971). Cytoplasmic microtubules are associated with the nucleus during division but do not interact directly with chromosomes. They, therefore, seem to serve only to define the polarity of the dividing nucleus, playing no immediate role in chromosome movement. Instead, the chromosomes become specifi- cally associated with the nuclear membrane during division; and it has been suggested that, as in prokaryotes, membrane growth or flow is responsible for chromosome distribution. Although mitosis in the hypermastigote flagellates has been considered orthodox (Hollande & Carruette-Valentin, 1971), certain features of their extensively described nuclear division (Cleveland, Hall, Sanders & Collier, 1934; Grasse, 1952; Hollande & Valentin, 1968a, b; Hollande & Carruette-Valentin, 1970, 1971) are unique. The mitotic spindle is entirely extranuclear and the nuclear membrane persists throughout division. The light-microscopical investigations of Cleveland et al. (1934) demonstrated the existence of specialized nuclear membrane-'chromosome fibre' interactions, through which engagement of chromosomes on the extra-nuclear mitotic spindle is established. More recently, Hollande & Carruette-Valentin (1971) have published numerous electron micrographs of these regions in various species of hypermastigotes, interpreting them as sites where kinetochores have become embedded in the nuclear membrane. Their work does not, however, provide a detailed ultrastructural description of the manner in which the kinetochore-membrane association develops. It is clear from the works of Hollande & Valentin (1968 a, b) and Hollande & Carruette- Valentin (1970, 1971) that microtubules converge at the sites of kinetochores. These authors, therefore, conclude that mitosis in hypermastigotes is conventional, with spindle fibres playing the major role in chromosome segregation. They provide no evidence, however, that chromosome movement takes place only after the kinetochore- microtubule association is established. In their light-microscopical investigations, Cleveland et al. (1934) did observe chromosome movement before chromosome- spindle engagement took place, but did not consider this to have any bearing on true chromosome segregation. The present study was undertaken to elucidate further the mitotic division mechanism in the hypermastigote flagellate Trichonympha agilis. Based on the study of serial sections of cells in various stages of division, the development of rostral structures Mitosis in Trichonympha 513 and the mitotic spindle, the differentiation of kinetochores and their engagement on the mitotic spindle, and aspects of kinetochore segregation will be described. On the basis of these findings, it is suggested that chromosome segregation in hypermastigotes involves the interaction of chromosomes with both membrane and microtubules, early segregation being influenced by membrane phenomena, while final segregation may be under the direct control of spindle microtubules. The mitotic mechanism thus may combine features of genophore segregation characteristic of both prokaryotes and eukaryotes.

MATERIALS AND METHODS Trichonympha agilis occurs as a member of the protozoan fauna in the hindgut of the termite ReticuUtermes flavipes. Termite cultures, established in small plastic chambers were generously provided by Dr G. R. Esenther (Forest Products Laboratory, Forest Service, USDA, Madison, Wisconsin 53705). These were maintained by the periodic addition of dampened aspen sawdust (Esenther, 1969).

Selection of dividing Trichonympha agilis Small termite cultures contain relatively few of the young forms or freshly moulted individuals in which divisions of Trichonympha agilis can be expected to occur naturally (Andrew & Light, 1929). For this reason, I chose to collect dividing cells from experimentally refaunated workers which had been defaunated by exposure to oxygen under pressure (Andrew & Light, 1929; Cleveland, 1925). For defaunation, termite workers, confined in a small wire cage, were placed in a pressure cylinder. After flushing with oxygen for 3 min, oxygen pressure within the cylinder was in- creased to 103 kN m~2 (15 lb in.~2) and carbon dioxide was added to yield a final pressure of 309 kN m~2 (45 lb in."2) (Bready & Friedman, 1963). Pressure was maintained for 45 min before the chamber was returned to atmosphere. All pressure changes were produced at a rate of approximately 7 kN m~2 s"1 (1 lb in.~2 s"1). After oxygen treatment, termites were kept in a humid atmosphere without food for 48 h. At the end of this time, the hindgut contents of several individuals were examined to verify that complete defaunation had been accomplished. Defaunated termites were refaunated by proctodeal feeding from normally faunated individuals (Cleveland, 1924). For refaunation, defaunated workers, marked for identification with a spot of red enamel applied to the head, were mixed with normal workers (at a ratio of 1 defau- nate to 3 normals) in 2-dram (7-4-1111) Titeseal vials (Lab Apparatus Co., Cleveland, Ohio 44128), 12 termites per vial. To maintain the necessary high humidity in these vials, the hollow plastic caps were perforated several times and the cavities stuffed with dampened cotton. A small amount of moist aspen sawdust was provided as food. Termites were maintained at approximately 25 °C in a room which was illuminated 14 h per day. Under these conditions, dividing Trichonympha appear in refaunated individuals approximately 15-17 days after refaunation is begun. During this interval, a variable proportion of refaunates harbour no dividing Trichonympha. Where divisions do occur, there may be as many as 40, but usually only 6—10 per gut.

Electron microscopy Termite hind guts were removed and placed in a small drop of very dilute Karnovsky (J965) fixative (0-13 % paraformaldehyde, 0-08% glutaraldehyde, 0006 M sodium phosphate buffer, pH 7-0) on a gel slide (acid-cleaned slides coated with 0-7% gelatin). The gut was opened with forceps, gut contents dispersed in the fixative, and a coverslip coated with Slip-Spray (E. I. DuPont de Nemours Co., Wilmington, Delaware, 19898) lowered gently over the preparation. In dilute fixative, Trichonympha is undistorted except for a very slight swelling, and when viewed at 50—100 x magnification, the flask-shaped non-dividing cells are easily distinguished 33 CEL 13 514 D.F.Kubai from the rounded, dividing individuals. After the positions of dividing cells were marked on the slide, the coverslip was gradually floated off by the addition of a more-concentrated Karnovsky fixative (2-0 % paraformaldehyde, 1-25 % glutaraldehyde, o-i M sodium phosphate buffer, pH 7-o). Fixation was continued for 2 h in the more-concentrated fixative, the slides were rinsed several times in o-1 M phosphate buffer and stored overnight in the same buffer at 4 °C. A second 2-h fixationwit h 2-0 % osmium tetroxide in o-i M phosphate buffer was followed by several washes in distilled water and a 2-h postnxation treatment with 0-5 % uranyl acetate in veronal-acetate buffer (Ryter & Kellenberger, 1958). Except as noted, all fixation procedures were carried out at room temperature. Slides were then passed through an ethanol series and propylene oxide-resin mixtures (Epon-Araldite) (Mollenhauer, 1964). For flat embedding, after final infiltration in the embedding mixture, excess plastic was drained from the slides and the marked areas inverted over Beem (Better Equipment for Electron Microscopy, Inc., Bronx, N.Y. 10468) capsule covers filled with the embedding mixture. Polymerization for approximately 3 days at 37 °C yielded soft but no longer tacky plastic disks which were easily separated from the slide by freezing on dry ice. Polymerization was then continued at 60 °C. Cells embedded in the 2-mm-deep plastic disks were examined for further selection in a phase-contrast microscope fitted with a long-focal-length condenser. Individual cells were serially sectioned (thickness = 50 nm as judged by interference colours) using the MT2-B 'Porter-Blum' Ultramicrotome (Sorvall, Norwalk, Conn.) and a DuPont (E. I. DuPont de Nemours, Co., Wilmington, Delaware, 19898) diamond knife. Ribbons of sections, picked up on Formvar films attached to wire loops, were transferred to single-hole grids (1x2 mm rectangular opening), stained with lead citrate (Reynolds, 1963) for 15 min or with uranyl magnesium acetate (Frasca & Parks, 1965) for 2 h followed by lead citrate. After staining, a thin carbon layer was evaporated over the sections. Micrographs were taken at original magnifications of 2200 times (serial sections) or 7500- 22000 times (individual micrographs) in a Siemens Elmiskop I at 80 kV with double condenser illumination. Kodalith LR Estar base roll film, developed in Kodak D-19 was used for serial electron micrographs, while Kodak Electron Image Plates developed in Kodak HRP were used for individual micrographs. Reconstruction of nuclei was as previously described (Kubai & Ris, 1969). No more than 2 consecutive sections were lost in any series of sections serving as basis for such reconstruction.

RESULTS In interphase, Trichonympha agilis is a pear- or spindle-shaped cell with a centrally situated nucleus. A structurally complex rostrum comprises the anterior-most portion of the cell (Kirby, 1932). During division, the posterior ends of the 2 hemirostra diverge and the cell assumes a spherical shape. As the hemirostra separate, an extranuclear spindle forms between them and the nucleus migrates anteriorly to assume a position just beneath the spindle. Chromosomes remain associated with the nuclear envelope at their kinetochores throughout the cell cycle. During division, these intranuclear kinetochores undergo a characteristic sequence of morphological changes, ultimately becoming inserted in openings of the nuclear envelope and making contact with extranuclear spindle microtubules. The more detailed description of this division which follows is based on the study of complete serial sections obtained from 32 cells. Table 1 summarizes the characteristics easily recognized through cursory examination of serial sections which I have used to classify various stages of division. Mitosis in Trichonympha 515 Rostrum The rostrum (Fig. 3) of interphase Trichonympha agilis has been described by Hollande & Carruette-Valentin (1970) and is essentially similar to the rostra of other species of Trichonympha (Pitelka, 1963; Grimstone & Gibbons, 1966). The apex of the cell, a dome-like outer rostral cap, is devoid of flagella; and a double layer of

Table 1. General features of cells at various stages of the cell cycle; numbers of cells sectioned and photographed at these stages

No. of cells No. of cells serially serially Stage Distinguishing features sectioned photographed Interphase Rostrum intact, no microtubules 4 — I Very slight rostrum separation; 1 — microtubules present but not organized into spindle II Well separated rostral halves; 4 1 well denned spindle; intranuclear kinetochores differentiated III Kinetochores enclosed in outpocketings 2 2 of intact nuclear envelope IV Some kinetochores as in stage III, 1 1 some as in stage V V Kinetochores inserted through openings 20 — in nuclear envelope and: a Spherical nucleus 1 b Central spindle in trough-like 1 deformation of the nucleus c Karyokinesis complete 1 microtubules reinforces the cell membrane in this region. Within the flagellated post- cap segment of the rostrum is the cross-striated rostral tube, a bilaterally symmetrical structure (conferring bilateral symmetry on the cell) consisting of opposed half- cylinders which originate anteriorly from 2 crescentic bodies. Kinetosomes of the rostral flagella abut on the rostral tube. Densely packed, finely fibrillar material, the central rod (the atractophore of Hollande & Carruette-Valentin, 1970), is found within the lumen in the anterior portion of the rostral tube. Fibres of the central rod fray out laterally and make intimate contact with the walls of the rostral tube. The inner rostral cap is an ill-defined mass of dense material lying between the outer cap and crescentic bodies. With regard to the disposition and structure of the rostral tube, rostral flagella, external cap and its reinforcing double layer of microtubules, my observations concur with those of Hollande & Carruette-Valentin (1970). However, the interrelationship between dense inner cap, anterior kinetosome(s) (cinetosome privilegie of Hollande & Carruette-Valentin, 1970), central rod and rostral cap is more compli- cated than was suggested by these authors. According to their description, 2 anterior kinetosomes are found in the rostral cap region, one lacking a flagellum and situated 33-2 516 D. F. Kubai in the external rostral cap, the other flagellated and lying in the cytoplasm separating the 2 rostral half tubes. In my preparations, one only anterior kinetosome can be recognized (Fig. 3), and in serial sections it is found to lack a flagellum. This anterior kinetosome is embedded in the dense amorphous inner cap, oriented with its long axis in the sagittal plane of the cell. Inner cap material is continuous, through the anterior opening of the rostral tube, with the central rod which in turn displays substantial connexions to the rostral tube near its junction with the crescentic bodies and with the crescentic bodies themselves (Fig. 3). Changes in the rostral structures of Trichonympha which occur at the time of nuclear division are well documented (Cleveland et al. 1934; Kirby, 1944; Hollande & Carruette-Valentin, 1970). In general, my observations of this process agree with the ultrastructural description which has been presented by Hollande & Carruette- Valentin (1970). In summary, in preparation for cellular reproduction, the bilaterally symmetrical rostrum splits in the sagittal plane into 2 hemirostra, with the concomitant breakdown of the rostral cap and its reinforcing microtubules. For each hemirostrum, which now includes a half-cylindrical rostral tube and associated kinetosomes, a second hemirostrum and a new rostral cap will be generated so as to provide each daughter cell with a complete set of rostral structures. Because examination of serial sections of whole cells at various stages of division allows a more detailed under- standing of these events and a closer correlation with nuclear division, a more detailed description is warranted here. Stage I is apparently the earliest stage of division encountered in my preparations, as judged by the appearance of microtubules destined to become organized into the mitotic spindle (see below, Spindle and Fig. 7). However, separation of the hemirostra has not yet begun, and the rostral cap is unchanged: a non-flagellated kinetosome is embedded in the dense inner cap (Fig. 4) and a still-intact double layer of microtubules underlies the membrane of the outer cap. In the earliest stage of division displaying well separated hemirostra (stage II), the dense inner cap and microtubules of the outer cap have disappeared (Fig. 5). Elabo- ration of the new rostral half tube together with associated kinetosomes has already begun (Figs. 1, 8). The new rostral tube material appears as a thin, curved striated lamella (lame parabasale of Hollande & Carruette-Valentin, 1970) extending from the anterior of each old rostral half tube. Thickening and elongation of the curved lamella proceeds throughout division (cf. Figs. 8, 10). Serial sections at stage Va indicate that the lamella is as long as the old rostral half tube, but it is obviously considerably thinner. Only at stage Vb (Fig. 11) is the new rostral tube lamella as wide and thick as that of the old hemirostrum. Because the central spindle extends between the posterior ends of the preexisting hemirostra (Figs. 10, 12 and see below, Spindle), the new curved rostral half tube does not develop parallel to the old (Fig. 24), a position it will assume in the daughter cell. Rather, the old and new hemirostra lie at an angle to each other, and are in proximity only at their anterior ends (Fig. 'i): New kinetosomes which are being laid down together with the daughter rostral half tubes are confined to the anterior, pre-bend portion of the new rostral half tubes as Mitosis in Trichonympha 517 was described by Kirby (1944). These kinetosomes remain non-flagellated (Fig. 10) until stage V, when growing flagella are seen to be associated with them (Fig. 11). A central rod is present in both daughter rostra in every division stage which I have examined (Figs. 6, 9, 11). In each daughter rostrum the central rod appears to be in continuity with the crescentic body and striated rostral half tube of only the old hemirostrum (Figs. 9, 11). No connexions are seen between central rod and newly

Microtubular Crescentic body reinforcement Preaxostyle Anterior kinetosome

New hemirostral tube

Central spindle

Fig. 1. Developing daughter rostrum in dividing Trichonympha agilis. The flagella which abut on the hemirostral tube have been omitted in this drawing. Two daughter rostra are found in each dividing cell, and the central spindle extends between them. forming hemirostrum. In early division, stage II, the central rod of each daughter rostrum appears to be smaller than in the interphase rostrum, but it is augmented during division, growing in length (cf. Figs. 6, 9). One anterior kinetosome surmounts each daughter rostrum, and, in contrast to the non-flagellated anterior kinetosome of the interphase cell, these are associated with well developed flagella (Figs. 5, 10). In division, then, the anterior kinetosome is distinguished from the flagellated kinetosomes in the remainder of the rostrum solely 518 D. F. Kubai by its position above the crescentic bodies. As in interphase, it is disposed with its long axis in the sagittal plane between old and new hemirostral tubes (Fig. i). Throughout division (II—V) each flagellated anterior kinetosome is associated with finely fibrous material, designated preaxostyle by Hollande & Carruette-Valentin (1970) (Figs. 1, 9, 10, 11). A thin sheet of this material is closely applied to the proxi- mal two-thirds of the anterior kinetosome; the sheet frays into fine fibrils which radiate towards the apex of the cell. The double layer of microtubules which constitutes a portion of the external cap of the non-dividing rostrum breaks down in early division and is clearly absent in stage II. These microtubules reappear at stage III, where a small bundle of short microtubules is found in the vicinity of each daughter rostrum, closely associated with the preaxostyle fibrils radiating from the flagellated anterior kinetosome (Figs. 1, 9, 10). It is not until stage Vb, however, that the clearly curved double layer of microtubules, destined to become the reinforcement of the external cap membrane, is fully elabo- rated (Fig. 11).

Spindle In dividing Trichonympha, the distinctive extranuclear spindle is organized between the posterior ends of the diverging hemirostra (Figs. 10, 12) (Cleveland et al. 1934; Kirby, 1944; Hollande & Carruette-Valentin, 1970). Microtubules appear before any changes in rostral structures are evident (stage I). At this stage, the cell retains the flask-shape and the centrally located nucleus characteristic of the interphase cell. However, the rostral half tubes diverge slightly at their posterior ends; and, at this level, microtubules are found in an unoriented array, filling the lumen of the rostral tube (Fig. 7). In addition, microtubules are scattered throughout the cytoplasm between rostrum and nucleus. At stage II, spindle structure is well defined and remains essentially the same throughout division (Figs. 10, 12): a massive bundle of microtubules, the central spindle, extends between the posterior ends of the well separated rostral half tubes. Radiating from each end of the central spindle are numerous microtubules which envelop a portion of the nuclear surface (Fig. 12). The microtubules which compose this latter portion of the spindle have been called ' chromosomal fibres' (Hollande & Carruette-Valentin, 1970). However, since they make no direct chromosome contact until relatively late in division (see below, Kinetochore differentiation) I shall call them pole-to-nucleus microtubules. In addition to the microtubules composing the central spindle and pole-to-nucleus microtubules, a few unoriented microtubules radiate from the poles of the central spindle toward the central rod in the newly forming rostra (Fig. 6) (cf. solenodesme of Hollande & Carruette-Valentin, 1970). No well defined structures (e.g. centrioles, centrosome) are seen at the spindle poles. During division, the nucleus moves anteriorly, so that in stages Il-Va it occupies a position just beneath the central spindle. Later, stage Vb, the central spindle lies in a groove on the nuclear surface (Fig. 13). The length of the central spindle was estimated in reconstructions of serial sections Mitosis in Trichonympha 519 (see below, Chromosome segregation). Though the measurements are only approximate, it appears that the pole-to-pole distance changes little through stage Il-Vb (being approximately 9-14 fim) but increases markedly thereafter, attaining a length of approximately 23 /tm at stage Vc.

Kinetochore differentiation In non-dividing cells and very early division (stage I) nuclei, kinetochores are not strikingly apparent. Careful examination of serial sections, however, allows the dis- crimination of small fibrillar masses situated immediately beneath the nuclear membrane (Figs. 14, 15). The constituent fibrils are clearly thinner than the remainder of the chromatin fibres which fill the nuclear volume. The fibrillar patches as seen in sections which are perpendicular to the plane of the overlying nuclear envelope measure about 375 nm in diameter and 75 run. thick. In stage I, two such masses were often found to lie side-by-side (Fig. 15). Because no nuclei at this stage were reconstructed, it is impossible to say if all are paired in this manner. Kinetochores become easily recognizable in stage II cells (Figs. 16-18). They now consist of a dense disk (180 nm in diameter and 30 nm thick) of granular appearance lying approximately 70 nm beneath the inner nuclear membrane, and a delicately fibrillar mass which occupies the space between disk and membrane. The fibrillar portion, 320 nm in diameter and 70 nm thick, is, to all appearances, identical with the fibrillar masses found underlying the nuclear envelope in interphase and stage I cells. At stage III (Figs. 19, 20) there is no evident change in the components of the kinetochore, though the disk is somewhat denser. The relationship of kinetochore to membrane is altered, however, in that a pouch-like evagination of the nuclear envelope encloses the fibrillar portion while the disk now lies in the plane of the nuclear envelope. There is no apparent differentiation of the nuclear envelope in regions of kinetochore-membrane association; typical nuclear pores are evident in these areas (Figs. 15, 16, 19,20). A still different type of association is found in stages IV and V (Figs. 21-24): the disk appears now to be inserted in a pore-like opening in the nuclear envelope and the fibrillar component lies in the cytoplasm. The disk measures 170 nm in diameter and 60 nm thick. It appears even denser than in previous stages. This 'inserted' kinetochore persists through the remainder of division, even after karyokinesis is complete (stage Vc). As I have not examined later stages of division, the subsequent fate of this structure remains unknown. By study of serial sections, it was determined that, except for stage IV, all kinetochores of a given nucleus are of similar structure and display an identical relationship to the nuclear envelope. The single stage IV nucleus which I encountered was characterized by the coexistence of 2 stages in kinetochore differentiation: kinetochores partially enclosed in outpocketings of the nuclear envelope and those inserted through openings (Fig. 31). This, then, is apparently the stage of transition from intranuclear to ' inserted' kinetochores. It is clear that until stage IV the intact nuclear envelope overlying intranuclear 520 D. F. Kubai kinetochores prevents direct interaction of chromosomes and spindle microtubules. At stages IV and Va, when kinetochores are first inserted in openings of the nuclear envelope, microtubule-kinetochore associations are still not obvious. In fact, in some sections, microtubules can be seen to lie parallel to the nuclear surface, passing over the kinetochore without establishing contact (Fig. 21). It is only at the later stages Vb and Vc that clear kinetochore-microtubule interactions are demonstrable (Figs. 22, 23), several microtubules of the pole-to-nucleus spindle contacting the extranuclear fibrillar mass of each kinetochore (Fig. 24). In my preparations, chromosomes are apparently swollen by the exceedingly dilute solution used for prefixation (see Materials and Methods)?and the individual chromosomes are consequently not recognizable in association with each kinetochore. How- ever, the insertion of chromatin beneath the kinetochore disk element is visible in Figs. 16, 19, and 20. This kinetochore-associated chromatin appears to have a regular structure in the latest stages of division (Figs. 22, 23). Other investigators have amply demonstrated that chromosomes are associated with kinetochores (Hollande & Valentin, 1968a; Hollande & Carruette-Valentin, 1970, 1971).

Kinetochore segregation The observation that kinetochores are often seen as pairs in serial sections of stages I and II (Figs. 15, 18) suggests that, at least in these stages, sister kinetochores can be recognized by their proximity. This, in turn, implies that examination of the distribution of kinetochores on the nuclear surface at various stages of division would allow a determination of the stage at which separation of sister kinetochores takes place. Accordingly, the positions of kinetochores were marked on models built from serial sections of several nuclei (Table 1, p. 515). From comparison of these models, it is clear that the initial separation of sister kinetochores takes place before the pole-to- nucleus microtubules establish direct contact with kinetochores. At stage II, when kinetochores lie beneath the nuclear envelope (Fig. 25) they are distributed over the entire nuclear surface and occur in distinct pairs (Fig. 26). No unpaired kinetochores are found. These pairs are obviously sister kinetochores. Later, stage III, when kinetochores have become enclosed in pouch-like evaginations of the nuclear envelope (Fig. 27), their distribution has changed. In the model depicted in Fig. 28, it is clear that kinetochores are now concentrated on that half of the nuclear surface facing the spindle. More significant, however, is the fact that discrete kinetochore pairs are no longer discernible, some of them (arrows, Fig. 28) being distinctly solitary. In another nucleus with similar kinetochores, Fig. 30, the unpaired disposal of kinetochores is again seen and, in addition, a group of closely packed kinetochores is found near each spindle pole. The above-mentioned 3 nuclei represent stages (II and III) with a well developed mitotic spindle (see above, Spindle). However, since the nuclear membrane remains interposed between intranuclear kinetochores and extranuclear spindle microtubules, it is evident that the originally-paired sister kinetochores move apart and become redistributed on the nuclear surface before there is any possibility for direct contact Mitosis in Trichonympha 521 between kinetochores and microtubules. Thus, at least the early segregation of sister kinetochores is accomplished without the immediate intervention of microtubules. In later stages, Ill-Va, no obvious changes in kinetochore distribution are discernible. At stage IV, however, as mentioned previously, the nuclear membrane opens at the sites of kinetochores. Thus, in such a nucleus (Fig. 32), both intranuclear and inserted kinetochores are present (Fig. 31). Generally speaking, it is those kinetochores farthest from the spindle which remain in the intranuclear condition.

Table 2. Number of kinetochores seen in reconstructions of nuclei at various stages of division

Division stage Kinetochore number II 78 (39 pairs) III 76 III 62 IV 70 Va 79 Vb 80 (in 2 groups, 39 and 41 respectively, one at each spindle pole)

At stage Vb, inserted kinetochores are in direct contact with pole-to-nucleus microtubules (Fig. 33). They are now clustered into 2 distinct groups (Fig. 34), leaving the remainder of the nuclear surface free of kinetochores. At this stage the central spindle lies in a depression on the nuclear surface and the 2 groups of kinetochores are on opposite faces of the nucleus, one toward each pole of the central spindle. Thus, kinetochore segregation is virtually complete. Although stage Vc nuclei were not reconstructed, serial sections show that the nucleus has split into 2 daughter nuclei with a compact grouping of kinetochores persisting at one pole of each daughter nucleus. Combining information from serial sections and nuclear models, it is possible to estimate the relative positions of the spindle poles and, thus, the length of the central spindle at various stages of division. The following values, in /tm, were obtained: stage II, 9-2; stage III, 9-2, 13-8; stage IV, 11-5; stage Va, 13-8; Vb, 13-8; Vc, 23. Such measurements can be considered only approximate, but they do indicate that the major spindle elongation takes place between stages Vb and Vc after kinetochore segregation is complete and in conjunction with the separation of daughter nuclei. Kinetochores were counted in the models built for study of kinetochore distribution (Table 2), but such determinations provide only a rough approximation of chromosome number: since each kinetochore is seen in only two to three sections, loss of one or two consecutive sections could lead to an underestimate of chromosome number. Chromosome counts obtained in the conventional manner are, to my knowledge, unavailable for this species. 522 D. F. Kubai

DISCUSSION Rostrum While the ultrastructure of the rostrum in interphase and dividing Trichonympha agilis has been described previously (Hollande & Carruette-Valentin, 1970), the present study is the first attempt to document the correlations between rostral and nuclear events during division. Because serial sections were used in my work, the determination that there is only one anterior kinetosome in each rostrum of T. agilis is made with reasonable confi- dence. This finding is in disagreement with Hollande & Carruette-Valentin (1970) who state that 2 anterior kinetosomes occur in each rostrum. It is possible that varie- ties of T. agilis differ in this regard. However, the micrographs presented by Hollande & Carruette-Valentin are based on random sections of dividing cells and never show 2 anterior kinetosomes in one section. Consequently, the illustrations on which they base their claim that there are 2 anterior kinetosomes, one flagellated and the other not, might also be interpreted as depicting longitudinal and cross-sections, respectively, of a single flagellated kinetosome. Unfortunately, my material did not include stages intermediate between I and II. Thus, the transition from one non-flagellated anterior kinetosome per cell (interphase and early division) to two flagellated anterior kinetosomes per cell (later stages of division, one in each newly forming rostrum) was not studied. It is, therefore, not yet possible to decide between the following alternatives: (1) the anterior kinetosome replicates and daughters subsequently become flagellated; (2) the anterior kinetosome breaks down early in cell division; the positions of anterior kinetosomes in the newly forming rostra are then assumed by pre-existing kinetosomes derived from the flagellated portion of the rostrum. Hollande & Carruette-Valentin (1970) speak of ' autoduplication' of anterior kinetosomes but present no evidence for this interpretation. Previous descriptions have indicated that the entire central rod is carried with one of the hemirostra at division. My observations that (a) the central rod is symmetrically attached to both hemirostral tubes in interphase and (b) the size of the central rod is comparable in both daughter rostra at early division stage II are inconsistent with such an interpretation. Rather, they suggest that central rod material is distributed equally to the 2 hemirostra.

Spindle The present study does not allow a detailed description of spindle formation in Trichonympha. However, the appearance of unoriented microtubules in the space between slightly divergent hemirostra at early division (stage I) suggests that elaboration of the central spindle results from the rearrangement of these microtubules and, further, that separation of hemirostra is dependent on this elaboration. In contrast, the central spindle in Trichonympha of cockroaches and in another hypermastigote, Barbulanympha, had been said to be formed by the overlapping of spindle fibres which grow out from the well separated centrioles (Cleveland et al. 1934). Mitosis in Trichonympha 523 Subdivision of the spindle into a separate central spindle and 2 pole-to-nucleus half spindles is characteristic of hypermastigotes. Hollande & Carruette-Valentin (1970) consider this the result of independent grouping of continuous (central spindle) and chromosomal (pole-to-nucleus) microtubules. If true, this would have bearing on the mechanism of chromosome movement, particularly in terms of current sliding-microtubule models of spindle function (Nicklas, 1971), which require the intermingling and interaction of continuous and chromosomal microtubules. However, it has not been possible to demonstrate that only chromosomal microtubules compose the pole- to-nucleus half spindle, and the significance of the unusual spindle structure in hypermastigotes remains obscure. My determinations of approximate length of the central spindle in Trichonympha at various stages of division suggest that no significant spindle elongation takes place until after kinetochores are segregated in 2 groups at opposite poles of the nucleus. It appears, then, that central spindle elongation is more closely associated with the separation of daughter nuclei than with daughter chromosome movement. Similarly, Cleveland et al. (1934) concluded that elongation of the central spindle has little influence on the separation of daughter chromosomes in the hypermastigotes Barbula- nympha and Trichonympha of the cockroach Cryptocercus.

Kinetochore structure and differentiation The kinetochores of Trichonympha agilis are multilayered disks, structurally identical to, but smaller than, the kinetochores of another hypermastigote, Barbulanympha ufalula (Hollande & Valentin, 1968a). Multilaminar disk organization has been proposed for the kinetochores of a number of organisms; these also range in diameter from 0-2 to 0-5 /im (Nebel & Coulon, 1962; Luykx, 1965; Jokelainen, 1967; Brinkley & Stubblefield, 1970). In both mammalian and hypermastigote kinetochores, microtubules impinge on the kinetochores in the low density outer layer. In hypermastigotes, as in Chinese hamster (Brinldey & Stubblefield, 1970), microtubules do not penetrate beyond this outer layer, ending at the dense disk component (Barbulanympha: Hollande & Valentin, 1968 a) or at the outer margin of the low density fibrous material (Trichonympha). In contrast, microtubules of certain mammalian cells are said to penetrate through the kinetochore, passing into the chromosome proper (Jokelainen, 1967). Typically, kinetochore regions of chromosomes are not identifiable during interphase, differentiated kinetochores becoming recognizable only during prophase when chromosomes condense in preparation for division (Brinkley & Stubblefield, 1970). In hypermastigotes, on the other hand, chromosomes do not decondense entirely in interphase and the kinetochores apparently retain their association with the nuclear membrane throughout the cell cycle: thus, in these organisms kinetochores are discernible underlying the nuclear envelope even in interphase, in Trichonympha as patches of low-density fibrous material (Fig. 14) and in Barbulanympha as dense 'calottes' (Hollande & Valentin, 1968). Differentiation of the kinetochore in Trichonympha agilis, summarized in Fig. 2, appears to proceed by its gradual condensation to form a dense, disk-shaped inner 524 D. F. Kubai kinetochore element associated with the low-density outer fibrous mass which had remained recognizable throughout interphase. This view, that kinetochore differentiation stems from condensation of kinetochore elements, suggests one possible interpretation of the manner in which the changing kinetochore-membrane relationship evolves in the course of nuclear division. If fibrils in the periphery of the interphase, low-density kinetochore patch are firmlyattache d to the membrane, their condensation during division to form the dense disk-like kinetochore element could deform the nuclear envelope. This would lead first to the enclosure of the outer fibrous element in a pouch-like evagination of the nuclear envelope and later, through continued

Fig. 2. Diagrammatic representation of the 4 stages of kinetochore development in Trichonympha agilis. A, during interphase the fibrillar kinetochore element is attached to the nuclear envelope. B, early in division the dense, disk-like kinetochore element becomes differentiated beneath the fibrillar element. Cytoplasmic spindle microtubules are separated from the kinetochore by the intact nuclear envelope. c, the disk element of the kinetochore now lies in the plane of the nuclear envelope and the fibrillar element is enclosed in an evagination of the nuclear envelope. Cyto- plasmic spindle microtubules are still separated from the kinetochore by the intact nuclear envelope. D, the nuclear envelope opens at the site of the kinetochore, leaving the disk element of the kinetochore inserted in a pore-like opening of the nuclear envelope and the fibrillar element in the cytoplasm. Spindle microtubules make intimate contact with the fibrillar element of the kinetochore. Mitosis in Trichonympha 525 condensation with resultant tension on the inner membrane of the nuclear envelope, to the opening of the nuclear envelope at the site of the kinetochore.

Chromosome movement It has been proposed that in certain organisms the direct action of a mitotic spindle is not necessary for chromosome movement. On the basis of light-microscopical investigations Grasse (1952) has maintained that the nuclear divisions of a number of protozoa, notably polymastigotes, hypermastigotes, dinoflagellates and foraminifera, must be considered as fundamentally different from conventional mitosis (orthomitosis). Consequently, he has defined pleuromitosis to categorize these divisions. Pleuromitosis is characterized by the absence of a typical metaphase configuration and, more importantly, by a mechanism of chromosome movement which does not involve the direct insertion of chromosomes in the spindle. In such divisions (Grass6, 1952, p. 112) chromosomes are fixed to the nuclear membrane and carried toward the spindle poles by intranuclear currents or some other unknown means, movement being possible even in the complete absence of spindle elements. Recent ultrastructural investigations of nuclear division in dinoflagellates (Lead- beater & Dodge, 1967; Kubai & Ris, 1969; Soyer, 1969, 1971) have reinforced the idea that chromosome movements may result from interaction between chromosomes and membrane. During division in these organisms, chromosomes are attached to well denned areas of the nuclear envelope. Since microtubules which appear during division are never seen to make direct contact with chromosomes, it has been concluded that the mechanism of chromosomes movement involves membrane growth or displacement (Kubai & Ris, 1969). Similarly, my study of nuclear division in Trichonympha agilis suggests that, at least in early phases of division, growth or flow of the nuclear membrane plays a role in chromosome distribution in hypermastigotes: comparison of the distribution of kinetochores in stages II and III (Figs. 26, 28) leads to the inescapable conclusion that sister kinetochores are separated and redistributed on the nuclear surface while no direct contact with microtubules is possible. That the movement of intranuclear membrane-associated kinetochores in Tricho- nympha agilis is a directed phenomenon leading toward separation of equal comple- ments of daughter chromosomes at opposite poles of the nucleus is by no means established. The observation of a gathering of a number of intranuclear kinetochores at each pole in stage III (Fig. 30) is, however, suggestive. Unfortunately, even if the paired kinetochores of stage II were redistributed in an orderly fashion prior to stage IV, this might not be discernible by the simple examination of the location of kinetochores on the nuclear membrane. For example, the elements of a kinetochore pair could, upon separation, come to lie in opposite hemispheres of the nuclear surface; such distribution would constitute an equipartition of the replicated chromosome complement, but the total distribution of kinetochores might still appear to be random. Since observations such as mine cannot establish the direction of kinetochore movement, the possibility remains that early kinetochore redistribution in Tricho- 526 D. F. Kubai nympha is uncoordinated or random and that the regular segregation of sister chromosomes occurs only after microtubules engage the kinetochores (stage V). If this is the case, however, it is difficult to conceive of a mechanism which would ensure that sister kinetochores become engaged with microtubules emanating from opposite spindle poles. In conventional mitosis, the necessary recognition of sister kinetochores is facilitated by the fact that they occupy opposing 'faces' of the metaphase chromosome and remain together until both establish contact with spindle microtubules (Brinkley & Stubblefield, 1970). If, however, the membrane-associated movements which separate sister kinetochores in T. agilis occur in a directed manner, so that the kinetochores of a pair come to lie in the domain of pole-to-nucleus microtubules radiating from opposite poles of the spindle, their engagement with microtubules of the appropriate pole would be ipso facto determined. In addition to the membrane-kinetochore relationship, direct association of kinetochores and spindle microtubules has been amply demonstrated for a number of species of hypermastigotes (Hollande & Carruette-Valentin, 1971; see Mazia, 1961, fig.45) . This, by analogy with the function of microtubules in conventional mitosis, has been considered to indicate that spindle microtubules here, also, participate actively in daughter chromosome distribution (Hollande & Carruette-Valentin, 1970, 1971)- In support of this view, elevation of the nuclear membrane at the site of inserted kinetochores is cited as evidence for traction by microtubules. Such indications are, of course, inconclusive (for example, kinetochores are at the same time found in depres- sions of the nuclear surface, Fig. 24) and, without experimental evidence, it is impossible to know whether in hypermastigote divisions the microtubules operate as in conventional mitosis. Alternatively, they may serve a more passive, guiding function, as, for example, in anchoring the already-separated chromosome to the appropriate poles during karyokinesis (see above, Spindle). Thus, until this question has been resolved, there is insufficient basis for the view presented by Hollande & Carruette-Valentin (1970, 1971) that nuclear division in hypermastigotes is a trivial variation of orthomitosis. Rather, my studies suggest, hypermastigotes may be another example of eukaryotes which retain primitive prokaryote-like features of chromosome segregation. Unlike dinoflagellates, however, where membrane phenomena seem to be the sole direct influence on chromosome movement, microtubules serving simply to establish the polarity of division (Kubai & Ris, 1969), hypermastigotes probably possess mechanisms of chromosome movement involving both membrane and microtubules. It is now evident from detailed ultrastructural analyses of divisions such as those of dinoflagellates and hypermastigotes that the 'pleuromitosis' of Grasse (1952) is not a well defined type of mitosis but encompasses widely divergent forms of unorthodox division.

I wish to thank Dr G. R. Esenther of the United States Department of Agriculture, Forest Service, Forest Products Laboratory, Madison, Wisconsin, who provided the termite cultures, Dr R. V. Smythe of the United States Department of Agriculture, Forest Service, Southern Forest Experiment Station, Gulfport, Mississippi, for his advice regarding defaunation and refaunation, Cheryle Hughes and John Dallman who made the drawings for this paper, and Dr Mitosis in Trichonympha 527 H. Ris in whose laboratory this investigation was carried out. This work was supported by a United States Public Health Service research grant (GM-04738) from the National Institutes of Health.

REFERENCES ANDREW, B. J. & LIGHT, S. F. (1929). Natural and artificial production of so-called 'mitotic flares' in the intestinal flagellates of Termopsis angusticollis. Univ. Calif. Pubh Zool. 31, 433-44°- BREADY, J. K. & FRIEDMAN, S. (1963). Oxygen poisoning of the termite, Reticulitermes flavipes (Kollar), and protection by carbon dioxide. J. Insect Physiol. 9, 337-347. BRINKLEY, B. R. & STUBBLEFIELD, E. (1970). Ultrastructure and interaction of the kinetochore and centriole in mitosis and meiosis. In Advances in Cell Biology, vol. 1 (ed. D. M. Prescott, L. Goldstein & E. McConkey), pp. 119-185. New York: Appleton-Century-Crofts. CLEVELAND, L. R. (1924). The physiological and symbiotic relationships between the intestinal protozoa of termites and their host, with special reference to Reticulitermes flavipes Kollar. Biol. Bull. mar. biol. Lab., Woods Hole 46, 178-227. CLEVELAND, L. R. (1925). Toxicity of oxygen for protozoa in vivo and in vitro: animals defaunated without injury. Biol. Bull. mar. biol. Lab., Woods Hole 48, 455-468. CLEVELAND, L. R. (1928). Further observations and experiments on the symbiosis between termites and their intestinal protozoa. Biol. Bull. mar. biol. Lab., Woods Hole 54, 231-237. CLEVELAND, L. R., HALL, S. R., SANDERS, E. P. & COLLIER, J. (1934). The wood-feeding roach Cryptocercus, its protozoa, and the symbiosis between protozoa and roach. Mem. Am. Acad. Arts Sci. 17, 185-342. ESENTHER, G. R. (1969). Termites in Wisconsin. Ann. ent. Soc. Am. 62, 1274-1284. FRASCA, J. M. & PARKS, V. R. (1965). A routine technique for double-staining ultrathin sections using uranyl and lead salts. J. Cell Biol. 25, 157-161. GRASSE, P. P. (1952). Traiti de Zoologie, t. I, fasc. 1. Paris: Masson. GRIMSTONE, A. V. & GIBBONS, I. R. (1966). The fine structure of the centriolar apparatus and associated structures in the complex flagellates Trichonympha and Pseudotrichonympha. Phil. Trans. R. Soc. Ser. B 250, 215-242. HOLLANDE, A. & CARRUETTE-VALENTIN, J. (1970). Interpretation g6n6rale des structures rostrales des Hypermastigines et modalit^s de la pleuromitose chez les Flagelles du genre Trichonympha. C. r. hebd. Sdanc. Acad. Sci., Paris 270, 1476—1479. HOLLANDE, A. & CARRUETTE-VALENTIN, J. (1971). Les Atractophores, l'induction du fuseau et la division cellulaire chez les Hypermastigines. Etude infrastructurale et revision systeinatique des Trichonymphines et des Spirotrichonympkines. Protistologica 7, 5-100. HOLLANDE, A. & VALENTIN, J. (1968a). Infrastructure des centromeres et deroulement de la pleuromitose chez les Hypermastigines. C. r. hebd. Se'anc. Acad. Sci., Paris 266, 367-370. HOLLANDE, A. & VALENTIN, J. (19686). Donn^es critiques sur la pleuromitose et affinit^s entre Trichomonadines et Joeniides. C. r. hebd. Seanc. Acad. Sci., Paris 267, 1383-1386. JOKELAINEN, P. T. (1967). The ultrastructure and spatial organization of the metaphase kinetochore in mitotic rat cells. J. Ultrastruct. Res. ig, 19-44. KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 1370-1380. KIRBY, H. (1932). Flagellates of the genus Trichonympha in termites. Univ. Calif. Pubh Zool. 37. 349-476. KIRBY, H. (1944). The structural characteristics and nuclear parasites of some species of Trichonympha in termites. Univ. Calif. Pubh Zool. 49, 185-282. KUBAI, D. F. & Ris, H. (1969). Division in the dinoflagellate Gyrodinium cohnii (Schiller). A new type of nuclear reproduction. J. Cell Biol. 40, 508-528. LEADBEATER, B. & DODGE, J. D. (1967). An electron microscope study of nuclear and cell division in a dinoflagellate. Arch. Mikrobiol. 57, 230-254. LEEDALE, G. F. (1970). Phylogenetic aspects of nuclear cytology in the algae. Ann. N. Y. Acad. Sci. 175, 429-453. LUYKX, P. (1965). The structure of the kinetochore in meiosis and mitosis in Urechis eggs. Expl Cell Res. 39, 643-657. 528 D. F. Kubai MAZIA, D. (1961). Mitosis and the physiology of cell division. In The Cell, vol. 3 (ed. J. Brachet & A. E. Mirsky), pp. 77-412. New York and London: Academic Press. MOLLENHAUER, H. H. (1964). Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, m-114. NEBEL, B. R. & COULON, E. M. (1962). Fine structure of chromosomes in pigeon spermato- cytes. Chromosoma 13, 272-291. NICKLAS, R. B. (1971). Mitosis. In Advances in Cell Biology, vol 2 (ed. D. M. Prescott, L. Gold- stein & E. H. McConkey), pp. 225-297. New York: Appleton-Century-Crofts. PITELKA, D. R. (1963). Electron-Microscopic Structure of Protozoa. New York: Macmillan. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212. RYTER, A. (1968). Association of the nucleus and the membrane of bacteria: A morphological study. Bact. Rev. 32, 39-54. RYTER, A. & KELLENBERGER, E. (1958). Etude au microscope electronique de plasmas contenant de l'acide desoxyribonucleique. I. Les nucleoides des bacteries en croissance active. Z. Naturf. 136, 597-605. SOYER, M. O. (1969). Rapports existant entre chromosomes et membrane nucleaire chez un Dinoflagelle parasite du genre Blastodinium Chatton. C. r. hebd. Se'anc. Acad. Set., Paris 268, 2082-2084. SOYER, M. O. (1971). Structure du noyau des Blastodinium (dinoflagell^s parasites). Division et condensation chromatique. Chromosoma 33, 70—114. STANIER, R. Y. (1970). Some aspects of the biology of cells and their possible evolutionary significance. In Organization and Control in Prokaryotic and Eukaryotic Cells, Symp. Soc. gen. Microbiol. 20, pp. 1-38. Cambridge University Press.

{Received 12 February 1973)

Fig. 3. Rostrum of interphase Trichonympha agilis, longitudinal section. A double layer of microtubules {mi) reinforces the membrane of the outer rostral cap (oc). The anterior kinetosome (ak) is embedded in the inner rostral cap {ic). The striated wall of the rostral tube (r) extends from a crescentic body (cb). Connexions between the central rod (cr) and rostral tube, crescentic body and inner rostral cap are indicated by arrows, x 26000. Fig. 4. Rostrum in division stage I, slightly oblique section. Inner rostral cap (ic) is present and surrounds a single non-flagellated anterior kinetosome (ak). x 42000. Mitosis in Trichonympha 529

34 530 D. F. Kubai

Fig. 5. Hemirostrum of stage II, sagittal section. Inner and outer rostral caps have disappeared (note absence of microtubules beneath the cell membrane). The anterior kinetosome (ak), here seen above the crescentic body (c6) of the old hemirostrum is now associated with a flagellum (/). x 37500. Fig. 6. Hemirostrum of stage II, longitudinal section. The central rod (er) present in the developing daughter rostrum is somewhat shorter than at later stages of division (cf. Fig. 9). Note microtubules radiating from the central rod. x 42000. Fig. 7. Posterior rostral area, stage I, cross-section. The hemirostral tubes (r) diverge slightly and unoriented microtubules fill the space between them, x 42000. Mitosis in Trichonympha 53*

34-2 532 D. F. Kubai

Fig. 8. Developing rostrum of stage II, oblique section. Spindle microtubules emanate from the base of the old hemirostrum (r^). Newly forming non-flagellated kinetosomes (k) are associated with the curved lamella of the new hemirostrum (r2). x 26000. Fig. 9. Developing rostrum of stage III, longitudinal section. The old hemirostral tube (rx) and its crescentic body (cb^ are connected (arrows) to the central rod (cr). The new hemirostrum (r,) is thinner than the old and its crescentic body (cbt) portion is poorly developed. New, non-flagellated kinetosomes (k) are associated with the new hemirostrum. The preaxostyle (p) is associated with the anterior kinetosome (ak). Short microtubules (mt), destined to become the reinforcement of the outer rostral cap, have appeared above the preaxostyle. x 42000. Mitosis in Trichonympha 533 M 534 D- F. Kubai

Fig. io. Developing daughter rostra in stage IV, sectioned longitudinally (dr{) and sagitally (drt). The curved lamella of the newly forming hemirostrum (r2) is associated with newly formed, non-flagellated kinetosomes (A) which are confined to its pre-bend anterior portion. A flagellated anterior kinetosome (ak) surmounts each daughter rostrum, and each is associated with a preaxostyle (p). A layer of short micrptubules (mi), destined to become the reinforcement of the outer rostral cap, lies just above each preaxostyle. Microtubules of the central spindle (cs) extend between the daughter hemirostra. x 26000. Mitosis in Trtchonympha 535

cs 536 D. F. Kubai

Fig. ii. Developing rostrum in stage Vb, slightly oblique section. The central rod (cr) is connected (arrows) to the old hemirostrum (rt) and its crescentic body (cbj. The crescentic body (cbt) of the new hemirostrum (r,) is better developed than in previous stages. Flagella (/) are growing out from the newly formed kinetosomes. A well developed double layer of microtubules {mt) overlies the frayed preaxostyle fibres (p). ok, anterior kinetosome. x 42000. Mitosis in Trichonympha 537 538 D. F. Kubai

Fig. 12. Extranuclear spindle, stage Va. The central spindle (cs) extends between old hemirostra of the 2 daughters rostra (dr1 and drt). Pole-to-nucleus microtubules radiate over part of the nuclear surface (pn). x 7700. Mitosis in Trichonympha 539 540 D. F. Kubai

Fig. 13. Extranuclear spindle, stage Vb. The central spindle («) occupies a trough- like depression of the nuclear surface. r1( old hemirostrum; r2, new hemirostrum. Note 'inserted' kinetochores at arrows. X 7700. Mitosis in Trichonympha

\ cs . .

13 542 D. F. Kubai

Fig. 14. Kinetochore in interphase cell. Kinetochore appears as a finely fibrillar mass underlying the nuclear envelope (arrows), x 75000. Fig. 15. Pair of kinetochores in early division stage I (arrows), x 75000. Fig. 16. Kinetochore in stage II. The kinetochore is a bipartite structure consisting of a finely fibrillar element (fe) lying just beneath the nuclear envelope and a dense disk (d). Chromatin mass (cm) is seen beneath the disk. Note intact nuclear membrane and microtubules in the cytoplasm, x 75000. Fig. 17. Kinetochore, stage II. Tangential section demonstrates the disk shape of the dense kinetochore element (d). The finely fibrillar kinetochore element surrounds the dense disk, x 75 000. Fig. 18. Paired stage II kinetochores, tangential section. Compare Fig. 26, a nuclear model with paired kinetochores. x 75000. Mitosis in Trichonympha 543 f.

t '

; *• .fa! 544 D. F. Kubai

Figs. 19, 20. Kinetochores in stage III. The dense disk element (d) of the kinetochore lies in the plane of the nuclear surface and the inner nuclear membrane is closely applied to the finely fibrillar kinetochore element (/«), enclosing it in a sac-like out- pocketing of the nuclear envelope. Note nuclear pores at arrows and microtubules in cytoplasm. Chromatin mass (cm) is seen beneath the disk, x 75 coo. Fig. 21. Kinetochore at stage Va. The kinetochore disk element (d) has been inserted in an opening in the nuclear envelope and the fibrillar element(/e) has come to lie in the cytoplasm. Microtubules are not yet in contact with the kinetochore. x 75000. Figs. 22, 23. Kinetochores at stage Vb. The dense disk element of the kinetochore (d) is inserted in openings of the nuclear envelope. Continuity between inner and outer membrane of the nuclear envelope is evident at the boundary of the openings (arrows). The fibrillar element (fe) of the kinetochore which now lies in the cytoplasm is in direct contact with microtubules. An orderly arrangement of the sub-disk chromatin mass (cm) is evident, x 75000. Mitosis inTrichonympha 545

'. '^ •! . * ' \ i A "V1 A..

h»,^ <§.

*§ CEL 13 546 D. F. Kubai

Fig. 24. Developing daughter rostrum, spindle and nucleus in stage Vb, oblique section. The central spindle (cs) extends from the old hemirostrum (r^. Note that the new hemirostrum (r,) does not develop parallel to the old. 'Inserted' kinetochores (arrows) are clustered at the ends of the trough-like depression of the nuclear surface (cf. Figs. 13, 34)- x 14700- Mitosis in Trichonympha

35-2 548 D. F. Kubai

Fig. 25. Drawing of intranuclear kinetochore of the type found in stage II reconstruction illustrated in Fig. 26. Fig. 26. Reconstruction of stage II nucleus. Kinetochores are indicated by black dots and are enlarged relative to the scale of the nucleus. They occur in distinct pairs and are distributed over the entire nuclear surface. Positions of spindle poles are indicated by X's. Fig. 27. Drawing of intranuclear kinetochore of the type found in stage III reconstruction illustrated in Fig. 28. Fig. 28. Reconstruction of stage III nucleus. Kinetochores are confined to that half of the nuclear surface closest to the spindle (spindle poles indicated by X's). Kineto- chores are no longer distinctly paired; solitary kinetochores are indicated by arrows. Mitosis in Trichonympha 549

25 26

27 28 55O D. F. Ktibai

Fig. 29. Drawing of intranuclear kinetochore of the type found in stage III reconstruction illustrated in Fig. 30. Fig. 30. Reconstruction of stage III nucleus. As in Fig. 28, kinetochores are confined to that half of the nuclear surface closest to the spindle and are not distinctly paired. In addition, clusters of kinetochores are found on the nuclear surface in the regions closest to the 2 spindle poles (X). Fig. 31. Drawing of the 2 types of kinetochores (intranuclear and 'inserted') found in stage IV reconstruction illustrated in Fig. 32. Fig. 32. Reconstruction of stage IV nucleus. Both intranuclear and 'inserted' kinetochores, as illustrated in Fig. 31, are found at this stage. Intranuclear kinetochores (arrows) are farthest from the spindle (spindle poles indicated by X's). Mitosis in Trichonympha

29 30

31 32 D. F. Kubai

33 34

Fig. 33. Drawing of the 'inserted', microtubule-associated kinetochore of the type found in stage Vb reconstruction illustrated in Fig. 34. Fig. 34. Reconstruction of nucleus at stage Vb. A trough-like depression on the nuclear surface contains the central spindle (spindle poles indicated by X's). Kinetochores are all 'inserted' and in contact with microtubules. They occur in 2 clusters, one toward each spindle pole.