Mitosis in the fungusBasidiobolus ranarum as revealed by electron microscopy

Protoplasma 70, 423--440 (1970)

�9 by Springer-Verlag 1970

M i t o s i s i n t h e F u n g u s Basidiobolus ranarum as R e v e a l e d

b y E l e c t r o n M i c r o s c o p y

KENJI TANAKA

Department of Bacteriology, University of Western Ontario, London, Canada

With 27 Figures

Received June 30, 1970

S u m m a r y

Mitosis of nuclei in vegetative hyphae of the fungus Basidiobolus ranarum has been studied by electron microscopy. Ceils fixed with glutaraldehyde and OsO~ were embedded in Vestopal. Sections were obtained of single cells whose mitotic status was known. Attention was paid to the behaviour of the microtubules, the nuclear envelope and the nucleolus. Nuclear division begins with the dilution and rearrangement of nucleolar material and the gradual breakdown of the nuclear envelope. At this stage the nucleus is surrounded by a sheet of closely packed microtubules. Some of these penetrate into the nucleus through gaps in the envelope. Dissolution of the envelope is followed or accompanied by the development of an extensive labyrinth of membranous cisternae which persists at the periphery of the division site through mitosis and probably contributes material to the envelopes of the daughter nuclei. The drum-shaped spindle of metaphase is composed of large numbers of microtubules aligned parallel to each other. Many of them are associated with chromosomes. Metaphase is soon followed by the movement of dense masses of nucleolar material and chromosomes to the poles of the division figure to form the socalled "end plates". Microtubules extend into the end plates but not beyond. Neither centrioles nor "centriolar plaques" have been seen.

1. I n t r o d u c t i o n

Basidiobolus belongs to the order Entomophthorales in the sub-class Zygo- mycetidae of the phytomycetes. I t is one of the few fungi in which the hyphal nuclei are not only large, measuring 15-20 by 5 microns, but can also be seen to divide by an ordinary form of mitosis. These aspects of BasidioboIus nuclei set the fungus apar t f rom many other and better known fungi in which the cells of vegetat ive hyphae contain m a n y small nuclei which divide by a modified form of mitosis. Earlier studies of Basidiobolus with the light microscope, reviewed by ROBINOW (1963), left several problems unsolved which, it was hoped, might yield to electron microscopy. The present work

28*

424 K. TANAKA: Mitosis in the Fungus Basidiobolus ranarurn

descr ibes e l e c t r o n m i c r o g r a p h s o f t h i n sect ions a n d has been m a i n l y c o n c e r n e d

w i t h the b e h a v i o u r o f the nucleus , t he n u c l e a r e n v e l o p e , t he m i t o t i c sp indle ,

t he o r i g i n o f t h e s p i n d l e f ibres a n d the search fo r cen t r io l e s o r e q u i v a l e n t

s t ruc tu res .

2. Materials and Methods

The organism used in this study was obtained from the Centraalbureau voor Schimmelcultures (CBS) at Baarn, Holland. It is the same strain that was used by Ro~Now (1963). It differs from freshly isolated strains of Basidiobolus ranarurn in having particularly long, straight spear-like terminal cells, in no longer forming either ballisro- or zygospores on yeast extract (0.5%)-glucose (2%) agar, a medium on whi& freshly isolated strains form both kinds of spores in abundance. Phase contrast microscopy of growing ceils was performed on slide cultures in 15% gelatin with glucose and yeast extract at room temperature. A Leitz Labor Lux II microscope was

used. For electron microscopy agar cultures were prepared on carbon-coated slides prepared by the method of ROBBINS and GON*TAS (1964 a). The carbon effectively prevented the agar film from becoming detached during subsequent handling and facilitated cleavage of the polymerized plastic from the slides. Thin films of agar were obtained by dipping carbon- coated slides into hot molten agar and draining off all but a thin layer. An explant of the volume of one cubic miitimeter was dissected out of the growing edge of a young cultllre under the binocular microscope and placed on the agar film. Cultures were kept in a moist atmosphere in a petri dish at room temperature. Eight to ten hours later cultures were fixed in shallow, single slide staining troughs in 3% glutaraldehyde solution in 0.1 M cacodylate buffer at pH7.0 in the presence of 0.005M CaCI2. Fixation was performed at room temperature for about half an hour and continued for another 12 to 18 hours at 4 ~ C. Fixed cultures were washed with several changes of the same buffer for 3 hours. During this time the cultures were scanned with a water immersion lens for cells in the desired stage of nuclear or cell division. The position on the slide of chosen cells was recorded in detailed maps which permitted their identification in the course of further handling. Postfixation was with 1.33% osmium tetroxide solution in 0.067 M collidine buffer containing 0.005 M CaC12 at pH 7.0 for 8 hours at 4 ~ C. After rinsing in water, cultures were soaked in a 0.5% aqueous solution of uranyl acetate for 2 hours, dehydrated in increasing concentrations of acetone and embedded in Vestopal W. Sections were cut on an MT-1 Porter-Blum microtome with glass knives. They were stained with lead citrate (VENABL~ and COGG~S~ALL 1965) and examined in a Philips "100" or "200" electron microscope.

Figs. 1 to 9 are phase contrast micrographs of the same living cell of Basidiobolus ranarurn

growing in 15 per cent gelatin with yeast extract and glucose Fig. 1. FuIi length view of a vegetative ceil. Note the difference in the texture of the cytoplasm distal to and proximal of the nucleus. X450 Figs. 2-9. Successive photographs of nuclear division. See text for details. X920. Fig. 2. Inter- phase nucleus. Fig. 3. Prophase. Nucleolus begins to fade. Fig. 4. Prophase. Rearrangement of nucleolar materials. Fig. 5. Pro-metaphase. Former nucleolar material concentrated in two "end plates". Fig. 6. Metaphase. End plates separated by a band of brightness which corresponds to the site of the &romosomes. Fig. 7. Anaphase. End plates are moving towards the poles. The bright material separating them now consists mainly of spindle fibers. Fig. 8. Telophase. Early stage in the reconstruction of daughter nuclei at the poles of the mitotic figure whi& appears as a rhomboid field of low density. Fig. 9. Daughter nuclei fully reconstituted. The formation of a transverse partition, initiating cell division, has begun

Figs. 1-9


3. Resul t s

3.1. P h a s e C o n t r a s t M i c r o s c o p y

A growing cell of B. ranarurn (Fig. 1) is long and thin and has a single nucleus with a huge nucleolus. The cytoplasm at the tip of the cell looks very different from that at the rear. The former is dense, the latter is broken up by large vacuoles and broad, liquid filled channels. Time lapse phase contrast micrographs of dividing nuclei have been published before. A fresh sequence (Figs. 2-9) obtained under better optical conditions is provided in the present paper to allow the reader to place the electron micrographs, which are the main concern of this study, in their proper context.

Fig. 10. Nuclei fixed with Helly, stained with Feulgen, and photographed mounted in acetocarmine. A = surface of resting nucleus. X1,720. B = Meta- phase. XI,720. C - Anaphase-telophase. X2,430. (By courtesy of Dr. ROBiNOW)

The chromosomes of living nuclei were indistinct but the changing distribution of nucleolar material could be followed with ease. Loss of density of the nucleolus at the beginning of mitosis (Fig. 3) is accompanied by the dis- appearance of the nuclear membrane (Fig. 4). These events are soon followed by the redistribution of nucleolar material into two thick masses of greater density than the original, intact nucleolus. Aligned parallel to each other these masses, which have an outer convex and inner plane surface (like a slice cut from the top of an egg), serve as "end plates" of the mitotic spindle which develops in the space between them (Figs. 5-7). During interphase the cytoplasm both forward of and to the rear of the nucleus contains many

Figs. 11-14 are sections obtained from the same nucleus at interphase. Fig. 11. Part of an interphase nucleus. The nucleolar region (NC), composed of dense material, occupies a large part of the nucleus. NE = nuclear envelope. X6,610. Fig. 12. More highly enlarged view of a part of Fig. 11. A few cytoplasmic microtubules (MT) are seen near the nuclear envelope (NE). X27,840. Fig. 13. Fibrillar matrix of the interphase nucleus. X82,000. Fig. 14. Grazing section of the surface of the nuclear envelope. Pores and cytoplasmic microtubules are clearly seen. )<49,200

Figs. 11-14

428 K. TANAKA

fluid-filled channels and lacunae. The lacunae in the rear portion of the cell are coarser and change shape more rapidly than those in front of the nucleus. During mitosis this pattern is temporarily replaced by a coarse granulation of the cytoplasm. At the same time the normally vivid streaming motion of the cytoplasm comes to an almost complete standstill. Normal streaming is resumed and the usual channels and lacunae reappear at telophase of mitosis. The time occupied by prophase was 7--8 minutes; metaphase took another 4-5 minutes. Formation of a transverse septum between the separating daughter nuclei invariably began 13 to 15 minutes after the metaphase plate had been clearly established and was completed in the course of seven minutes (Fig. 9).

3.2. E l e c t r o n M i c r o s c o p y

3.2.1. The Resting Stage

One of the most striking features of the resting nucleus of Basidiobolus is the huge nucleolus which is easily recognized under the light microscope. Fig. 11 shows part of a longitudinal section of a resting nucleus. The nucleus is bounded by the nuclear envelope. The nucleolus contains electron opaque cords embedded in a less dense granular matrix. Higher magnification of the same section of the nucleus in Fig. 12 reveals two components, fine threads and granules 180 to 200 • in diameter in the nucleolar region. The denser portions of the nucleolus are probably composed of aggregates of these granules. A seemingly homogeneous zone of low density which intervenes between the nucleolus and the nuclear envelope is filled with fine fibrils approximately 50A in diameter (Fig. 13). Electron micrographs of the interphase nucleus do not reveal the definite localization of chromatinic material, which appears particulate and scattered about in the nucleolus in light microscopy on Feulgen-stained preparation (Fig. 10 A). The cytoplasm surrounding an interphase nucleus contains long mitochondria, a few microtubules (Fig. 12, MT) and close to the envelope, a few vesicles. Grazing sections of the envelope (Fig. 14) reveal numerous microtubules in close proximity to but not in direct contact with the envelope and arranged parallel to the long axis of the nucleus and the long axis of the cell. Grazing sections also provide good views of pores in the nuclear envelope. These measure 800 A in outside diameter, seem to be composed of subunits 200_~ in diameter arid are plugged with a dense granule.

3.2.2. Prophase

Nuclear division of Basidiobolus begins with swelling and increased trans- parency of the nucleolus. The dense cords of the nucleolar matrix now break down into more evenly dispersed granules. This dilution of the nucleolar material is accompanied by the partial breakdown of the nuclear envelope

Mitosis in the Fungus Baddiobolus ranarurn 429

Fig. 15. Early prophase. The nuclear membrane has begun to disintegrate. Nucleolar material (NC) is being diluted. )<7,260 Fig. 16. Closely pa&ed microtubules (MT) close to a prophase nucleus. Some of them (arrow) enter the interior of the nucleus through gaps in the disintegrating envelope (NE).)<39,000

430 K. TANAKA

Fig. 17. Another superficial section through the prophase nucleus of Fig. 16. Remnants of the nuclear envelope at top left and bottom right. MT = microtubuies. X56,000 Fig. 18. Cross section of a nucleus at a stage of prophase comparable to that illustrated in Fig. 15. Cross sections of microtubules are seen both inside and outside the nuclear envelope (NE). X65,600

Fig. 19. Longitudinal section of a nucleus in metaphase. Chromosomes (CHR) are arranged as an equatorial plate. Nucleolar material (NC) is concentrated at the poles. The nucleus is flanked by an extensive spongework of cisternae (LC). 5<13,970 Fig. 20. High-power view of another section of the same nucleus. Spindle microtubules (SMT) pass through the nucieolar material (NC) and end near the membrane (arrow). 5<35,475 Fig. 2I. Microtubules of the spindle (SMT) in contact with chromosomes (CHR). 5<46,400

Mitosis in the Fungus Basidiobolus ranarum 431

Figs. 19-21

432 K. TANAKA

(Figs. 15 and 16). The number of microtubules surrounding the nuclear envelope is now much greater than it is during interphase. Some of them seem to run parallel to each other and more or less parallel to the envelope. However, grazing sections demonstrate that all the microtubules are not aligned in the same direction. Examination of serial sections suggests that the microtubules are arranged in the shape of two caps which enclose and extend some way around the prophase nucleus. A view of the surface of the envelope of a disintegrating prophase nucleus is provided in Fig. 17. One sees numerous microtubules and the beginnings of a membranous perinuclear labyrinth which becomes increasingly voluminous during later stages of mitosis. At this stage spindle fibers have not yet formed in the interior of the nuclear region where chromosomes are not clearly differentiated from nucleolar material but some of the external microtubules are now beginning to enter the nuclear region through openings in the disintegrating nuclear envelope (Figs. 16 and 18). Following this stage, microtubules, free or associated with chromosomes, appear in the nucleus. At a later stage, when the nuclear envelope has almost completely disintegrated, the nuclear region is, as it were, walled off from the cytoplasm by the aforementioned labyrinthine membranous spongework which is at this time maximally developed (Figs. 19 and 20). At this stage it becomes clearly apparent that some of the microtubules are associated with the chromosomes (Fig. 21).

3.2.3. Metaphase

At metaphase dense clumps of granules and fibers regarded as profiles of chromosomes are arranged in an equatorial plane and many of the microtubules cross this plane at right angles to it (Figs. 10 B and 19). In many instances microtubules are seen in close contact with profiles of chromosomes. Alongside them are others which may be in contact with chromosomes above or below the plane of the sections or which may represent the continuous fibers of the mitotic spindle. The microtubules traverse the dense masses of nucleolar material that have now concentrated at the poles and end close to the perinuclear spongework of membranous cisternae. No direct connec- tions of the microtubules of the spindle with the membranes have been observed (arrow in Fig. 20) and no organized structure resembling a centriole has been seen in any of the numerous sections of dividing nuclei that we have examined. The microtubules of the spindle have diameters of approximately 200 _~. Most of them are straight but wavy tubules have also been seen.

3.2.4. Anaphase

At anaphase the chromosomes have moved or have been pushed towards the poles of the mitotic figure (Fig. 10 C). Fig. 22 shows an example of this stage at which most of the chromosomes have already become embedded in


Fig. 22. Anaphase. Dense "end plates" consisting of nucleolar material and chromosomes are separated by a wide interzonal space which is free from mitochondria but still contains many microtubules. Note reconstructed envelopes (NE) around the outer aspect of the end plates. X8,670 Fig. 23. More highly enlarged view of another section of the same nucleus. Spindle microtubules in contact with chromosomes (CHR). X51,000

434 K. TANAKA

dense caps ("end plates") of nucleolar material. A portion of one polar region of an anaphase nucleus is shown at high magnification in Fig. 23. The spindle microtubules lie in channels of matter of low density bordered by the dense matrix of the "end plates". Here and there the microtubules are seen to end squarely in contact with bars of especially dense matter which may represent parts of chromosomes. The end plates are no sooner formed than they are surrounded on their outer aspect by a newly formed close fitting envelope which is in some places continuous with the spongework of cisternae which at this stage is still present in the cytoplasm outside the mitotic figure (Fig. 22, lower left).

3.2.5. Telophase

At telophase the end plates move or are moved still further away from each other and at the same time become rounded and gradually completely enclosed by a nuclear envelope; become, in fact, daughter nuclei. Fig. 24 shows part of a longitudinal section through a cell at this stage of mitosis. Another section through the same nucleus is shown at higher magnification in Fig. 25. The plane of the section is inclined to the plane of the mitotic figure; consequently, only one of the daughter nuclei is shown. The other nucleus is represented by profiles of the labyrinth of cisternae which persists far into telophase. The interzonal region between the separated nuclei is still devoid of cytoplasmic organelles but full of microtubules and ribosomal granules. Part of a telophase nucleus is shown at high magnification in Fig. 26. At this stage it is not possible to distinguish chromosomal from nucleolar material since both have similar density. Telophase nuclei are almost completely surrounded by a smoothly fitting envelope of normal dimensions except for a central gap facing the interzonal region. The envelope at this point appears to close concentrically in the manner of an iris dia- phragm. Large numbers of microtubules are converging towards the gradually narrowing gap. Scattered among them are aggregates of dense granules destined to be included in the new nucleolus. Across the cone of converging microtubules lie others which may have strayed into this region from the surrounding cytoplasm. A young, completely reconstituted nucleus is shown in Fig. 27. The cell to which it belongs had already begun to be subdivided by a transverse partition. In the nucleus chromatin and nucleolar material

Fig. 24. Telophase nucleus sectioned at an oblique angle to the spindle axis. One of the daughter nuclei with nearly completed envelope is at the top of the figure. Profiles of the labyrinth of perinuclear cisternae (LC) are seen at the upper left and at the lower pole of the division site. There is a broad interzonal region. X7,630 Fig. 25. Another section of the nucleus of Fig. 24. MT = cytoplasmic microtubules. X20,880 Fig. 26. Another portion of the section of Fig. 25. Numerous spindle micrombules (SMT) are diverging from a gap in the envelope of a telophase nucleus. X20,880


Figs. 24-26

436 K. TANAKA

have been sorted out; the nucleolar material is more loosely packed than it is in mature nuclei, Remnants of the labyrinthine cisternae are still to be seen close to the nucleus, Remnants of the mitotic spindle were still present in the interzonal region of this specimen but invasion of the region by mitochondria had begun.

Fig. 27. Reconstituted nucleus. The nuclear envelope is complete but the nucleolus has not yet acquired the dense texture characteristic of the interphase stage. The cell containing this nucleus (and its sister nucleus) had begun co lay down a sepmm between the two nuclei. X9,340

4. D i s c u s s i o n

Basidiobolus is counted among the Entornophthorales~ Other members of this order, e~g. Empusa and Conidiobolus, have coenocytic hyphae filled with innm-aerab~e smali n~clei wifl~ minute chromosomes. Mitosis is intranuclear and its phases are not easily described because a distinct metaphase stage is la&ing and anaphase and telophase are accomplished by elongation and constriction of the intact nucleus. Basidiobolus differs markedly from its congeners in having a more or less conventional type of mitosis in the course of which the nuclear envelope breaks down, and the &romosomes align them- selves on a metaphase plate and move to opposite poles with the help of a massive spindle apparatus. Only unusual is the persistence of the nucleolar material which is discussed below. The process of mitosis is readily followed


in living nuclei and were it not for the small size of its chromosomes and their invisibility in the living nucleus, Basidiobolus would be a particularly rewarding object for the study of mitosis in a living cell. Among fungi that distinction is at present held by Fusarium oxysporium in which AIST (1969) has recorded with admirable clarity the behaviour of chromosomes and spindle in living dividing nuclei. The conservation of nucleolar material during mitosis and its close association with the chromosomes in Basidiobolus, previously seen in the light microscope and now, more clearly, in electron micrographs is reminiscent of chromosome behaviour in Spirogyra (JORDAN and GODWARD 1969). Many instances have been recorded of the presence of microtubules close to but outside the envelope of nuclei in early prophase of mitosis (HARRIS 1952, MANTON 1964 a, b, ROBBINS and GONATAS 1964 b, PICKETT-HEAPS and NORTHCOTE 1966, CRON- SHAW and EsAo 1968). Remarkable is the sudden increase in their numbers at this stage. In wheat meristematic cells about to divide a band of microtubules is seen close to the cell wall encircling the nucleus in a plane at right angle to the long axis of the future mitotic spindle (PICKETT-HEAPS and NORTHCOTE 1966). This band of tubules disappears at the start of prophase concomitant with the emergence of a new set of microtubules aligned to form the spindle of mitosis. The authors have suggested the possibility that the microtubules of the preprophase band may somehow contribute to the deveiL opment of the proper spindle apparatus which begins at prophase. The suc- Cessive emergence of sets of microtubules during early stages of division has also been reported for other plant cells (CRONSHAW and EsAu 1968). How- ever, despite these and kindred observations, the sudden emergence of ordered arrays of microtubules at prophase in Basidiobolus and other cells remains an unsolved problem. In the mitosis of the nuclei of animal cells and of fungi that have a motile phase such as Albugo (BERLIN and BOWEN 1964), Allomyces (RENAUD and SWIFT 1964, ROBINOW and BAKERSPIGEL 1965), and Saprolegnia (HEATH and GREENWOOD 1969), the continuous fibers of the mitotic spindle are usually regarded as direct or indirect products of the activity of centrioles. In the evocative phrase of D~ HARVEN and BERNHARD (1956) "Le centriole apparait ainsi comme la v&itable fili~re du cytoplasme". However, spindle tubules can apparently be formed without the presence of centrioles. In higher plants they seem to be lacking altogether. In fungi that lack a motile phase, such as ascomycetes and basidiomycetes proper, centrioles are also absent, but in dividing nuclei of these forms the tubules of the intranuclear spindle diverge from disc-shaped organelles ("plaques") closely attached to or set into the nuclear envelope at points opposite to each other. Regarded as homologous to centrioles by RoI31NOW and MARAK (1966) and AIST (1969) they are believed to be equivalents of kinetochores by GIRBARDT (1968) on the basis of detailed observations on nuclear behaviour during mitosis of the basidio- Protoplasma 70/8--4 29

438 K. TANAKA

mycete Polystictus versicolor. Oddly enough, in Basidiobolus nuclei, either ordinary or modified centrioles or kinetochore equivalents have not been found in the present work. Little is known and less can be suggested about the origin of microtubules. De novo synthesis in the region where microtubules are expected to be observed should not be ruled out. Apart from the problem of the origin of spindle fibers there is the question of the identity of the tubules found outside of the nucleus at prophase with those which later compose the fibers of the mitotic spindle. In conventional mitosis microtubules in the vicinity of a prophase nucleus seem to pass through gaps in the nuclear membrane to condensed portions of the chromosomes (PIcKETT-HzAI'S and NORTHCOTE 1966). Events seem to be following a similar course in Basidiobolus. At any rate, microtubules appear to be in firm association with chromosomes in the dividing nucleus by the time the nuclear envelope has disintegrated. Connec- tions between spindle fibers and chromosomes have been seen in meiotic and mitotic figures in several fungi (ALDRICH 1967, Lu 1967, [MOTTA 1967, ICHIDA and FULLER 1968) but neither in Basidiobolus nor in other fungi have distinct components of chromosomes been seen which might represent proper kinetochores. In this respect fungal chromosomes resemble those described in several protozoa (Roach and DANIELS 1962, JENKINS 1967, TUCKER 1967). The behaviour of membranous structures during the breakdown and reconstruction of the nuclear envelope has been discussed by several authors (PORTER and MACHADO 1960, ROm3INS and GONATAS 1964 b, KRISHAN and BUCK 1965). In many, though not all, fungal cells that have been examined from this point of view, the nuclear envelope persists during mitosis and daughter nuclei are produced by elongation and constriction of the mother nucleus. In Basidiobolus the breakdown of the nuclear envelope at prophase is accompanied by the development of an extensive spongework of membranes at the periphery of the mitotic figure. New envelopes begin to be laid down early in anaphase. The interval between breakdown of the nuclear envelope and its reconstruction around the daughter nuclei is thus very brief. It deserves to be pointed out that the perinuclear spongework of cisternae is composed not only of flat sheets but also of tubular structures. Seen as a whole the perinuclear system appears to provide a barrier around the site of mitosis against the intrusion of cytoplasmic organelles and serves also as a source of material for the reconstruction of the nuclear envelopes. In the nucleolus of Basidiobolus fibrillar and granular components are inter- Mngled, in contrast to the nuclei of plant and animal cells where these two kinds of material are more clearly set apart from each other (LAFoNTAINE and CHOUINARD 1963, BRINKLEY 1965, STEVENS 1965). The nucleoius is diluted out but, contrary to what is seen in higher organisms, only partly dissolved during mitosis. Experiments with labelled nucleoli will be required to decide how much nucleolar material is merely rearranged and how much is


n e w l y syn thes ized du r ing mitosis . A t p resen t i t seems t ha t much o ld nuc leo la r

m a t e r i a l r emains in t ac t in the f o r m of smal l aggregates of dense granules

which, ea r ly in mitosis , coalesce to f o r m the dense " e n d p l a t e s " of the mi to t i c

figure. O n the basis of l igh t m i c r o g r a p h s i t has been suggested (ROBINOW

1963) t ha t sp ind le fibers arise t h rough the conver s ion of nuc leo la r m a t e r i a l

bu t no evidence o f such a process has been encoun te red in the presen t w o r k .

A c k n o w l e d g e m e n t

The author thanks Prof. C. F. ROBINO\V for guidance and encouragement throughout the course of this work, for reading the manuscript, and for granting permission to use his photographs in Fig. 10. He is also grateful to Mr. JOHN MARAK and to Mrs. MICKY HALL for their technical assistance. This investigation was carried out while the author held a post- doctoral fellowship of the Medical Research Council of Canada in 1967-1968.

R e f e r e n c e s

AIST, J., 1969: The mitotic apparatus in fungi, Ceratocystis fagacearurn and Fusarium oxysporum. J. Cell Biol. 40, 120--135.

ALDRICH, H. C., 1967: The ultrastructure of mitosis in three species of Physarurn. Myco- logia 59, 127--148.

BERLIN, J. D., and C. C. BO-,VEN, 1964: Centrioles in the fungus Albugo candida. Amer. J. Bot. 51, 650--652.

BRINKLEY, B. R., 1965: The fine structure of the nucleolus in mitotic divisions of chinese hamster cells in vitro. J. Cell Biol. 27, 411--422.

CRONSHAW, J., and K. EsAu, 1968: Cell division in leaves of Nicotiana. Protoplasma 65, 1--24.

DE HARVEY, E., et W. B~RNHARD, 1956: ]~tude au microscope 61ectronique de l'ultrastructure du centriole chez les vert6br6s. Z. Zellforsch. 45, 378--398.

GIR~3ARDT, M., 1968: Ultrastructure and dynamics of the moving nucleus. Symp. Soc. exp. Biol. 22, 249--259.

HARRIS, F., 1962: Some structural and functional aspects of the mitotic apparatus in sea urchin embryo. J. Cell Biol. 14, 475--487.

HEATH, I. B., and A. D. GREENWOOD, 1969: Communication by Dr. ROBINOW. ICmDA, A. A., and M. S. FULLER, 1968: Ultrastructure of mitosis in the aquatic fungus

Catenaria anguillulae. Mycologia 60, 141--155. JENKINS, R. A., 1967: Fine structure of division in ciliate protozoa I. Micronuclear mitosis

in Blepharisma. J. Cell Biol. 34, 463--481. JORDAN, E. G., and M. B. E. GODWARD, 1969: Some observations on the nucleolus in

Spirogyra. J. Cell Sci. 4, 3--15. KRISHAN, A., and R. BucK, 1965: Structure of the mitotic spindle in L-strain fibroblasts.

J. Cell Biol. 24, 433--444. LAFONTAINE, J. G., and L. A. CHOUINARD> 1963: A correlated light and electron microscope

study of the nucleolar material during mitosis in Vicia faba. J. Cell Biol. 17, 167--201. Lu, B. C., 1967: Meiosis in Coprinus lagopus: a comparative study with light and electron

microscopy. J. Cell Sci. 2, 529--536. MANTON, L, 1964 a: Observations with the electron microscope on the division cycle in the

flagellate Prymnesium parurn Carter. J. roy. micr. Soc. 83, 317--325. - - 1964b: Preliminary observations on spindle fibers at mitosis and meiosis in Equisetum.

J. roy. micr. Soc. 83, 471--476.

29*


MOTTA, J. J., 1967: A note on the mitotic apparatus in the rhizomorph meristem of Armil- Iaria mellea. Mycologia 56, 370--375.

PmKETT-HEAvs, J. D., and D. H. NORTI~COTE, 1966: Observation of microtubules and endoplasmic reticulum during mitosis and cytokinesis in wheat meristems. J. Cell Sci. 1, 109--120.

PORTER, K. R., and R. D. MAC~IADO, 1960: Studies on the endoplasmic reticulum IV. Its form and distribution during mitosis in cells of onion root tip. J. biophys, biochem. Cytol. 7, 167--180.

R~NAUD, F. L., and H. SwIvT, 1964: The development of basal bodies and flagella in Allomyces arbusculus. J. Ceil Biol. 23, 339--354.

ROBBINS, E., and N. K. GONAT•S, 1964 a: In vitro selectio~x of the mitotic cell for subsequent electron microscopy. J. Cell Biol. 20, 356--359.

- - - 1964b: The ultrastructure of a mammalian cell during the mitotic cycle. J. Cell Biol. 21, 429--463.

RO~INow, C. F., 1963: Observations on cell growth, mitosis, and division in the fungus Basidiobolus ranarum. J. Cell Biol. 17, 123--152.

- - and A. BA~eRSPmEL, 1965: Somatic nuclei and form of mitosis in fungi. In: The fungi (C. C. AINSWORTH and A. S. S~SSMAN, eds.), New York: Academic Press, Inc. 1, 119.

- - and J. MAI~AK, 1966: A fiber apparatus in the nucleus of the yeast cell. J. Ceil Biol. 29, 129--151.

ROTH, L. E., and E. W. DANIELS, 1962: Electron microscopic studies of mitosis in amoebae. II. The giant ameba Pelornyxa carolinensis. J. Cell Biol. 12, 57--78.

STEWNS, B. J., 1965: The fine structure of the nucleolus during mitosis in the grasshopper neuroblast cell. J. Cell Biol. 24, 349--368.

TUCKER, J. B., 1967: Changes in nuclear structure during binary fission in the ciliate Nassula.

J. Ceil Sci. 2, 481--498. VeNABLe, J. H., and R. COGGESHALL, 1965: A simplified lead citrate stain for use in electron

microscopy. J. Cell Biol. 25, 407--408.

Author's address: Dr. KENJI TANAK& Institute of Applied Microbiology, The University of Tokyo, Bunkyo-Ku, Tokyo, Japan.

Documents

Mitosis in the fungusBasidiobolus ranarum as revealed by electron microscopy