MITOSIS IN THE FISSION YEAST SCHIZOSACCHAROMTCES … · confirm and extend the observations made with light microscopy. Without first arranging themselve on a metaphasse plate, mitotic

J. Cell Sci. 9, 475-507 (197O 475Printed in Great Britain

MITOSIS IN THE FISSION YEAST

SCHIZOSACCHAROMTCES POMBE: A

COMPARATIVE STUDY WITH LIGHT AND

ELECTRON MICROSCOPY

E. KATHLEEN McCULLY AND C. F. ROBINOWDepartment of Bacteriology and Immunology, University of Western Ontario,London, Canada

SUMMARYMitosis in Schizosaccharomyces pombe has been followed in living cells by phase-contrast

microscopy and studied in fixed and suitably stained preparations by light microscopy. Success-ful preservation of nuclear fine structure in this yeast, not previously achieved, has allowed us toconfirm and extend the observations made with light microscopy.

Without first arranging themselves on a metaphase plate, mitotic chromosomes becomegrouped in 2 clusters radiating, finger-like, from 2 points of attachment at opposite poles of anelongating nucleus. At these 2 sites electron microscopy reveals the presence of disk-shapedelectron-dense organelles which we have called kinetochore equivalents (KCE). At mitosis theKCEs are connected across the nucleus by a narrow bundle of parallel microtubules which werefer to as the spindle.

Integration of our observations has led us to propose that at mitosis the separation of theKCEs and their attached chromosomes is initiated by a differential expansion of the nuclearenvelope restricted to the region between recently divided KCEs and that expansion of thenuclear envelope later becomes general, resulting in a marked elongation of the nucleus.Displacement of the nuclear contents to the ends of the elongated nucleus gives it the shape ofa dumbbell.

The elongation of the microtubule bundle keeps in step with the elongation of the nucleusbut does not appear to be the cause of it. It may have the function of keeping the separatedKCEs rigidly apart.

During mitosis the nucleolus persists and stretches out within the unbroken envelope of thenucleus as it elongates. Towards the end of division equal amounts of nucleolar material arefound in the rounded ends of the dumbbell-shaped nucleus.

The break up of the dumbbell shape into daughter nuclei seems to involve the breaking ofits tenuous middle part and a pivoting of its 2 ends in opposite directions.

In the course of our work on mitosis we have become aware of features in the cytoplasm ofgrowing 5. pombe cells which are described here for the first time. The cells invariably containseveral prominent vacuoles containing an extremely electron-dense material which stainsmetachromatically with toluidine blue and may be polyphosphate. The mitochondria are ofspecial interest for 2 reasons. First, because they have unique mesosome-like membraneinvaginations and secondly, because a mitochondrion is regularly associated with the singleKCE by the side of the interphase nucleus, as well as with each one of the 2 KCEs that occupyopposite ends of the intranuclear spindle during mitosis.

INTRODUCTIONObservations on dividing fungal nuclei with the electron microscope are not yet

extensive, and the literature based on light microscopy is filled with contradictions.Nevertheless, the evidence available seems to indicate that in many instances, the

476 E. K. McCully and C. F. Robinow

geometry of mitosis in fungi differs from the 'classical' pattern. Pickett-Heaps (1969)has suggested that variations in the patterns of mitosis which are encountered indifferent groups of fungi may represent 'relic' stages in the evolution of the morecomplex mitotic mechanisms in cells of plants and animals. Detailed studies of a widerange of fungi are needed for an assessment of this interesting generalization.

The fission yeast, Schizosaccharomyces pombe, a fungal organism now increasinglystudied by cell physiologists and geneticists (Mitchison, 1970; Leupold, 1970) is alsowell suited for observations on mitosis. Large numbers of dividing cells are readilyobtained, and the mitosis of the single central nucleus is related in a regular mannerto the cell's cycle of growth and division (Mitchison, 1970). Dividing nuclei can thusbe expected in cells exceeding a certain length.

Previous studies of mitosis in S. pombe have been carried out exclusively with thelight microscope. Early literature on the subject has been reviewed by Schopfer,Wustenfeld & Turian (1963). These authors, on the basis of observations made oncells stained with Giemsa solution after hydrolysis, concluded that 'mitosis appearsto be accomplished without the help of a spindle apparatus and without the formationof typical metaphase plates'. Using acid fuchsin, one of us (C. R.) has previouslydemonstrated the presence of an intranuclear fibre in dividing nuclei of this yeast.A photomicrograph of this fibre, together with several illustrations of chromosomeconfigurations at mitosis similar to those shown by Schopfer et al. (1963), and a time-lapse sequence of living dividing nuclei with phase-contrast microscopy, have beencontributed by C.R. to a recent monograph on S. pombe by Mitchison (1970).

Until now, fixatives used in studies of the fine structure of S. pombe (MacLean,1964; Schmitter & Barker, 1967; Osumi & Sando, 1969; Oulevey, Deshusses &Turian, 1970; Heslot, Goffeau & Louis, 1970), have been unsuitable for the pre-servation of nuclear organization. We have now made observations with the electronmicroscope which confirm and add to the previous knowledge of mitosis in thisorganism based on light microscopy. We have also made new observations on thestructure and behaviour of the mitochondria and certain cytoplasmic inclusions.

MATERIALS AND METHODS

Material

Most of our observations have been made on cells of 2 morphologically indistinguishablediploid strains of S. pombe which were kindly supplied by Dr H. Gutz of the University ofTexas. Because diploids are considerably larger than haploids, they were easier to examine andphotograph with light microscopy. However, we have satisfied ourselves that nuclear structureand behaviour in normal wild-type haploids is the same as in diploids. The yeasts were main-tained and propagated for all purposes at room temperature on a single medium consisting ofDifco yeast extract 0-5 g, glucose 2-0 g, and agar 1-5 g per 100 ml of water.

Phase-contrast microscopy of living cells

We have used 'spreading drop' slide cultures in 21 % gelatin prepared according to Robinow& Marak (1966).

Mitosis in S. pombe 477

Fixation for light microscopy

After initial difficulties, the following reliable method was adopted for transferring sufficientlylarge numbers of growing and dividing cells from monolayers on agar to coverslips. A fairlyheavy innoculum was streaked 2 or 3 times over a peripheral segment of a Petri dish and leftovernight at room temperature. The next morning the cells were spread over the rest of thedish using a bent glass rod wetted with water. Eight hours later, some of the fresh crop of cellswas scraped into a narrow ridge with the edge of a coverglass. The strip of agar covered by theheaped up cells was cut out, lifted from the dish, and placed, yeasts down, on the edge of a22 x 22 mm coverslip. A small amount of fresh egg white was streaked alongside the slab ofagar with a fine wire loop. The agar strip was then swiftly pushed sideways across the coverslip,leaving a thin film of cells dispersed in egg white on the coverslip which was immediatelyplunged into fixative contained in Columbia staining jars.

Two fixatives were used: Helly's (mercuric chloride 5 g, potassium dichromate 3 g, water100 ml; to 10 ml of which o-6 ml of 37% formaldehyde (formalin) is added just before use);and formalin-acetic acid-alcohol (FAA) (formalin 5 ml, glacial acetic acid 5 ml, 95 % ethanol50 ml, water 40 ml). Fixation with Helly's mixture, which is unstable, was not extended beyond10-20 min but preparations were sometimes left for longer periods in FAA. After both fixationsthe preparations were rinsed and stored in 70 % ethanol.

Staining for light microscopy

Chromosomes. The HCl-Giemsa procedure recommended by Robinow in Mitchison (1970)proved satisfactory provided that decolorization with acidified water was carried far enoughand the buffer-mounted specimen was adequately flattened with the help of the device de-scribed by Miller & Colaiace (1970). Transparent Giemsa preparations gave the same informa-tion as can be obtained with the aceto-orcein technique described in Mitchison (1970) butyielded more distinct photographs than the latter. Separation of the chromosomes was mosteasily achieved in material fixed in FAA, but the small portions of chromosomes that extendinto the nucleolus (see below) are more obvious after Helly fixation.

Nucleoli and spindles. These were selectively stained with acid fuchsin in 1% acetic acid(Robinow & Marak, 1966). After 4 min in a 1140000 dilution of this stain, spindles and nucleolistood out sharply against the unstained chromatin regions of nuclei in Helly-fixed preparations.Stained specimens were mounted over a drop of 1 % acetic acid. Cells fixed in FAA had noappreciable affinity for acid fuchsin, even when the dye was used in relatively high concentra-tions (e.g. 1:1000 for 10 min).

Cytoplasmic inclusions. Helly-fixed cells were stained in two ways: (i) In toluidine blue, for1 min in a 0005 % aqueous solution which was acidified just before use with 1 drop of o-oi NHC1 per 10 ml of stain. Stained specimens were mounted over a drop of acidified water (onedrop of o-oi N HC1 per 10 ml water), (ii) In Sudan black B, 0-5 % in ethylene glycol. Unstainedspecimens were directly mounted over a drop of the stain.

Microscopy and photography

We used a Bausch and Lomb tungsten ribbon lamp and a Carl Zeiss microscope equippedwith a VZ condenser (n.a. 1-4) adjustable for both phase-contrast and ordinary microscopy.A Kodak Wratten filter No. 11 was used in phase-contrast microscopy. For the photography ofstained preparations this was replaced by an interference filter maximally transmitting at546 nm. Photographs were taken with x 100 objectives (fluorite and apochromat for phase-contrast and ordinary microscopy respectively) in conjunction with a x 16 compensatingeyepiece, on cut film (Kodak Super-Panchropress for phase-contrast; Ektapan for stainedpreparations) carried by a bellows at a distance above the eyepiece which provided an initialmagnification of x 1800.

478 E. K. McCtdly and C. F. Robinow

Preparation for electron microscopy

We used a modification of the method of Karnovsky (1965). Monolayers of growing cells inPetri dishes were flooded with Karnovsky's formaldehyde-glutaraldehyde mixture. The cellswere scraped off the plates, centrifuged, and resuspended in fresh fixative for 14 h at roomtemperature. The fixative was removed with 8-10 washes of o-i M cacodylate buffer at pH 72.The cells were post-fixed for 6 h at room temperature in 1-33 % OsO4 in collidine buffer. Theywere then washed twice in collidine buffer and twice in distilled water before being stained in0-5 % aqueous uranyl acetate for 2-5 h. The cells were next suspended in molten 1-5 % wateragar at 47 °C and quickly centrifuged into a pellet before the agar could solidify. After cooling,the pellet of cells embedded in solid agar was cut into o-5-mm cubes. These were dehydratedin a graded ethanol series followed by anhydrous acetone and embedded in Araldite 6005(Richardson, Jarett & Finke, i960). Silver or grey sections, cut with a diamond knife, weremounted on carbon films and double-stained for 20 min with 1 % aqueous uranyl acetatefollowed by 8 min with lead citrate (Reynolds, 1963). Specimens were viewed with a PhilipsEM 200 electron microscope at 60 kV.

OBSERVATIONS

Cytoplasmic features

The cytoplasm of log-phase cells is closely packed with ribosomes and has manyvacuoles scattered throughout (Figs. 20, 21, 39). With the electron microscope it canbe seen that these vacuoles contain an extremely electron-dense material and arebounded by a unit membrane (see especially Fig. 38). The dense material does notfill the vacuole completely and loose coils of unit membrane are often present in therest of the vacuole space. Phase-contrast microscopy of living cells shows the vacuolesas dark spheres about 0-4-0-6 /im in diameter (Figs. 1-8). After fixation for lightmicroscopy, the vacuole contents stain red with toluidine blue at a low pH, a charac-teristic which may indicate the presence of polyphosphate (Keck & Stich, 1957). Thevacuoles do not stain with Sudan black B (compare Figs. 31, 32) suggesting that nounbound lipid is present.

The cytoplasm of growing cells also contains a few spherical inclusions about0-2-0-3 /im in diameter which tend to be in clusters at the ends of the cell. Thesehave a low electron density and are not membrane-bound (Figs. 21, 39). Inclusionsof the same size and relative quantity p£r cell are recognizable in the light microscope.With phase-contrast microscopy these appear as bright refractile droplets (Fig. 1).With bright-field microscopy it can be seen that these spheres stain blue with Sudanblack B (Fig. 31). They are presumably lipid inclusions.

The mitochondria are short in cells of the early growth phase (Fig. 20) and becomelonger as the cell grows. In long cells (which are close to nuclear division), the mito-chondria may stretch the entire length of the cell (Fig. 21), or they may be highlybranched. They remain long during mitosis and appear to divide just before theinward growth of a transverse septum results in the formation of 2 daughter cells.Continued fragmentation of the mitochondria after closure of the septum (Fig. 41)seems to produce the short mitochondria that are characteristic of cells in the earlygrowth phase.

The mitochondria in cells from growing cultures have a few long convoluted cristae


surrounded by a fine grey matrix and ribosome-like particles (see especially Figs. 25-27). They also have mesosome-like membrane invaginations (Figs. 22-24). Thesemembranes often seem to be covered with amorphous electron-dense material andthey usually encircle an electron-transparent region. There may be several of thesemembranous inclusions in one mitochondrion (Fig. 21).

The regular association of a mitochondrion with the nucleus is dealt with below inthe section which describes nuclear features seen with electron microscopy.

Nuclear features

Observations on the nuclei of Living cells with phase-contrast microscopy. Phase-contrast microscopy has provided information about the sequence and timing ofmitotic events and a standard by which to judge the quality of preservation achievedby the fixation method used for electron microscopy.

The nucleus in long cells just before division is spherical or ovoid and has a darkgrey, excentrically placed nucleolar region and a transparent region which appropriatestaining shows to be the site of chromatin (described below), although we see noindication of chromosomes (Fig. 1).

The first visible sign of the beginning of mitosis is a slight decrease in the densityof the nucleolus. Next, a faint grey line appears across the chromatin region (Fig. 2).Electron microscopy and the evidence from stained preparations, to be describedbelow, allows us to identify this grey line as the intranuclear spindle. Subsequently,there is a marked nuclear elongation into a dumbbell shape through intermediatestages as shown in Figs. 3-5 and 7. The nucleolus persists and is stretched out in theinterior of the elongated nucleus.

Shortly after the slender dumbbell stage has been reached, there is a period of lowvisibility which may be due partly to a change in the consistency of the nuclear con-tents at this time, and partly to nuclear movements in addition to a straight glidingapart of the dumbbell ends. During this indistinct period, separation into daughternuclei apparently takes place.

By the time 2 separated nuclei finally become clearly visible, their volume seems tobe much greater than were the rounded ends of the dumbbell (compare Figs. 5, 7,with 6, 8). The nucleoli in these daughter nuclei are both excentrically placed but aretilted towards opposite sides of the cell (Figs. 6, 8). The latter observation is consistentwith our conclusions based on electron microscopy, that nuclear separation involvespivoting movements of the dumbbell ends in opposite directions. The sense of direc-tion of these proposed movements is indicated by arrows in Fig. 7.

Observations by light microscopy on cells stained with Giemsa. Giemsa's stain showsclearly that the nucleolus and the bulk of the chromatin occupy distinctly separatenuclear regions. In sufficiently flattened, transparent preparations of FAA-fixed cells,the main mass of chromatin in resting nuclei can be resolved in most instances intoa cluster of 5-7 short, thick bent rodlets which we regard as chromosomes (Figs. 9, 10).Their number seems to be the same in resting nuclei of the cells of haploid and diploidstrains.

31 C E L 9


The intranuclear spindle which is revealed by phase-contrast microscopy in livingdividing nuclei is not visible in hydrolysed Giemsa preparations.

The chromosomes are found contracted and aggregated into 2 groups already at anearly stage of mitosis when the nucleus just begins to elongate and the shape of thenucleolus starts to become irregular (Figs. 11, 12). The details of the process ofchromosome segregation which must be taking place at this time have remainedobscure.

The rest of mitosis seems to involve increasingly wider separation of the 2daughter chromosome clusters which, during this phase, are usually seen to divergelike spread fingers from 2 points of origin (in the geometrical sense) at opposite poles ofthe elongated nucleus (Figs. 13-15). At the same time the nucleolus is pulledapart into 2 tapering masses which remain in contact with the chromosomes at theirbase.

At all stages of the nuclear cycle the region occupied by the nucleolus contains somechromatin in the form of either short rods, as in the recently reconstituted nuclei ofFig. 16, or winding threads as in Fig. 17. We regard these objects as intranucleolarchromosomes or portions of chromosomes embedded in the nucleolus. They areinvariably less chromatinic than the main mass of chromosomes aggregated besidethe nucleolus, except in the relatively small compact nuclei of non-dividing cells(from cultures several days old) where the nucleoli are regularly seen to contain2 distinct, presumably contracted chromatinic bodies which are as deeply stainable(if not more so) as the main mass of chromatin (Figs. 18, 19).

Observations on tlie nucleus by electron microscopy. The following account describeschanges in nuclear morphology which occur during the growth cycle of S. pombe asseen with electron microscopy. We have used 2 criteria for arranging these observa-tions on fixed cells into a life-like sequence. First, characteristics such as cell lengthand the presence or absence of a septum which, in S. pombe are correlated with thegrowth cycle (Mitchison, 1970), and secondly, our knowledge of mitosis in livingcells.

The early interphase nucleus is spherical, with an excentrically placed nucleolarregion containing ribosome-like particles dispersed among amorphous electron-densematerial, and a chromatin-containing region which is of uniformly low electrondensity, showing nothing recognizable as chromosomes (Fig. 20). Located on theoutside of the nuclear envelope, adjacent to the chromatin-containing region androughly opposite the position of the nucleolus is a structure which, for reasons to beoutlined in the Discussion, we call the KCE (kinetochore equivalent). The positionof this organelle at interphase is always closer to one of the side walls of the cell thanit is to the cell's longitudinal axis, and it is always lying in a narrow ribosome-freezone which is bounded by the outer nuclear membrane on one side and a mito-chondrion on the other. Serial sections of the early interphase KCE show that it isa curved disk about 220 nm in diameter with a bulge in the centre of its concave sidewhich is in contact with the nuclear envelope (Figs. 25, 26). This curved disk has2 regions: an electron-dense portion consisting of at least 3 layers plus the bulge, anda fuzzy grey portion covering the convex surface of the electron-dense region. There

Mitosis in S.pombe 481

is also some amorphous electron-dense material inside the nuclear envelope, beneaththe site where the KCE appears to be attached, which has the effect of making thisregion stand out from the rest of the envelope.

At late interphase, the nucleus is enlarged and usually ovoid (Fig. 21). The KCEat this stage is in the same position but its morphology is different from that of thepreviously described interphase KCE. It has become longer and more bar-shaped andit lacks the fuzzy grey surface portion (Fig. 27). Like the early interphase KCE, thistype of KCE is accompanied by amorphous electron-dense material underlining theinner aspect of the nuclear envelope.

On rare occasions we have observed what appears to be a divided KCE locatedbeside an otherwise normal-looking late interphase nucleus. This structure can bestbe described as 2 short electron-dense bars which lie very close and almost parallel toeach other in a ribosome-free identation of the nuclear envelope, with their long axesat right angles to the membrane surface (Fig. 28).

Nuclei in the earliest stages of mitosis that are comparable to what we have seen inthe light microscope, look similar to interphase nuclei but are characterized by thepresence of a short peripheral spindle which runs through the chromatin region andjoins 2 KCEs located a short distance apart on the outside of the envelope; the shortsegment of envelope between the KCEs always appears to be convoluted or bulged(Figs. 29, 30, 33). However, over the greater part of their surface, these nuclei stillretain the rounded or oval contours that we see in interphase nuclei. The peripheralspindle seems to be a narrow bundle of parallel microtubules which is slightly curvedwhen the KCEs are a short distance apart (Fig. 29), and straight when the KCEs aremore widely separated (Figs. 30, 33). At this peripheral spindle stage and at all sub-sequent stages of spindle formation, the KCEs have a non-layered, disk-like appear-ance and seem to be closely pressed to the nuclear envelope. As in interphase, KCEsat the ends of intranuclear spindles are located in ribosome-free zones and each isclosely associated with a mitochondrion. These KCEs have approximately the samediameter as the curved, disk-shaped KCEs of early interphase but are considerablylonger than the 2 component bars of the proposed divided KCE described above. Thefact that we have not observed any of the intermediate steps which must occur betweenthe divided KCE stage (Fig. 28) and the short, curved, peripheral spindle stage(Fig. 29), may indicate that this change occurs rapidly.

A later stage in mitosis is characterized by a nucleus which has a narrow, elongated,almost rectangular shape (Fig. 35). The nucleolus persists and is stretched out in theinterior of the nucleus. The spindle microtubules occupy the longitudinal axis andseem to run parallel to each other, without interruption, between 2 KCEs located atopposite poles of the nucleus.

Subsequently the rectangular-shaped nucleus becomes further elongated into adumbbell shape, with 2 rounded ends joined by a long narrow channel containing thespindle microtubules (Figs. 37-39). These microtubules are stretched in a long,straight, parallel bundle between the widely separated KCEs located at oppositedumbbell ends. Before the long dumbbell stage (Fig. 39) is reached, the nucleolusapparently becomes divided between the rounded daughter ends so that no nucleolar

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482 E. K. Mc&dly and C. F. Robinow

material is found in the narrow connecting channel. The whole dumbbell-shaped unitis surrounded by an intact nuclear envelope. This membrane is typically double inmost places, but along the envelope of the spindle channel there are several minuteregions of high electron density which can be resolved into fold-like arrangements,with 4 unit membranes instead of 2 (arrows Fig. 37 and insets). As reported byOulevey et al. (1970), and shown in Fig. 39, the cell septum is sometimes beginningto form at this stage, before nuclear separation is complete.

Two of the final steps in the division process are the breaking down of the spindlechannel, and the return of each KCE from its central position at a pole of the divisionaxis to its interphase position on the surface of the nucleus facing a side wall of thecell (as in Figs. 20, 21). Our observations seem to indicate that both these steps areachieved at the same time by a pivoting movement of each daughter nucleus. Thismovement may be inferred from the shape and position of daughter nuclei in cellswith an almost completed or a fully completed septum. Whereas the daughter ends ofthe dumbbell-shaped unit are elongated in the pulling-out direction, with each portionof the divided nucleolus located nearly opposite its respective polarly positionedKCE, a newly separated daughter nucleus is usually seen to be elongated almostperpendicularly to the division axis, with the nucleolus now opposite the KCE in itsnew interphase position at the side of the nucleus (compare shape of nuclei and thepositions of the nucleoli and KCEs in Figs. 39 and 41). The 2 KCEs (and nucleoli)of recently separated nuclei are always seen to lie facing opposite side walls of the cell,suggesting that final separation of daughter nuclei may involve pivoting movements inopposite directions by end portions of the dumbbell shaped nucleus.

After separation has occurred, mitochondria which accompany the polar KCEs inthe dumbbell stage are now consistently located in close apposition to the KCEs intheir new interphase positions (compare again Figs. 39 and 41). At this stage the KCEonce more becomes the curved, clearly layered disk which we have described as acharacteristic of early interphase cells.

Observations with the light microscope on nucleoli and spindles stained with acidfuchsin.The relative positions of the spindle and nucleolus at every stage in the division processare closely similar to those we have observed in the electron microscope (compareFigs- 33. 34: 35» 36: 39, 4°)- This technique confirms our electron-microscope observa-tions that the spindle in S.pombe is, in reality not spindle-shaped but is instead aparallel bundle of microtubules, since, the 'spindle' stained with acid fuchsin has theshape of a thin wire. This is in contrast to the spindles in Aspergillus (Robinow &Caten, 1969) which have a distinct, tapered, cigar-like shape.

At stages when the nucleus is elongated and the spindle occupies the longitudinaldivision axis, the wire-like spindle of 5. pombe has prominent knobs on the ends. Thesize of these knobs seems to correspond closely to the size of the KCE plus the ribo-some-free zone as seen with electron microscopy at comparable stages of nuclearelongation.

The small bead-like 'spindle initials' which are a regular feature of the restingnuclei of Saccharomyces and Aspergillus (Robinow & Marak, 1966; Robinow & Caten,1969) were not seen in the resting nuclei of S.pombe.


DISCUSSION

Why ' kinetochore equivalent' ?

Since the osmophilic structures that have now been seen several times at the polesof mitotic and meiotic intranuclear spindles in ascomycetes and basidiomycetes havebeen given different names by different authors, as set out in Table 1, we must justifyour use of Girbardt's term 'kinetochore equivalent' (KCE) for the spindle-associatedorganelle in S. pombe. All the names, except KCE, imply that the spindle-associated

Table 1. Names used for spindle-associated organelles

Name givenStudies of somatic

nuclear division Studies of meiosis

Modified or poorlypreserved centrioles

Centriolar plaques

Centriole ahnlicheKOrper

Centrosomal plaques

Centrosome

Disque centrald'un aster

KCE

Motta (1967)Armillaria

Robinow & Marak (1966)Saccharomyces

Robinow & Caten (1969)Aspergillus

Motta (1969)Armillaria

Girbardt (1968)Polystictus

Zickler (1969)Ascobolus,Podospora

Wells (1970)Ascobolus

Lerbs & Thielke (1969)Coprirws

Zickler (1970)Ascobolus, Podospora

Lu (1967)Coprinus

Westergaard & von Wettstein(1970)

NeotiellaSchrantz (1967)Pustularia

structures at the poles of dividing fungal nuclei are analogous to centrioles, since eventhe term 'centrosome' originally stood for a specialized region in the cytoplasm ofanimal cells containing a pair of centrioles (see Heidenhain, 1907, for a critical reviewof this once much-discussed subject). To consider these spindle-associated organellesin ascomycetes and basidiomycetes as modified centrioles one must assume that theyhave the same function as centrioles. As Pickett-Heaps (1969) has persuasively argued,the widely held belief that centrioles form spindles is based largely on circumstantialevidence and is unsound, since many cells without centrioles are able to form spindles.Then again, in certain cells that have large numbers of centriole-like basal bodiesavailable to them, such as the ciliated protozoa, centrioles have never been observed atthe poles of either mitotic or meiotic spindles. Moreover, even in those cells where acentriole is associated with the spindle, the microtubules are never directly connectedto it but appear to arise either from small bar-shaped 'centriolar satellites', strikingly


illustrated by Szollosi (1964) and de Harven (1968), or from the amorphous electron-dense material which has been frequently noted in the vicinity of centrioles (e.g. byBuck, 1967). All this evidence seems to indicate that the function of the centriole isnot directly related to spindle formation, and it would seem inappropriate to useterms relating to centrioles for the organelles in fungi which are more consistentlyand more intimately associated with microtubules than are centrioles and do thereforeseem to play a more likely role in spindle formation.

Pickett-Heaps (1969) proposes that the organelles in fungi are more accuratelydescribed as 'Microtubule organizing centres', a general term which he also used forsimilar electron-dense microtubule-associated regions in plant and animal cells in-cluding, among many examples, the kinetochore regions of chromosomes and thesmall electron-dense 'nucleating centres' in ectodermal cells of sea-urchin blastulae(Tilney, 1968). Experimental proof that the 'nucleating centres' are indeed sites ofmicrotubule assembly has since been provided by Tilney & Goddard (1970).

Although ' microtubule organizing centre' appears to be an acceptable general termfor spindle-forming organelles in fungi, we propose that use of the specific term'kinetochore equivalent' is justifiable in S.pombe for the following reasons: (i) Theorganelle in S. pombe appears to organize microtubules. Many observations (reviewedby Luykx, 1970) suggest that this is also true of the kinetochore region of chromosomes,(ii) The structure of the organelle resembles that of a true kinetochore. The layeredappearance of the organelle in 5. pombe, and even more so of the comparable ' centri-olar plaque' in Saccliaromyces (see fig. 28 in Matile, Moor & Robinow, 1969) isreminiscent of the distinctly layered kinetochores of certain animal chromosomes(Jokelainen, 1967; Brinkley & Stubblefield, 1966, and others) and of the chromosomesin Oedogonium (Pickett-Heaps & Fowke, 1969). (iii) The organelle in S. pombe isattached to the chromosomes. Our evidence for this statement is, admittedly, notconclusive since the chromosomes of S. pombe cannot be seen in the electron micro-scope. However, comparison of the chromosome configurations in stained preparationswith electron micrographs of dividing nuclei (e.g. compare Fig. 13 with Fig. 35), hasleft us with the impression that the chromosomes move apart in 2 clusters becausethey are attached to 2 opposing points on the nuclear envelope, and that these pointsare the spindle-associated organelles. Therefore we suggest that chromosomes areattached to the organelle, just as chromosome material is attached to a true kineto-chore.

Support for this hypothesis is provided by our observations that the spindle in thelight microscope appears to have the shape of a thin wire instead of a spindle shape,and that in the electron microscope it seems to consist of microtubules which runparallel to each other without interruption between the 2 organelles. Apparently, nodiscontinuous microtubules are present like the ones shown by Heath & Greenwood(1970) in Saprolegnia (in which, as in S. pombe, chromosomes are not visible withelectron microscopy). One would expect to see similar discontinuous microtubules atanaphase in 5. pombe if the chromosomes were not, as we suggest, directly attached tothe terminals of the microtubule bundle.

Thus, in the light of these arguments, we have adopted Girbardt's term' kinetochore


equivalent' for the electron-dense organelles found at the ends of the intranuclearspindle in S. pombe.

This term is definitely not applicable to the spindle-associated organelles of all fungi.There is no doubt from the observations of Lu (1967) on Coprinus and of Wells (1970)and Zickler (1970) on Ascobolus that during meiosis in these fungi, separate chromo-somes and chromosomal fibres exist. We consider that their so-called ' centrosomes'and 'centriolar plaques' may be best termed 'microtubule organizing centres' andmay be analogous to centriolar 'satellites' instead of to kinetochores.

Chromosomes

The number of linkage groups in S. pombe is 6 according to the extensive studiesof Flores da Cunha (1970). This is in good agreement with the number of chromatinicrodlets in interphase nuclei (5-7), which we regard as chromosomes. The fact thatapproximately the same numbers are seen in the nuclei of diploid and haploid cellsrecalls the experience of Robinow & Caten (1969) with diploid and haploid Aspergillusnuclei, and suggests that in S. pombe as in Aspergillus, there may be somatic pairingof chromosomes. For a detailed treatment of this subject, which is beyond the scope ofthe present paper, the reader is referred to the extended discussions by Brown & Stack(1968) and Stack & Brown (1969).

We do not know whether the nucleolar chromosomes are separate from, or parts of,the main mass of chromosomes which we believe to be attached to the KCE. We feelcertain however, that they are identical with the paired chromatinic elements whichSchopfer et al. (1963) described as 'possible centrosomes'. They are obviously not thesame as the spindle-forming organelles discussed in the present work.

Membrane growth, microtubule assembly and ' cytoplasmic forces' in nuclear elongationand division

To clarify our discussion of these matters, the stages of mitosis in 5. pombe aresummarized in Table 2.

We propose that the 2 KCEs which are seen at opposite poles of short peripheralspindles in Stage II nuclei arise from the division (via the parallel-bar stage) of thesingle KCE associated with the interphase nucleus, and that they have been movedapart to their Stage II positions by a differential membrane growth of a region ofnuclear envelope between them. We base this conclusion on the convoluted orbulging appearance of the short stretch of nuclear circumference between the KCEsat Stage II, which seems to indicate that this region has expanded, while the rest ofthe nuclear envelope has not and has therefore retained its round or oval interphasecontours.

Further, we propose that this differential membrane growth could also achieve thechange from Stage II to Stage III, since membrane growth exclusively in the regionbounded by the peripheral spindle would ultimately move the KCE attachment pointsinto polar positions and shift the excentrically placed nucleolus into a central positionwithout any actual nucleolar movement. This is suggested by our phase-contrastobservations (compare the position of the nucleolus in Figs. 1-3).


After Stage III has been achieved by differential membrane growth, Stage IV maybe the expression of a general expansion of the nuclear envelope. This could cause afurther separation of the KCEs since pressures in the cytoplasm resulting from theclose proximity of the cell's side walls would ensure that the growing nucleus ex-panded towards the ends of the cell. If it is assumed that the chromosomes and per-haps the nucleoli, too, via the nucleolar chromosomes, are in some way all linked tothe 2 KCEs then, as the KCEs are moved farther and farther apart, there is eventuallynot enough nuclear material left in the central region of the nucleus to prevent thecollapse of the nuclear envelope into a tight-fitting sleeve around the central part ofthe spindle.

Table 2. Characteristics of stages of mitosis in S. pombe

Stage ofnuclearcycle

Interphase

MitosisStage I(duplicationof the KCE)

Stage II

Stage III

Stage IV

Stage V(after separa-tion ofdaughternuclei butbefore cellseparation)

Shape ofnucleus

Spherical

Spherical oroval

Spherical oroval with abulging seg-ment

Elongated(roughlyrectangular)

Elongated(dumbbellshaped)

2 sphericaldaughternuclei

Position ofnucleolus

Excentric,nearly oppositeposition of theKCE

Excentric

Excentric

Central

Distributedto daughterend of thedumbbell

Both excentriceach one in apositionnearly oppo-site its re-spective KCE

Position of spindleand of the KCE

No spindle: KCElocated on thesurface of thenucleus facing aside wall of thecell

No spindle:duplicated KCEs ininterphase positionas above. They areparallel bars whichlie perpendicular tothe nuclear envelope

KCEs are at the polesof a peripheral intra-nuclear spindle

Spindle now occupiesthe long axis of thenucleus. KCEs areat the spindle poles,i.e. the ends of thenucleus

Spindle occupies thelong axis of thenucleus. KCEs areat the spindle poles,i.e. the ends of thenucleus

No spindle: KCEs ininterphase positionfacing opposite sidesof the cell

Shown inFig. no.

1, 20, 21

28

2, 29, 30, 33,34

4. 35. 36

37-4O

6, 8, 41


The fold-like regions along the nuclear envelope of the spindle channel at Stage IVcan be interpreted in 2 ways. They could be localized regions of membrane growthwhich, had the cell not been fixed, would soon have slid apart into an elongatedstretch of normal double membrane. However, we do not know of any publisheddescription of membrane growth in this way, and we do not see these fold-like areasat any other stage in the elongation process. Therefore it seems more likely that in-creased resistance to movement encountered by the daughter ends of the nucleus atStage IV as they approach the ends of the cell may cause the membrane, which iscontinuing to expand in the spindle channel region, to buckle back on itself.

We have rejected the possibility that the spindle is generating the force to cause thechange from Stage I to Stage IV. Our observations show that the KCE especiallyduring spindle formation, is closely associated with the nuclear envelope and appearsto be attached. In Saccharomyces, attachment of the comparable 'centriolar plaques'has been even more clearly established by Robinow & Marak (1966), who showed thatthey were 'set into enlarged pores of the nuclear envelope'. If the growing spindlewere generating the force to separate the membrane-attached KCEs, the shape of thenucleus at each stage would be different from what we have observed. At Stage II wewould expect the segment of envelope bounded by the peripheral spindle to be flatter,not more bulging than the rest of the nuclear envelope. At Stage III and IV we wouldexpect the growing spindle to push out the poles of the nucleus into sharp points. Onthe contrary, we have observed that the ends of nuclei at Stage III and IV are rounded,with the KCEs sitting in slight membrane depressions.

We consider that spindle growth is keeping in step with rather than causing KCEseparation, and that the spindle microtubules hold the separated KCEs rigidly apart.Microtubules are known to provide rigidity in many types of cells and, indeed, inmitosis of the dinoflagellate nucleus, there is good evidence that this is their chieffunction, while the chromosomes are moved apart by growth of the nuclear envelope(Kubai & Ris, 1969). Since the chromosomes seem to be attached to the KCE inS. pombe, the proposed membrane growth which moves KCEs apart may be causinggenome separation just as it does in dinoflagellates and in the primitive bacterialmitotic process (Ryter, 1968).

Between Stage IV and Stage V, an interval of poor visibility intervenes in livingcells seen with phase-contrast microscopy and we are therefore unclear about thedetails of daughter nuclei separation. It seems very likely that the microtubule pre-cursors may have become depleted by the time the long spindle of Stage IV has be-come assembled and that the spindle may therefore start to break down shortly afterthis stage has been reached. However, the consistent pivoting of daughter dumbbellends in opposite directions, as evidenced by the shape of daughter nuclei and thepositions of their KCEs and nucleoli in our electron micrographs of Stage V, to-gether with our phase-contrast observations at this stage which show the nucleoli atopposite sides of the daughter nuclei, suggest that other forces besides spindle break-down must be at work between Stages IV and V. Girbardt (1968) has been led bycinematographic evidence to postulate the existence of' cytoplasmic forces' which acton the KCEs of dividing hyphal nuclei of Polystictus. Perhaps such forces also act on


the KCEs of 5. pombe after spindle breakdown. It needs only the further assumptionof an intrinsic polarity of the organization of the KCEs to account for their beingmoved in opposite directions by the putative cytoplasmic forces.

Our view of the KCE attached to an expanding nuclear envelope as the main instru-ment of chromosome separation, leads us to support Pickett-Heaps (1969) in hisbelief that among the fungi 'relics of primitive ultrastructure' may be found pre-served and functioning.

Unanswered questions about mitochondria

Mitochondria have not been seen in close association with the spindle-formingorganelles of other fungi. In contrast, every KCE that we have seen in 5. pombe, bothin interphase and at mitosis, has been located in a narrow ribosome-free zone which isseparated from the rest of the cytoplasm by a mitochondrion. Since there is no reasonto suspect that the mitotic process in 5. pombe requires more energy than it does inother fungi, we believe that the KCE-associated mitochondria may be playing anotherrole in addition to energy production. At present, we cannot suggest what this rolemight be.

It is difficult to know whether the unique mesosome-like membranes inside themitochondria of log-phase S. pombe cells are real structures or artifacts of fixation.We feel certain, however, that they correspond to the electron-transparent' nucleoid'regions described by Osumi & Sando (1969) in S. pombe mitochondria after per-manganate fixation, because these 'nucleoids' are similar in size and position to our'mesosomes' and it was not clearly demonstrated that DNA was present in them.A full consideration of this subject is beyond the scope of the present work. However,it is interesting to note that these structures are not present in the mitochondria ofnormal stationary-phase cells (our own observation; and Heslot et al. (1970)). Thisseems to indicate that these mitochondrial regions have some functional significance.

This work has been supported by a grant to C. Robinow by the Medical Research Council ofCanada.

The authors thank Miss Julia Wang for reliable and enthusiastic technical assistance andMr John Marak for his skill and resourcefulness in keeping the electron microscope workingat peak efficiency.

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malian cell in vitro. Chromosoma 19, 28-43.BROWN, W. V. & STACK, S. M. (1968). Somatic pairing as a regular preliminary to meiosis.

Bull. Torrey hot. Club 95, 369-378.BUCK, R. C. (1967). Mitosis and meiosis in Rhodnius prolixus: the fine structure of the spindle

and diffuse kinetochore. jf. Ultrastruct. Res. 18, 489-501.FLORES DE CUNHA, M. (1970). Mitotic mapping of Schizosaccliaromyces pombe. Genet. Res. 16,

127-144.GIRBARDT, M. (1968). Ultrastructure and dynamics of the moving nucleus. In Aspects of Cell

Motility, 22nd Symp. Soc. exp. Biol. (ed. P. L. Miller), pp. 249-259. Cambridge UniversityPress.


HARVEN, E. DE (1968). The centriole and the mitotic spindle. In Ultrastructure in BiologicalSystems, vol. 3 (ed. A. J. Dalton & F. Haguenau), pp. 197-227. New York and London:Academic Press.

HEATH, I. B. & GREENWOOD, A. D. (1970). Centriole replication and nuclear division in Sapro-legnia. J. gen. Microbiol. 62, 139-148.

HEIDENHAIN, M. (1907). Centriol, Cytocentrum oder Mikrocentrum, Centrosom und Spharein ihrem gegenseitigen Verhaltnis. In Plasma und Zelle, Abt. I (ed. K. von Bardeleben),pp. 231-236. Jena: Fischer.

HESLOT, H., GOFFEAU, A. & Louis, C. (1970). Respiratory metabolism of a "petit negative'yeast Schizosaccliaromyces pombe 972h-1. J. Bact. 104, 473-481.

JOKELAINEN, P. T. (1967). The ultrastructure and spatial organization of the metaphase kineto-chore in mitotic rat cells. J. Ultrastruct. Res. 19, 19—44.

KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for usein electron microscopy. J. Cell Biol. 27, 137A (Abstr.).

KECK, K. & STICH, H. (1957). The widespread occurrence of polyphosphate in lower plants.Ann. Bot., N.S. 21, 611-619.

KUBAI, D. F. & Ris, H. (1969). Division in the dinoflagellate Gyrodinium cohnii (Schiller).J. Cell Biol. 40, 508-528.

LERBS, V. & THIELKE, C. (1969). Die Entstehung der Spindel waharend der Meiose vonCoprinus radiatus. Arch. Mikrobiol. 68, 95—98.

LEUPOLD, U. (1970). Genetical methods for Schizosaccharomyces pombe. In Methods in CellPhysiology, vol. 4(ed. D. M. Prescott), pp. 169-177. New York and London: Academic Press.

Lu, B. C. (1967). Meiosis in Coprinus lagopus: a comparative study with light and electronmicroscopy. J. Cell Set. 2, 529-536.

LUYKX, P. (1970). Cellular Mechanisms of Chromosome Distribution. Int. Rev. Cytol., Suppl. 2(ed. G. H. Bourne & J. F. Danielli). New York and London: Academic Press.

MACLEAN, N. (1964). Electron microscopy of a fission yeast, Schizosaccharomyces pombe.J. Bact. 88, 1459-1466.

MATILE, P., MOOR, H. & ROBINOW, C. F. (1969). Yeast cytology. In The Yeasts, vol. 1 (ed.A. H. Rose & J. S. Harrison), pp. 219-302. New York and London: Academic Press.

MILLER, M. W. &COLAIACE, J. D. (1970). Elimination of material that obscures stained chromo-somes in squashes of Viciafaba root tips. Stain Technol. 45, 81-86.

MITCHISON, J. M. (1970). Physiological and cytological methods for Schizosaccharomycespombe. In Methods in Cell Physiology, vol. 4 (ed .D . M. Prescott), pp. 131-165. New York andLondon: Academic Press.

MOTTA, J. (1967). A note on the mitotic apparatus in the rhizomorph meristem of Armillariamellea. Mycologia 59, 370-375.

MOTTA, J. (1969). Somatic nuclear division in Armillaria mellea. Mycologia 61, 873-886.OSUMI, M. & SANDO, N. (1969). Division of yeast mitochondria in synchronous culture.

J. Electron Microsc. 18, 47-56.OULEVEY, N., DESHUSSES, J. & TURIAN, G. (1970). Etude de la zone septale de Schizosaccharo-

myces pombe en division a ses 6tapes succesives. Protoplasma 70, 217-224.PICKETT-HEAPS, J. D. (1969). The evolution of the mitotic apparatus: an attempt at comparative

ultrastructural cytology in dividing plant cells. Cytobios 3, 257-280.PICKETT-HEAPS, J. D. & FOWKE, L. C. (1969). Cell division in Oedogonium I. Aust.J. biol. Sci.

22, 857-894.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.RICHARDSON, K. C , JARETT, L. & FINKE, E. H. (i960). Embedding in epoxy resins for ultrathin

sectioning in electron microscopy. Stain Technol. 35, 313-323.ROBINOW, C. F. & CATEN, C. E. (1969). Mitosis in Aspergilkis nidulans.jf. Cell Sci. 5, 403-431.ROBINOW, C. F. & MARAK, J. (1966). A fiber apparatus in the nucleus of the yeast cell. J. Cell

Biol. 29, 129-151.RYTER, A. (1968). Association of the nucleus and the membrane of bacteria: a morphological

study. Bact. Rev. 32, 39-54.SCHMITTER, R. E. & BARKER, D. C. (1967). A new fixation method for Schizosaccharomyces

pombe. Expl Cell Res. 46, 215-220.

490 E. K. McCully and C. F. Robinmo

SCHOPFER, W. H., WUSTENFELD, D. & TURIAN, G. (1963). La division nucleaire chez Sclrizo-saccharomyces pombe. Arch. Mikrobiol. 45, 304-313.

SCHRANTZ, J. P. (1967). Presence d'un aster au cours des mitoses de l'asque et de la formationdes ascospores chez l'Ascomycete Pustularia cupularis. C. r. liebd. Sianc. Acad. Set., Paris264D, 1274-1277.

STACK, S. M. & BROWN, W. V. (1969). Somatic pairing, reduction and recombination: Anevolutionary hypothesis of meiosis. Nature, Lond. 222, 1275-1276.

SZOLLOSI, D. (1964). The structure and function of centrioles and their satellites in the jelly-fish Phialidium gregariwn. J. Cell Biol. 21, 465-479.

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TILNEY, L. G. & GODDARD, J. (1970). Nucleating sites for the assembly of cytoplasmic micro-tubules in the ectodermal cells of blastulae of Arbacia punctulata. J. Cell Biol. 46, 564-575.

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WESTERGAARD, M. & WETTSTEIN, D. VON (1970). The nucleolar cycle in an ascomycete. C. r.Trav. Lab. Carlsberg 37, 195-237.

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{Received 18 March 1971)

Figs. 1-8. Time-lapse phase-contrast micrographs of living cells.

Figs. 1-4 are of the same cell. Figs. 5, 6 are of another cell, x 3600. The interphasenucleus (Fig. 1) has 2 distinct regions. One is a dark grey, excentrically placednucleolus (ri) and the other is a region of low density which is known from stainedpreparations to be the site of chromatin (chr, see Figs. 9, 10). Figs. 2-4 illustrate thetransformation of this interphase nucleus into an elongated, almost rectangularnucleus with the nucleolus stretched out in a central position. In Fig. 2 the spindle(s/>-between arrows) can be seen as a fine grey line crossing the nuclear region of lowdensity. Fig. 2 was taken 13 min after Fig. 1. The intervals between Figs. 2 and 3and between Figs. 3 and 4 are 4 and 6 min, respectively. Fig. 5 shows an early dumbbellstage. The nucleolus is stretched out in the constricted central portion, and 2 regionsof low density (chr, see Fig. 13) can be seen at opposite ends of the nucleus. In Fig. 6nuclear division has been completed and a transverse septum (s) has started toform (between asterisks). Note how the nucleoli of the 2 daughter nuclei are facingopposite side walls of the cell. The time interval between Figs. 5 and 6 is 52 min.

Two types of cytoplasmic inclusions are visible in these living cells: refractile lipiddroplets (I) and dense spherical vacuoles (v).

Figs. 7, 8. Interpretive drawings of Figs. 5, 6, respectively. The arrows indicate howwe think the ends of the dumbbell will pivot when final separation of daughter nucleitakes place (see text).

Mitosis in S. pombe

8


Figs. 9-19. Distribution of chromosomes in resting and dividing nuclei of 5. pombeshown by Giemsa staining after hydrolysis. For Figs. 9—17 the fixative was Helly's, forFigs. 18, 19 it was FAA. x 3600.

Fig. 9. Resting nuclei in cells representing 3 different stages of the growth cycle;5-7 chromosomes (chr) can be seen adjacent to the faintly staining nucleolus («).

Fig. 10. A resting nucleus, but the length of the cell as well as the size and distinctnessof the chromosomes suggest that mitosis is imminent.

Figs. 11-15. Dividing nuclei. The chromosomes separate as 2 clusters. Where theyareclearly resolved, chromosomes of the polar clusters appear to diverge finger-like fromsingle points of origin at the ends of elongated nuclei. These points coincide with thepositions occupied by the KCEs at the poles of intranuclear spindles (not visible withthe HCl-Giemsa procedure). Compare Figs. 11-15 with Figs. 35, 39. The nucleolus(n) is lightly stained. Note how it is pulled out between the chromosome clusters (clir)in such a way that a considerable portion of the nucleolar material remains in contactwith the chromosome clusters.

Figs. 16—19. Resting nuclei stained more deeply than those in the other figures onthis plate to reveal the chromosomes that extend into the nucleolus (n.chr). Fig. 16 showsthese chromosomes in a recently divided pair of nuclei. Fig. 17 shows them in theinterphase nuclei of growing cells. Figs. 18, 19 are of cells from a 3-day-old culture.The volume of the nucleolar material is much reduced and the intranucleolar chromo-somes now appear as a pair of distinct round bodies by the side of the main mass ofchroma tin.


V

chr

9 10 ^ 11 12

chr

p 4

I i 413 14 15 16 19

n.chrM _ 17


Fig. 20. Early interphase (as determined by the length of the cell). The KCE which has2 regions of different electron density is located on the outside of the nuclear envelopeadjacent to the electron-transparent chromatin region (clir) on the side opposite thenucleolus (n). A few short mitochondrial profiles can be seen (m). One of these lies nextto a ribosome-free zone around the KCE (kce). The cytoplasm also contains vacuoles(D), partially filled with electron-dense material, x 17500.

Fig. 21. Late interphase. At this stage the nucleus is larger and the KCE has a moreuniform electron density than in the cell shown by Fig. 20. The position of the KCErelative to the chromatin region (chr), the nucleolus (n), and the cell's side wall is similarto that seen in Fig. 20. A long mitochondrion (m) runs almost the complete length ofthe cell and seems to separate the ribosome-free zone around the KCE from the restof the cytoplasm. Inside this mitochondrion, several inclusions are present which atthis low magnification appear to consist of a central transparent region and an electron-dense periphery. Prominent vacuoles (v) and clusters of lipid droplets (/) are presentin the cytoplasm, x 17500.

Figs. 22-24. T h e mesosome-like appearance of the mitochondrial inclusions seen athigh magnification. Fig. 22 shows one of the inclusions in the long mitochondrion seenin Fig. 21 (indicated by the arrow), x 88300.


*

t

21 20

C E L 9


Fig3. 25-29. Sequence showing the change in KCE morphology between early inter-phase and the initial stages of spindle formation at mitosis. A portion of the nucleuscan be seen towards the bottom of each micrograph. The electron-dense KCEs ateach stage are located on the outside of the nuclear envelope in a ribosome-free zoneand are accompanied by a mitochondrion (m) on their cytoplasmic side, x 80700.

Figs. 25, 26. Adjacent sections of an early interphase KCE showing that it is a curvedlayered disk with a bulge in the centre of its concave surface which touches the nuclearenvelope. Amorphous electron-dense material underlines the inner surface of thenuclear envelope in the region where the KCE is in contact with the membrane.

Fig. 27. KCE in late interphase. In contrast to the KCE in Figs. 25, 26, this type ofKCE is longer and flatter and does not have a pronounced layered appearance.Electron-dense material on the inside of the nuclear envelope also accompanies theKCE at this stage.

Fig. 28. This micrograph shows what we interpret as a recently divided KCE. Note the2 short nearly parallel bars which lie in an indentation of the nuclear envelope. They areflanked by accumulations of osmophilic material which may be spindle precursors.

Fig. 29. Shows two KCEs joined by a slightly curved bundle of microtubules (sp),and separated by a convoluted stretch of nuclear envelope. At this stage the KCEsappear to be closely pressed to the nuclear envelope and do not have a pronouncedlayered appearance. The length of the spindle shown in this micrograph is 1-15 /im.

Mitosis in S. pombe

<*•?••

V

»3B^^R

497

tv-

26

. . • 9 - *

m

32-2


Fig. 30. Slightly later stage of spindle formation than in Fig. 22. In this nucleus thenucleolus (n) and the chromatin (chr) have the same relative positions to each other asthey do in interphase. A straight bundle of parallel microtubules (sp) crosses thechromatin region. Because it is sectioned slightly obliquely, only the KCE at thespindle end near the top of the micrograph is visible, but the ribosome-free zonearound the other KCE (lower arrow) can be seen at the spindle end, near the bottomof the micrograph. The close association between mitochondria and the ribosome-free zones around the KCEs can be clearly seen in this micrograph. The shortsegment of nuclear envelope between the KCEs and bounded by the peripheralspindle has a markedly convoluted appearance in contrast to the smooth contours of therest of the nucleus, x 53800. The length of the spindle is 1-55 /tm.

Fig. 31. Shows the size, quantity and position of granules which stain blue with Sudanblack B in log-phase cells, x 3600.Fig. 32. A cell from the same culture as the one in Fig. 24, stained with acidified tolui-dine blue to show the size, position, and relative quantity of the metachromatic cyto-plasmic inclusions, x 3600.


30

32


Figs. 33, 34. Closely comparable electron and light micrographs showing the relativepositions of nucleoli (n) and spindles (sp) in dividing nuclei which are not yet markedlyelongated and still have excentrically placed nucleoli, as in interphase.

Fig. 33. The nucleus in this micrograph is at a slightly later stage of mitosis than theone in Fig. 30. The length of the peripheral spindle (sp) is 1-75 /(m. Note how thespindle seems to be a bundle of parallel microtubules running through the chromatinregion (chr) between 2 terminal KCEs, and how the short segment of nuclear envelopethat is bounded by this spindle bulges out from the more or less oval contours of therest of the nucleus. Although the KCE (kce) nearer the bottom of the micrographappears not to have an associated mitochondrion, further sections of this nucleusshowed that one was present, m, mitochondrion, x 67250.

Fig. 34. Acid-fuchsin-stained cell. Note that the spindle, which is slightly inclinedto the plane of focus, appears to be of equal diameter along its length, x 4500.


n

34


Figs. 35, 36. Closely comparable light and electron micrographs showing the relativepositions of the nucleolus (n) and spindle (sp) in dividing elongated nuclei which havea more or less rectangular shape.

Fig. 35. Note how the chromatin (chr) is located at the ends of the nucleus while thenucleolus is stretched out in the central region. Compare with light micrographs ofGiemsa-stained cells at similar stages of mitosis (e.g. Fig. 13). The spindle which is3-75 fim. in length, occupies the longitudinal axis of the nucleus. Note how the KCEs(kce) at the ends of the spindle are each associated with a mitochondrion (m). x 43 900.

Fig. 36. Acid-fuchsin-stained cell. Note how the spindle has the shape of a thin wirewith knobs on the ends, x 4500.

Mitosis in S. pombe

sp

n

36


Figs. 37, 38. Two portions of a dumbbell-shaped nucleus shown whole in Fig. 39.Fig. 37. Shows part of the spindle channel. Note that the microtubule bundle is

bounded by an intact nuclear envelope composed of the usual 2 membranes, except forseveral small regions of high electron density (arrows). If these regions are sectioned at afavourable angle, it can be seen that they are fold-like arrangements of 4 unit mem-branes continuous with the 2 membranes on each side of them (inset and interpretivedrawing), (sp, spindle.) x 107600; inset x 228700.

Fig. 38. Shows a section of one of the daughter dumbbell ends. The curved arrowsnear the bottom of the micrograph indicate where the envelope narrows down into thespindle channel. Note how the microtubules run from themouthof thespindlechannel,through the nucleolus (n) and the chromatin region (chr) towards that region ofthe nuclear envelope where the KCE (kce) appears to be externally attached. Amitochondrion is associated with the ribosome-free zone around the KCE. Theribosome-like particles in the nucleolus are clearly visible at this magnification. Notealso the unit membrane (vm) around the vacuole (v) at the top right of the micrograph.(sp, spindle.) x 80700.

Mitosis in S. pornbe 5°5

m

sp

chr

37J

i


Figs. 39, 40. Closely comparable light and electron micrographs showing the relativepositions of the nucleolus (n) and the spindle (sp) in dividing nuclei which haveelongated into a dumbbell shape. The nucleolar material is divided between thedaughter dumbbell ends which are joined by a long spindle.

Fig. 39. The small arrows in the central portion of the cell indicate the narrowspindle channel. Note how the daughter dumbbell ends are slightly elongated in thepulling-out direction and how the spindle-associated KCEs are at the opposite polesof the nucleus (i.e. near the ends of the cell). Each of the KCEs is associated with amitochondrion (m). The length of the spindle (from kce to kce) in this nucleus is9-25 fim. Several vacuoles (v) and a few lipid droplets (/) can be seen in the cytoplasmof the cell. Asterisks indicate where the cell septum is starting to grow inwards fromthe side walls of the cell, chr, chromatin region, x 13800.

Fig. 40. Acid-fuchsin-stained cell. Note the long wire-like spindle with knobs onthe ends, x 4500.Fig. 41. One daughter end of a cell with a completely formed septum (s). Incontrast to the daughter dumbbell ends in Fig. 39, the newly separated daughternucleus seen in this micrograph is elongated almost at right angles to the formerpulling-out direction. The nucleolus (n) now faces the cell wall on the right sideof the micrograph, while the KCE {kce) is located beside the nucleus near the left cellwall. Note the mitochondrion (m) which appears to have recently become pinched offinto 2 short portions. One of these is associated with the KCE at the side of thenucleus. At this stage the KCE has 2 regions of different electron density, as it does inearly interphase cells. The prominent electron-transparent region inside the mito-chondrion close to the position of the KCE is one of the mesosome-like inclusions.chr, chromatin region; v, vacuole. x 31 300.


40

chr

kce

n

kceandm

n

39 41

Documents

MITOSIS IN THE FISSION YEAST SCHIZOSACCHAROMTCES … · confirm and extend the observations made with light microscopy. Without first arranging themselve on a metaphasse plate, mitotic