J. Cell Sci. 8, 557-571 ("970 557Printed in Great Britain

MICROTUBULES AND A CONTRACTILE RING

OF MICROFILAMENTS ASSOCIATED WITH

A CLEAVAGE FURROW

J. B. TUCKERDepartment of Zoology, The University, St Andrews, Fife, Scotland*,and Department of Zoology, Cambridge, England

SUMMARY

A ring composed largely of microfilaments is situated underneath the pellicle and at the baseof the cleavage furrow in the ciliate Nassula during binary fission. The microfilaments havediameters ranging from 4 to 10 run. There are substantial indications that the ring activelyconstricts in a sphincter-like fashion and is the main contractile agent causing furrowing. Ascleavage proceeds the ring thickens and the dense layer of the pellicle becomes progressivelymore deeply folded. The longitudinal axes of the folds are at right angles to the longitudinalaxes of the microfilaments and the plane of the ring. Folds form only where the pellicle overliesthe ring. Two distinct phases of cleavage have been distinguished. The furrow constricts theorganism at a progressively more rapid rate until the cleavage constriction has a diameter ofabout 5 /im and the microfilaments plug the constriction. After this furrowing proceeds muchmore slowly. A girdle of several thousand microtubules embedded in a densely stainingmaterial forms between the ring and the pellicular folds during the final stages of cleavage.Constriction and severance of the narrow cleavage constriction joining daughter oiganismsduring the final phase of cleavage involve mechanisms different from those acting during theearlier phase of furrow development.

INTRODUCTION

Active contraction occurs in the furrow region of cleaving sea-urchin eggs (Wolpert,1966). Portions of cortex excised from an incipient furrow region in the ciliate Stentorand grafted elsewhere on the organism's surface subsequently exhibit contractionsand form furrows (Tartar, 1968). Thus it appears that the contractile elementseliciting cleavage are located in the immediate vicinities of furrows. The participationof a contractile ring passing around the base of the cleavage furrow has often beenconsidered (for example, Wolpert, i960). A constricting ring which apparently pullsthe furrow inwards has been described in living grasshopper spermatocytes (Mota,1959). Rings (or bands, where cleavage is unilateral) of microfilaments, of diametersranging from 3 to 10 nm, positioned against and running along the furrow base havebeen described for the ciliate Paramecium (Jurand & Selman, 1969), the cleavingeggs of a diversity of animals (Schroeder, 1968; Tilney & Marsland, 1969; Arnold,1969; Szollosi, 1970; Selman & Perry, 1970) and dividing epithelial cells of mousemammary glands (Scott & Daniel, 1970). Densely staining material concentratedalong the furrow bases of dividing HeLa (Robbins & Gonatas, 1964) and chick

• Present address.

558 J. B. Tucker

mesenchyme cells (Allenspach & Roth, 1967) probably has the same microfilamentouscomposition.

The investigation reported here deals mainly with the functional role of a ring ofmicrofilaments situated at the base of the cleavage furrow in the ciliate Nassula duringbinary fission. Evidence suggesting that this ring actively constricts and that its con-traction is the prime cause of the deformations of the organism's pellicle which occur inthe vicinity of the cleavage furrow is presented and discussed. Thousands of micro-tubules are concentrated at the base of the furrow between the pellicle and the ringduring the final stages of cleavage; their origin, interaction with the microfilamentousring, and functional involvement during cleavage are considered.

MATERIALS AND METHODS

The species of Nassula used, the procedure for culturing it, and the methods used forelectron microscopy and silver staining have already been described (Tucker, 1967, 1970).

The diameters of the cleavage constrictions of several organisms were measured to the nearest5 /im at intervals after the start of fission (see Tucker, 1970) using a dissecting microscope fittedwith an eyepiece micrometer. The organisms remained in the culture dishes at room tempera-ture (22 °C) while these measurements were made, because isolating organisms into depressionslides or confining them on microscope slides and beneath coverslips induced abnormalcleavage rates. The sides of furrows are so closely apposed during the later stages of cleavagethat furrow bases are not visible using this method. The final measurement in each series wasmade to the nearest 07 /tm after the organism had been fixed in an aqueous solution of 25 %glutaraldehyde and 06% sucrose buffered at pH 78 with phosphate buffer (18 mM). Thesides of furrows separated slightly after fixation so that furrow bases could be seen (Fig. 11).

Dividing organisms usually lie motionless on the bottoms of culture dishes. The pointat which separation of sister organisms occurred was assessed by directing a current of waterat them just sufficient to move the organisms. Separated sisters drifted apart.

The amount of time which had elapsed since the start of fission when organisms were fixedfor electron microscopy or silver staining was estimated by comparing the diameters of theircleavage constrictions with those of living organisms measured at known times after the startof fission.

RESULTSThe cleavage furrow constricts the organism at a progressively more rapid rate

for about 90 min following the start of fission until the cleavage constriction has adiameter of about 5 /tm (Fig. 1). Then furrow advance slows abruptly, and for a periodof about 25 min the diameter of the constriction either remains unchanged or slowlydecreases (Fig. 1). These 2 phases of furrowing will be referred to as the rapid andslow phases of cleavage, respectively. Finally the cleavage constriction suddenlystretches, narrows, and breaks (Figs. 4-6).

During the rapid phase of cleavage, and at the start of the slow phase, a ring ofmicrofilaments (J) is positioned underneath the dense pellicular layer (d) at the base ofthe furrow (Figs. 2, 9). The microfilaments are fairly straight and their longitudinalaxes run around the ring. They have diameters ranging from 4 to 10 nm. The diameterof an individual microfilament sometimes varies slightly at different points along itslength. Fine bridges (arrows) connect adjacent microfilaments; other dense material ofirregular shape and arrangement is also situated between filaments (Fig. 9). Cross-

Microtubules and microfilaments 559

sections of portions of the ring have a reticular appearance; irregularly spaced denselystaining nodes are interconnected by strands of less densely staining material (Fig. 7).The nodes are probably microfilaments.

120 r

100E=».co.3 80

f 608

40

20

0 -

20 40 60 80Time after start of fission, min

100 120

Fig. 1. A graph showing the diameter of the cleavage constriction from the start offission until daughter organisms separate. Each point represents a measurement madeon the same organism.

About 100 ciliary rows {kineties) extend between anterior and posterior poles ofNassula. Kineties are drawn closer together in the cleavage constriction as furrowingproceeds (Fig. 8). A bundle of subpellicular microtubules extends along the length ofeach kinety at the start of fission; the tubules have diameters of about 24 nm. Eachbundle consists of about 30 tubules arranged in a series of closely stacked straight rowssituated just beneath the dense pellicular layer (d) (Fig. 10). There are no subpelliculartubules in the regions between kineties at the start of fission. Stacks of tubule rows areno longer present in the furrow region by about 85 min after the start of fission. Thedense pellicular layer (d) is slightly folded and a curved row of tubules (m) is positionedbelow each of the larger folds. Densely staining material (i) is associated with thetubules; it forms a more or less continuous layer between the dense pellicular layer andthe microfilamentous ring (/) and extends between tubule rows (Fig. 9). The tubules,diameter about 24 nm, have slightly curved longitudinal profiles; they curl around thebase of the furrow. Less densely staining regions are included in the layer of denselystaining material where it extends between tubule rows. These regions (x) are circularin cross-section and have the same diameters as the cores of the tubules (m) (Fig. 9).They may be developing tubules, their walls at this point having the same density as

560 J. B. Tucker

the surrounding dense material, because at this time, as will be shown below, largenumbers of microtubules are forming in the regions occupied by the dense material.

At the end of the rapid phase of cleavage the dense pellicular layer is much moreextensively folded than it was previously (compare Figs. 2, 9). Large numbers of sub-pellicular microtubules (m) and associated dense material (1) are concentrated insidethe folds forming a girdle between the dense pellicular layer (d) and the microfila-mentous ring (/) (Fig. 2). Folds (d) occur only where the dense pellicular layer overliesthe ring; their longitudinal axes are at right angles to the longitudinal axes of the micro-filaments (/) in the ring (Fig. 13). The vast majority of the girdle tubules have thesame lengths as the folds (about i-8 /tm) and do not protrude beyond the ends of thefolds. The cleavage constrictions of 2 organisms fixed during the slow phase ofcleavage possessed about 14000 and 12000 girdle tubules (Figs. 2 and 3, respectively).Nassula has about 100 kineties and sections show about 30 tubules in each kinetytubule bundle. Hence bunching of kinety tubules in the cleavage constriction canaccount at most for only about 3000 girdle tubules. Whether or not any of the kinetytubules contribute to the girdle has not been ascertained. However, even if all thekinety tubules are included in the girdle, development of several thousand newtubules must occur during girdle formation. No spindle tubules are included in thegirdle. Spindles (s) still span the cleavage constriction 86 min after the start of fissionbut they are inside the ring (/) (Fig. 12) and divide before the slow phase of cleavagebegins (see Tucker, 1967), so that none of them traverses the constriction during theslow phase of cleavage (Fig. 2).

Portions of the ring (/) have saddle-shaped cross-sectional profiles during the rapidphase of cleavage (Fig. 12). The ring thickens throughout the later stages of the rapidphase of cleavage; it has not been examined during earlier cleavage stages. For example,the ring has a maximum thickness of about 0-15 /tm 85 min after the start of fission(Fig. 9) and about 10 min later a maximum thickness of about 0-4/6111 (Fig. 2).Whether or not the breadths of the saddle-shaped profiles of the ring also change hasnot been ascertained. At the beginning of the slow phase of cleavage a considerableproportion of the microfilaments lining the inner surface of the ring (arrows) areno longer oriented with their longitudinal axes passing around the ring (/) (Fig. 2).These regions of the ring have a reticular appearance in cross-sections of the constric-tion, indicating that the filaments have been cut in transverse or oblique section. Mostof the microfilaments (/) have lost their annular arrangement during the later stagesof the slow phase of cleavage, although a few (arrows) still pass around the inner sur-face of the girdle; the microfilaments form a plug completely filling the cleavageconstriction interior to the girdle (Fig. 3).

DISCUSSION

The contractile nature of the microfilamentous ring

The orientation and positioning of pellicular folds with respect to the ring areprecisely those which would be expected if the ring were actively contracting andattached to the overlying pellicle. The progressively more rapid rate at which con-

Microtubules and microfilaments 561

striction occurs during the rapid phase of cleavage also indicates that the micro-filamentous ring is the main contractile agent involved in furrow development. Forthe ring thickens as it constricts and there is no increase in the lateral spacing of micro-filaments as this occurs. If the breadth of the ring does not decrease, then as the ringconstricts the number of microfilaments per unit segment of the ring increases. If thetension generated in the ring is proportional to the number of microfilaments per unitring segment then ring tension and the rate of furrowing should increase progressivelyas observed (Fig. 1), provided that there is no marked change in the resistance of thepellicle to its deformation, or change in the total number of ring filaments after thestart of fission. These observations are also compatible with the view that slidinginteractions between overlapping filaments are an essential feature of the tension-generating process and that the rate of furrowing accelerates because more filamentsoverlap one another per unit ring segment as the ring constricts and thickens.

Microfilaments are associated with cleavage furrows in eggs and tissue cells (seeIntroduction) where furrowing probably involves mechanisms similar to those examinedhere in a ciliate. If furrow microfilaments operate in the same way in all animal cells,furrowing may frequently proceed at a progressively more rapid rate. Both Wolpert(1966) and Dan (1963) have recorded an increase in the rate of furrowing during theearly stages of cleavage in sea urchin eggs. However, Szollosi (1970) has noted that thethickness of the microfilamentous band at the base of furrows in eggs of the cnidarianAequorea does not change appreciably during cleavage.

Microfilamentous rings are associated with sphincter-like contractions in othersituations. For example, in Nassula a microfilamentous annulus encircles the top of thecytopharynx which constricts when the organism feeds (Tucker, 1968) and bands ofmicrofilaments encircle the accessory hearts of the ascidian Botryllus (de Santo &Dudley, 1969). There is an abundance of reports for a wide range of protozoa,metazoan tissue cell types, and even for certain algae (Drum & Hopkins, 1966; Nagai& Rebhun, 1966; Halfen & Castenholz, 1970) dealing with microfilaments situated incytoplasmic regions where some form of contraction or movement is taking place. Thedescription of microfilaments, diameter about 7 nm, exhibiting ATPase activity in theslime mould Physarum (Wohlfarth-Bottermann, 1964) is especially important becausematerials which markedly resemble actin and myosin have been isolated from thisorganism (Hatano & Tazawa, 1968; Nachmias, Huxley & Kessler, 1970). There areindications both for (Rappaport, 1967) and against (Wolpert, 1963) the possibilitythat an actomyosin-like complex is involved in furrow formation.

The two phases of cleavage

Mitchison (1952) noted that if a contracting ring was involved in furrowing then itwould either have to constrict to nothing, which would involve something more than astraightforward contraction of the ring, or the final nipping off of sister cells when thecleavage constriction attained a diameter of less than 5 /im might be due to somemechanism different from that involved in ring constriction. It is clear that in Nassulathe ring does not constrict to nothing, but forms a plug in the constriction, and thereare 2 distinct phases in the cleavage process, rapid and slow, distinguished by an abrupt

36 CEL S

562 J. B. Tucker

decrease in the rate of furrow advance, the onset of pellicular folding and considerablechanges in the arrangement and composition of fibrous elements associated with thefurrow base when the cleavage constriction has a diameter of about 5 /.ova.

An abrupt slowing of furrowing and the consequent existence of a relatively per-sistent cytoplasmic bridge during the final cleavage stages has been reported for arange of animal cell types (Hughes & Swann, 1948; Chambers, 1951; Lewis, 1951). Individing HeLa cells cleavage proceeds for about 26 min to form a cytoplasmic bridgewith a diameter of about 1-5 /im, which then persists for 2 h or longer before a complexsequence of events, quite different from anything observed during earlier cleavagestages, finally severs the bridge (Byers & Abramson, 1968). Very probably, specialevents quite distinct from, or at least additional to, those concerned with the majorityof a furrow's advance are introduced during the final cleavage stages in most animalcells to expedite the final separation of sister cells and ensure that a fairly durable andimpermeable barrier at all times partitions liquid cytoplasm from the extracellularmedium in the region of final severance. In Nassula the microfilamentous plug and themicrotubular girdle may be involved in sealing the broken ends of the cytoplasmicstrand when severance occurs.

No indications of the activity responsible for the final constriction, elongation, andseverance of the cleavage constriction during the slow phase of cleavage have beenobtained. Active sliding of girdle tubules in the manner suggested for extension of themicrotubular km fibres of Stentor (Bannister & Tachell, 1968) and/or attempts bysisters to swim apart may be involved.

Role of the microtubular girdle

Microtubules are generally believed to be fairly rigid structures. This is certainlythe case for certain microtubular components in the cytopharynx of Nassula (Tucker,1968). Hence the formation of the microtubular girdle in the cleavage constrictionprobably impedes furrow advance to some extent. The girdle starts to develop at theend of the rapid phase of cleavage but considerable constriction and pellicular foldingoccur after the tubules have formed. Since the girdle lies between the ring and thepellicle any contractions causing furrowing and originating in the ring must be trans-mitted through the girdle to the pellicle. The densely staining material which liesbetween girdle tubules and extends between the ring and the pellicle may effect suchtransmission. While binding tubules, ring and pellicle together a substantial amount ofshearing and/or flowing occurs in the dense material permitting the ring to constrictand the pellicle to fold. In this respect the dense material behaves as a sticky gel.Densely staining material of similar appearance occupies intertubular spaces andapparently plays a similar role in regions of the cytopharynx of Nassula where thegreatest shearing stresses are believed to occur; here too considerable deformations ofthe dense material sometimes take place (Tucker, 1968).

Accumulation of girdle tubules is not simply a consequence of their being trapped inthe cleavage constriction by the advancing furrow, as is apparently the case duringformation of the microtubular spindle mid-body in the late cleavage constriction ofsome tissue cells (for example, AJlenspach & Roth, 1967). The development of girdle

Microtubules and microfilaments 563

tubules is precisely correlated in both space and time with final stages in the formationof the cleavage constriction. The girdle therefore probably has an important role con-cerned with events bringing about the final narrowing and severance of the constric-tion. Special cleavage tubules are formed in the flagellate Chlamydomonas and it hasbeen suggested that they may provide a rigid framework influencing the form of thefurrow (Johnson & Porter, 1968). Girdle tubules in Nassula may act similarly. It hasbeen argued that the greatest contractile forces develop in the ring at the end of therapid phase of cleavage; this is precisely the time at which the girdle develops. Therole of the girdle may be to distribute ring tension in a longitudinal direction relativeto the organism's polar axis, so that a cleavage constriction of some length is formedand the base of the furrow has a U-shaped rather than a V-shaped profile. The girdlemay ensure that the cleavage constriction has considerable resistance to breakingbefore procedures such as the formation of the microfilamentous plug seal the moreliquid cytoplasmic portions of the sister cells from each other. The combined actionsof the microtubular girdle and the contractile microfilamentous ring may be like theapplication of a corset in which a system of constricting cords draws in a circularpalisade of stays to shape and support a slender waist.

Part of this investigation was undertaken during the tenure of an S.R.C. Research Fellowshipwhich is gratefully acknowledged. I should like to thank Dr M. A. Sleigh for critically readingthe manuscript.

REFERENCES

ALLENSPACH, A. L. & ROTH, L. E. (1967). Structural variations during mitosis in the chickembryo. J. Cell Biol. 33, 179-196.

ARNOLD, J. M. (1969). Cleavage formation in a telolecithal egg (Loligo pealii). J. Cell Biol. 41,894-904.

BANNISTER, L. H. & TATCHELL, E. C. (1968). Contractility and the fibre systems of Stentorcoeruleus.J. Cell Sci. 3, 295-308.

BYERS, B. & ABRAMSON, D. H. (1968). Cytokinesis in HeLa: post-telophase delay and micro-tubule-associated motility. Protoplasma 66, 413-435.

CHAMBERS, R. (1951). Micrurgical studies on the kinetic aspects of cell division. Ann. N.Y.Acad. Sci. 51, 1311-1326.

DAN, K. (1963). Force of cleavage of the dividing sea urchin egg. Symp. int. Soc. Cell Biol. 2,261-276.

DRUM, R. W. & HOPKINS, J. T. (1966). Diatom locomotion: an explanation. Protoplasma 6a,i-33-

HALFEN, L. N. & CASTENHOLZ, R. W. (1970). Gliding in a blue-green alga: a possible mechan-ism. Nature, Lond. 225, 1163-1164.

HATANO, S. & TAZAWA, M. (1968). Isolation, purification and characterization of myosin Bfrom myxomycete plasmodium. Biochim. biophys. Ada 154, 507-519.

HUGHES, A. F. & SWANN, M. M. (1948). Anaphase movements in the living cell. A study withphase contrast and polarized light on chick tissue cultures. J. exp. Biol. 25, 45-70.

JOHNSON, U. G. & PORTER, K. R. (1968). Fine structure of cell division in Chlamydomonasreinliardi. Basal bodies and microtubules. J. Cell Biol. 38, 403-425.

JURAND, A. & SELMAN, G. G. (1969). The Anatomy of Paramecium aurelia. London: Macmillan.LEWIS, W. H. (1951). Cell division with special reference to cells in tissue cultures. Ann. N.Y.

Acad. Sci. 51, 1287-1294.MITCHISON, J. M. (1952). Cell membranes and cell division. Symp. Soc. exp. Biol. 6, 105-127.MOTA, M. (1959). Karyokinesis without cytokinesis in the grasshopper. Expl Cell Res. 17,

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NACHMIAS, V. T., HUXLEY, H. E. & KESSLER, D. (1970). Electron microscope observations onactomyosin and actin preparations from Physarum polyceplialum, and on their interactionwith heavy meromyosin subfragment I from muscle myosin. J. molec. Biol. 50, 83-90.

NAGAI, R. & REBHUN, L. I. (1966). Cytoplasmic microfilaments in streaming NitelJa cells.J. Ultrastruct. Res. 14, 571-589.

RAPPAPORT, R. (1967). Cell division; direct measurement of maximum tension exerted byfurrow of echinoderm eggs. Science, N.Y. 156, 1241-1243.

ROBBINS, E. & GONATAS, N. K. (1964). The ultrastructure of a mammalian cell during themitotic cycle. J. Cell Biol. 21, 429-463.

SANTO, R. S. DE & DUDLEY, P. L. (1969). Ultramicroscopic filaments in the ascidian Botrylhisschlosseri (Pallas) and their possible role in ampullar contractions. J. Ultrastruct. Res. 28,

SCHROEDER, T. E. (1968). Cytokinesis: filaments in the cleavage furrow. Expl Cell Res. 53,272-316.

SCOTT, D. G. & DANIEL, C. W. (1970). Filaments in the division furrow of mouse mammarycells. J. Cell Biol. 45, 461-466.

SELMAN, G. G. & PERRY, M. M. (1970). Ultrastructural changes in the surface layers of thenewt's egg in relation to the mechanism of its cleavage. J. Cell Sci. 6, 207-227.

SZOLLOSI, D. (1970). Cortical cytoplasmic filaments of cleaving eggs: a structural elementcorresponding to the contractile ring. J. Cell Biol. 44, 192—209.

TARTAR, V. (1968). Micrurgical experiments on cytokinesis in Stentor coeruleus. J. exp. Zool.167, 21-35-

TILNEY, L. G. & Marsland, D. (1969). A fine structural analysis of cleavage induction andfurrowing in the eggs of Arbacia punctulata. J. Cell Biol. 42, 170-184.

TUCKER, J. B. (1967). Changes in nuclear structure during binary fission in the ciliate Nassula.J. Cell Sci. 2, 481-498.

TUCKER, J. B. (1968). Fine structure and function of the cytopharyngeal basket in the ciliateNassula. J. Cell Sci. 3, 493-514.

TUCKER, J. B. (1970). Morphogenesis of a large microtubular organelle and its association withbasal bodies in the ciliate Nassula. J. Cell Sci. 6, 385-429.

WOHLFARTH-BOTTERMANN, K. E. (1964). Cell structures and their significance for ameboidmovement. Int. Rev. Cytol. 16, 61-131.

WOLPERT, L. (i960). The mechanics and mechanism of cleavage. Int. Rev. Cytol. 10, 163-216.WOLPERT, L. (1963). Some problems of cleavage in relation to the cell membrane. Symp. int.

Soc. Cell Biol. 2, 277-298.WOLPERT, L. (1966). The mechanical properties of the membrane of the sea urchin egg during

cleavage. Expl Cell Res. 41, 385-396.(Received 22 July 1970)

Unless otherwise stated, all figures are electron micrographs of Nassula fixed withglutaraldehyde, post-fixed with osmium tetroxide and stained with uranyl acetate andlead citrate. The estimated period of time elapsing after the start of fission when anorganism was fixed, or in the case of a living organism when it was photographed, isindicated in the accompanying legend by the period in question followed by theabbreviation 'a.s.f.'.

Fig. 2. A cross-section through the mid-point of the cleavage constriction of theorganism shown in Fig. 11 which was fixed about 95 min a.s.f. at the beginning of theslow phase of cleavage. The girdle of microtubules (m) and densely staining material (j)is positioned between the deeply folded dense pellicular layer (d) and the microfilamen-tous ring (/). Microfilaments near the inner surface of the ring (arrows) no longerfollow circular courses around the ring at this stage. The fragmented and poorlypreserved vesicular pellicular layer (v) lies outside the dense layer.

Microtubules and microfilaments 565

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566 J. B. Tucker

Fig. 3. A cross-section of the cleavage constriction of an organism fixed about105 min a.s.f. during a late stage of the slow phase of cleavage. A girdle of micro-tubules (m) and dense material (i) is positioned inside the vesicular (u) and folded denselayers (d) of the pellicle. The remainder of the cleavage constriction is filled by aplug of microfilaments. Filaments at the periphery of the plug follow circular coursesaround the edge of the plug (arrows); filaments (/) nearer the centre of the plug areless compactly and regularly arranged.

Figs. 4-6. Nomarski interference contrast micrographs of living organisms takenusing microflash, each showing a portion of 2 incipient daughter organisms and thecleavage constriction linking them during the slow phase of cleavage, x 1800.

Fig. 4. About 90 min a.s.f. at the start of the slow phase of cleavage.Fig. 5. About 112 min a.s.f., approximately 3 min before the final separation of the

daughter organisms, and shortly after the final rapid narrowing of the cleavageconstriction had started. The appearance of the dense id) and vesicular (v) pellicularlayers in living organisms is also shown.

Fig. 6. The cleavage constriction broke in the middle about 1 s after this micro-graph was taken.

Microtubules and microfilaments 5<>7

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568 J. B. Tucker

Fig. 7. A portion of the microfilamentous ring (/) in cross-section in an organismfixed during the rapid phase of cleavage about 86 min a.s.f. Microtubules («/) areforming in the layer (g) between the ring and the dense pedicular layer (d).Fig. 8. Lateral view of a dividing organism stained with silver and fixed 95 min a.s.f.during the slow phase of cleavage. The rows of intensely stained small circles show thelinear arrangement of basal bodies in kineties. The kineties converge as they approachthe cleavage constriction (arrow) which is out of the plane of focus.Fig. 9. Part of the microfilamentous ring (/) sectioned longitudinally in a sectionpassing transversely through an organism at right angles to its longitudinal axis andthrough the mid-point of its cleavage constriction. The organism was fixed about85 min a.s.f. during the rapid phase of cleavage. The dense pellicular layer (rf) hasstarted to fold. Curved rows of microtubules (n/) lie beneath the larger folds.Densely staining material (1) is associated with these tubules and with regions (A)where microtubules are developing. Fine bridges (arrows) connect filaments in thering.Fig. 10. Cross-section through a bundle of kinety microtubules positioned justbelow the dense pellicular layer (d) approximately 30 /an from the furrow about 30 mina.s.f. x 148000.

Fig. 11. Lateral view of an organism after it had been fixed (about 95 min a.s.f.) andembedded in Araldite. The fine structure of its cleavage constriction is shown inFig. 2. Phase-contrast.

Microtubules and microfilaments

570 J. B. Tucker

Fig. 12. Median longitudinal section of a cleavage constriction fixed about 86 mina.s.f. during the rapid phase of cleavage. Microtubules and densely stained materialare developing to form a girdle (g) between the dense pellicular layer (d) and thesaddle-shaped profiles of the microfilamentous ring (/). Micronuclear separationspindles (s) traverse the cleavage constriction.Fig. 13. The section grazes through the outer layers of a cleavage constriction. Thelongitudinal axes of the microfilaments in the ring (/) and the pellicular folds (rf) areapproximately at right angles to each other. 86 min a.s.f.

Microtubules and microfilaments 571

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