CONTRACTILITY AND THE FIBRE SYSTEMS OF STENTOR COERULEUS

J. Cell Sci. 3, 295-308 (1968) 295

Printed in Great Britain

CONTRACTILITY AND THE FIBRE SYSTEMS

OF STENTOR COERULEUS

L. H. BANNISTER AND E. C. TATCHELLDepartment of Biology, Guy's Hospital Medical School, London, S.E. 1

SUMMARY

Observations of the microtubular 'km' system and filamentous ' M ' systems in livingspecimens of Stentor coerulens show that on contraction of the body both systems shorten,thicken and remain straight, but that on initial relaxation they behave differently: the km fibresbegin to lengthen later than the M fibres, the latter being thrown into sinuous folds. Electronmicroscopy of specimens cooled before fixation appears to confirm this difference in behaviourin relaxing specimens.

The M fibres of the stalk region are discrete and prominent bundles, but in the adoral halfof the body they are extensively linked together by side branches. There are diffuse filamentousattachments between the M fibres and the kinetosomes.

The vertical microtubular stacks which make up the km fibres are each linked with a pairof kinetosomes. Each stack contains about 21 microtubules grouped in a 2 + 19 pattern. Cross-bridges are present between the microtubules of adjacent stacks. The number of stacks in eachkm fibre cross-section is smaller in extended specimens than in contracted ones, indicating thatthe stacks slide upon one another as the body changes its length.

INTRODUCTION

One of the outstanding problems connected with the heterotrich ciliates of the genusStentor concerns their ability to make rapid contractions. Ultrastructural studies(Faure-Fremiet, Rouiller & Gauchery, 1956; Faure-Fremiet & Rouiller, 1958;Randall & Jackson, 1958; Inaba, 1961) have shown that there are two distinct typesof cortical fibre systems which might be responsible for contraction, one of themmicrotubular in composition and the other consisting of fine fibrils. These arerespectively the 'myoneme ectoplasmique' and 'myoneme endoplasmique' of Faure-Fremiet et al. (1956) and the 'km' and ' M ' fibres of Randall & Jackson (1958). Bothof the two last mentioned groups of workers considered it likely that both sets offibres were contractile, although proof of this was not available (see Discussion).

This paper reports some additional findings which throw further light on thestructure and functions of the fibre systems of Stentor coeruleus. The terminologywhich will be used is that of Randall & Jackson (1958). Accordingly, the superficiallongitudinal fibres composed of microtubules will be called the km fibres, and thedeeper set of fibrillar fibres will be named the M fibres, thus, we hope, avoidingproblems of confused terminology.

Cell Sci. 3

296 L. H. Bannister and E. C. Tatchell

MATERIALS AND METHODS

Stentor coeruleus was obtained from the culture collection maintained at the BotanyDepartment of the University of Cambridge. They were kept in a variety of differentculture media, including that used by Sleigh (1956).

Living specimens were examined by low-power phase-contrast microscopy, bothwhilst they moved freely and whilst restrained in a weak solution of methyl cellulose.For closer examination they were lightly compressed beneath thin coverslips so thata x 100 oil-immersion phase-contrast objective could be used. In this way singlemicrographs of good quality could be obtained, and for further analysis cinephoto-graphic records were made at 16 and at 24 f.p.s. It was found to be particularly valuableto film whilst changing the focus with the microscope fine adjustment from the surfaceof the pellicle down into the deeper regions of the cytoplasm. In this way superficialstructures could be distinguished from deeper ones by later analysing the film at 16or at 2 f.p.s.

Specimens of Stentor were stimulated to contract either by mechanical means(tapping the microscope stage) or electrically. The latter was achieved by passingpulses (12 V, 0-2 mA, o-i sec) of direct current between two silver electrodes insertedbetween slide and coverslip.

For structural examination of fixed specimens, sections were prepared for electronmicroscopy and light microscopy. For electron microscopy, animals were fixed at0-2 °C for 30-40 min in either a i-o% osmium tetroxide solution buffered withveronal acetate and containing 45 mg/ml sucrose (Elliott & Bak, 1964) or in a 2-5 or6 % glutaraldehyde solution buffered with phosphate and containing 60 mg/mlsucrose. The pH of all solutions was in the range 7-2-7-4. After glutaraldehydefixation specimens were washed for 12 h to 3 days in several changes of phosphatebuffer (Millonig, 1961) and then post-fixed with osmium tetroxide for 30-60 min.Some were then embedded in a 2% solution of agar to facilitate further handling(Haller, Ehret & Naef, 1961). Specimens prepared in all the above ways were sub-sequently embedded in Araldite, and individual specimens were then selected andsectioned with a glass knife mounted in a Huxley ultramicrotome. Sections werestained first either in a saturated solution of uranyl acetate in 50 % ethanol or in a 1 %solution in methanol, and subsequently in 0-4 % lead citrate.

Animals fixed in the above manner were invariably fully contracted but it was foundthat this difficulty could be avoided in two ways. First, free-swimming specimens werecooled to just above o °C, when they adopted a pear-shaped form but continued toswim very slowly (backwards). They were then allowed to remain in this conditionfor 5-10 min, after which they were fixed in the 1 % osmium tetroxide solutiondescribed above. Most of them retained their semi-relaxed form subsequent to thefixation. It was found that whilst glutaraldehyde fixation alone always caused con-traction to the spherical form, exposure of previously cooled specimens to osmiumvapour for 5 min followed by glutaraldehyde fixation prevented this full contraction.The second method is that described by Boggs (1965) for the fixation of extendedspecimens of Spirostomum. This technique entails allowing the ciliates to extend

Fibre systems of Stentor 297

fully in a weak solution of gelatin which is then cooled so that it sets around themfirmly. This prevents the organisms from changing shape when they are fixed, althoughof course it cannot be claimed that they are truly relaxed. We have found that thistechnique works well for Stentor whether the fixative employed is osmium tetroxide orglutaraldehyde.

To correlate light and electron microscopy observations, sections of fixed Stentorwere examined by light microscopy. Sections were taken either from Araldite-embedded material prepared for electron microscopy, at 2-5 fi, and stained with tolu-idine blue-pyronin, or else were cut at 2 ju, from wax-embedded specimens (osmium-fixed) and stained by Mallory's triple technique.

Measurements taken from electron micrographs were checked with micrographsof carbon replicas of optical diffraction gratings ruled at 32000 lines to the inch.Sections were viewed with an RCA EMU-3 E electron microscope with a 50 or 30 fiobjective aperture.

RESULTS

Light microscopy

General observations. The appearance of the body will first be described as seen insemi-contracted (pear-shaped) forms. A series of longitudinal bands is present in thecortical region of Stentor coeruleus, consisting of alternate clear and pigmented stripes(Figs. 3-10). The longitudinal fibres which are the subject of this paper lie beneaththe pellicle of the clear stripes. Between adjacent clear stripes lie the blue-green bodieswhich give the pigmented stripes their characteristic colour. These pigment bodiesmake a suitable reference point for the identification of the km fibres which lie at aboutthe same depth beneath the pellicle (Figs. 5, 7).

In the aboral (stalk) half of the body the M fibres are found beneath the km fibres(Figs. 8, 10) at the same depth as a prominent cortical layer of mitochondria. Twoother means of identification must also be used to distinguish the two fibre systemsfrom each other. Along the left-hand margin of the km fibre (looking towards the oralend) is a row of cilia. The bases of these cilia are seen most clearly when the km fibrepasses over an internal vacuole, and under these conditions it is seen that there aretwo rows of kinetosomes attached to the km fibre, and that the kinetosome nearest thekm fibre is devoid of a cilium (Fig. 5). This confirms the electron microscope findingsof Kennedy (1965) for Stentor. The M fibres are characterized by a scattering of smalldense granules along their sides. These distinguishing features are found in theelectron micrographs of Randall & Jackson (1958).

If the M fibres are traced from the stalk region where they are clearly visibletowards the adoral end, they are seen gradually to taper, and then disappear. The onlytype of fibre which is visible in the adoral half of the body is therefore the km fibrewith its attendant kinetosomes.

Behaviour of fibres during contraction and relaxation. When Stentor coeruleus is in thepartially contracted state the organism is pear-shaped, the pellicle is unwrinkled, andthe km and M fibres are straight. The bases of the cilia are spaced at 1-1-5 /* intervals.

19-2


When a suitable stimulus is given, a very rapid contraction takes place and it is notpossible to follow its course in detail without some means of high-speed analysis.However, after contraction the pellicle is very wrinkled, with the pigment bodies lyingin its folds giving the pigment stripes a banded appearance (Fig. 4). The km fibresare seen to have remained straight and to have thickened (Fig. 7), and the bases of thecilia have moved so close that they frequently present the illusion of another fibrelying alongside the km fibre (Fig. 6). The spacing of such kinetosomes may be as littleas 0-5 fi from centre to centre, indicating that the km fibres have shortened.

The M fibres are seen to have thickened considerably and to have remained straight(Fig. 8) after body contraction, likewise indicating that they have shortened.

Soon (about 5-20 sec) after contraction and before any other change is seen in theappearance of the pellicle or of the km fibres (Fig. 9) a change occurs in the form ofthe M fibres (Figs. 3, 10). These are seen to be thrown slowly but progressively intolateral convolutions. Eventually, as the body begins to extend, these convolutionsbecome at first more pronounced, but as body extension continues, the M fibresgradually re-straighten. With the re-extension of the body, the wrinkles in the pelliclealso disappear and the km fibres, which are straight at all times, become thinner untilin the super-extended form they are not visible with the light microscope. The basesof cilia also move apart. Measurements made in the stalks of animals extended to 75 %of their maximum length give a distance of about 25 ju, between cilia.

If whilst the body has begun to elongate it is stimulated to contract again, thesinuosities in the M fibres immediately disappear and the condition reverts to that offull contraction already described.

The events described above have been observed in animals restrained undercoverslips, but similar events can be seen in unrestrained specimens (Fig. 3), althoughthey cannot, of course, be analysed in detail because of the lower magnificationswhich must be employed.

Electron microscopy

The M fibres. The anatomy of the M fibres varies along the length of the body. Inthe aboral (stalk) half, they are conspicuous structures, elliptical in cross-section, butin the adoral half they are narrower and are extensively linked together by a network ofside branches, thus forming a nearly continuous cortical sheet around the body. TheM fibres are generally bounded in all regions on at least one side by a membrane, andin some sections in the aboral half the M fibre is partly enclosed in a membrane-linedcanal (Fig. 12). Mitochondria are often found closely associated with the M fibres.Densely staining bodies composed of aggregations of small (50 A) particles are presentalongside and apparently attached to the membrane of the M fibre (Figs. 11, 12, 17).These are more prominent in osmium-fixed material than after glutaraldehyde fixation.

The substructure of the M fibre is seen most clearly after fixation with glutaraldehyde(Figs. 12, 13). The major component consists of filaments with a diameter of 80-100 A.In transverse section these filaments are seen to be tubular, the wall of the tube beingabout 30 A thick (Fig. 13). In longitudinal sections of M fibres the filaments veryrarely show sharp bends, but are oriented with their long axes more or less parallel


to the long axis of the M fibre which they constitute. This parallel orientation is notstrictly adhered to, however, and in any section numbers of filaments oriented at anangle with the longitudinal axis of the fibre are seen (Fig. 13). Another type of fibrousmaterial is also found associated with the 100-A filaments, consisting of thin (40 A)diffusely arranged filaments, connecting the M fibres to the overlying kinetosome/km-fibre complex and to the pellicle on either side of the km fibre (Figs. 12, 17). Thesesmaller filaments appear actually to penetrate the M fibre and to run among the larger(100 A) filaments. Whether or not they extend into the M fibre beyond its peripheryis as yet uncertain.

Adoral end

Subpellicularmicrotubules

Ciliatedkinetosome

Interkinetosomalfibres

Anteriorfibre sheet

Prongof densematerial

Fig. 1. Diagram showing the relationship between the kinetosomes and associatedstructures in the body (based on a series of longitudinal sections).

The microtubular systems associated with the kinetosomes. These have been describedby Kennedy (1965) for Stentor (and the closely related Blepharisma) and our findingsin general confirm his observations. Additional details, however, necessitate somereinterpretation (see Discussion).

The kinetosomes are found in pairs of one ciliated and one non-ciliated ('barren')kinetosome (Figs. 14-16). The barren kinetosome is nearest the km fibre, and attacheddirectly to it, whereas the ciliated one is attached directly to a group of about8 microtubules ('subpellicular microtubules', Fig. 15) which run beneath the pellicleat right angles and away from the km fibre. Both kinetosomes are connected to eachother by means of densely staining fine fibres which run between their kinetosomaltriplet fibres (Figs. 14, 15).


Our findings differ from those of Kennedy with respect to the mode of junction ofthe barren kinetosome with the km fibre (Fig. i). The km fibre consists of a series ofparallel groups of microtubules 200-220 A in diameter. In turn, each of these groupsconsists of a stack of microtubules arranged one above the other (see Randall &Jackson, 1958). Each of these stacks lies in contact with a barren kinetosome, whichmarks its anterior end. The junction between the two structures is shown in Figs. 14-16, and diagrammatically in Fig. 1. The stack is twisted in its initial course so thatthe microtubule which eventually forms its peripheral edge lies in contact with andparallel to the outer microtubule of one of the kinetosome triplets (Figs. 14, 17). The

2 1

0-V

noKinetosomes

Fig. 2. The organization of the km fibre microtubule stacks, seen in cross-section.The direction of the kinetosomal edge of the km fibre is indicated by arrows. (a) Tracingof part of the cross-section shown in Fig. 19, showing cross-bridges between micro-tubules. (b) Diagram of the organization of a single stack, with the microtubulesnumbered from the peripheral (pellicular) to the central edge. Microtubules 1 and 2are distinct from the remaining 19. A complete lateral cross-bridge is indicated onmicrotubule 3, and incomplete ones are illustrated on other microtubules.

stack curves towards the aboral foot on leaving the kinetosome. Densely staining fibreswhich spring from the barren kinetosome triplets run between the kinetosome and thekm fibre microtubules, in a series of 4-5 prongs on either side of the stack (Figs. 12,14-16). These prongs apparently fuse at their tips with the microtubules.

In addition to these structures, another set of fine fibres comes from the triplets ofthe ciliated kinetosome, swinging adorally (away from the foot) to join a vertical sheetof fibrous material ('anterior fibre sheet') resting against the km fibre (Figs. 1, 14, 15).This vertical sheet is traversed vertically by a number of ribs (Fig. 15). It correspondsto Kennedy's 'anterior kinetodesmal fibre'. In each stack there are up to 22 micro-tubules (Figs. 12, 19, 20). In a cross-section of a km fibre, the stacks on the kinetosomalside of the fibre have a more or less constant number of microtubules (out of 165cross-sections of such stacks, representing 5 km fibres in one specimen of Stentor, 1 had22 microtubules, 109 had 21, 54 had 20 and 1 possessed 18). For convenience of des-cription these are numbered 1-21 from the peripheral edge to the central (see Fig. zb).


Towards that side of the km fibre which is the further from the kinetosomal row,the number of microtubules per stack becomes smaller (Fig. 12), by the progressiveloss of microtubules from the central edge. All the microtubules in a stack are joinedtogether by short cross-bridges (Figs. 18,19). Between microtubules 3-20 (or 21) theseare about 50 A long, so that the microtubules are spaced at 260-A intervals fromcentre to centre. In longitudinal section these cross-bridges are seen to be about 125 Aapart. Cross-bridges are also found between microtubules 1, 2 and 3, but these arelonger than the others, measuring about 100 A, so that these microtubules are about310 A from centre to centre. If a line is drawn through the centres of all the micro-tubules in a stack (seen in cross-section) a gentle curve is formed through micro-tubules 21-3, concave towards the kinetosomal side of the km fibre, but then itabruptly alters course away from the kinetosomal row through microtubules 2 and 1.Microtubules 2 and 1 are therefore distinct from the other microtubules both in theirspacing and in their positioning (Figs, 2 a, b).

When a km fibre is viewed in fairly precise cross-section it is seen that cross-bridgesare also present between the microtubules of adjacent stacks. These are clearest at theperipheral (subpellicular) edge of the km fibre (Figs. 12, 19, 20). In each stack,microtubule 3 is always involved at one end of such bridges. Occasionally two adjacentstacks are separated from each other, and then the incomplete cross-bridge is seen tobe joined at one end (that end which is the further away from the kinetosomal side) tomicrotubule 3 (Fig. 19). Sometimes complete cross-bridges appear to be composed oftwo filaments (Fig. 19). Fainter cross-bridges between adjacent stacks are also foundfurther away from the pellicle, although only a few of these can be identified with anycertainty in any km fibre cross-section. A tracing of part of a cross-section (takenfrom Fig. 19) is shown in Fig. 2 a to make some of these clear. Typical cross-bridgesare about 150 A long and 40 A thick. They usually run between the wall of onemicrotubule and the wall of another, and very seldom to the connective between twomicrotubules in a stack. Where bridges are incomplete they are almost always attachedat their ' anti-kinetosomal' end to one of the microtubules. Such incomplete cross-bridges are often seen to have a slight enlargement at their free ends. Cross-bridgesare found not only between microtubules in stacks which are topographically adjacent,since where the usual orderly sequence of stacks is broken, cross-bridges may alsooccur between microtubules which are close together but not serially adjacent (Fig. 20).

Cross-bridges running between adjacent stacks have also been found in longitudinalsections of km fibres, although they are much more difficult to identify. Preliminarymeasurements suggest that microtubule 3 has cross-bridges spacing at 250-A intervalswhereas elsewhere, cross-bridges may be up to 750 A apart. This would account forthe greater density of the image of the former cross-bridges, since 2 or 3 of them maybe superimposed in a single cross-section.

The number of stacks in each km fibre cross-section varies according to the positionin the body. Although no precise measurement of number in relation to absoluteposition in the body is yet available, the general picture is that those in the stalk regionnear the foot of Stentor have the greatest number, reaching up to 66 in a singlecross-section. It is here also that the closest spacing of kinetosomes (0-3 fi) is found,


and where in living animals the greatest contraction is observed. At the extreme adoralend there is a rapid drop in the number of stacks in a cross-section, with a minimumof one at the adoral termination of the km fibre (Fig. 21). There are no microtubularconnexions with the adoral membranelle apparatus.

Specimens fixed in the extended state. Using the gelatin method specimens can befixed in the extended position. The M fibres are very conspicuous in transversesections of the stalks of such animals (Fig. 22), and are seen to lie close against thepellicle. The filaments constituting the M fibres present the same appearance as incontracted animals. The km fibres on the other hand are seen to have a very smallnumber (as low as 1) of vertical microtubule stacks (Figs. 22, 23) when seen in cross-section. In some positions where one would expect to find them (that is, immediatelyabove an M fibre) no microtubular stacks could be found at all.

Up to 20 microtubules have been counted in these stacks, which is within the rangefound in the stacks in contracted animals; the diameter of these microtubules showsno difference from those of contracted specimens. It would seem then that the numberof stacks per km fibre is the only point of difference between contracted and extendedspecimens with respect to the arrangement of the microtubules.

Figure 23 shows that the microtubule stacks of the km fibre are, in extended animals,almost enclosed within the M fibre.

Specimens cooled before fixation. These specimens show a condition resembling thatof animals relaxing after contraction. Horizontal sections cutting the surface of thestalk region show a wrinkled pellicle, straight km fibres, and unmistakably wrinkledM fibres (Fig. 24).

DISCUSSION

The fibre systems of Stentor have been extensively studied by light microscopy(see the reviews of Taylor, 1941; Tartar, 1961), but because the anatomical basis wasuncertain, no firm conclusions as to possible contractile functions were reached.

Faure-Fremiet et al. (1956) and Faure-Fremiet & Rouiller (1958) were the first tostudy the electron-microscope appearance of Stentor. Of the two systems of fibreswhich they found, the more superficial one (corresponding to the km fibre) was equatedwith the prominent fibre system which they saw in the stalks of contracted animals.Since the component fibres became straight and thick when Stentor contracted, andthen became folded as relaxation took place, they concluded that this was a contractilefibre system. Electron micrographs appeared to confirm this conclusion, since sectionsthrough the pellicular region showed distorted km fibres and straight M fibres. It maybe noted, however, that their electron micrographs are ambiguous in this respect,since the apparent buckling of the km fibres could easily have resulted from a bucklingof the whole pellicle, such as often occurs in processing for electron microscopy. TheM fibres were also (Faure-Fremiet & Rouiller, 1958) thought to be contractile onaccount of the similarity of their fine structure to that of the contractile spasmonemesof some peritrich ciliates (see, for instance, Randall & Hopkins, 1962).

Randall & Jackson (1958) reported that two fibre systems could be observed in


living specimens, corresponding to the two fibre systems of their electron micrographs.Both systems were found to behave in an identical manner in contraction and relaxa-tion: on contraction, both fibre types shortened and remained straight, whereas onrelaxation, both were thrown into sinuous folds. Therefore it was thought that bothwere likely to be contractile.

Our own findings suggest that both groups of workers were probably mistaken intheir identification of the two fibre systems in living animals. By far the most obviousof the two fibres in the stalks of contracted living animals is the M fibre and it is thiswhich becomes folded in relaxing animals, the km fibre remaining straight at all times.This difference of behaviour is verifiable in living animals by the analysis of cinema-tographic records made under conditions where both sets of fibres are clearly seen,and their characteristic markers (for example, kinetosomes for the km fibres, irregulargranules for the M fibres) are taken into account. Further evidence is afforded by theelectron micrographs of pre-cooled specimens, where straight km fibres and con-spicuous folded M fibres are seen. It is likely that the prominent fibres in the stalkregion of living animals which Faure-Fremiet et al. (1956) took to be the km fibres werereally M fibres, and that they did not see the overlying km fibres in these specimens.It is also possible that Randall & Jackson failed to identify living km fibres, and thatthey mistook the central and peripheral edges of a single M fibre for two distinctfibres, which therefore appeared to behave in an identical manner.

The behaviour of the M fibres in body contraction and relaxation in the stalkindicates that they are contractile elements: they shorten and thicken on bodyshortening, and they begin to elongate immediately afterwards, before the rest of thebody, being thrown into lateral folds. The diffuse connexions between the M fibresand the overlying kinetosomes and pellicle could constitute structural attachmentswhich would bring about changes in body length with changes in the M fibres. TheM fibres in the adoral half of the body form a nearly continuous sheet around theanimal, and it is interesting that the pellicle in contracted specimens shows con-siderable bulging in the pigment stripe region. This could be caused by a lateralcontraction of the M fibre material, thus drawing adjacent km fibres towards eachother and causing the pellicular bulging. The presence of such a continuous sheet isfound in other contractile ciliates such as Spirostomum (Finley, Brown & Daniel, 1964)and in other ciliates which can contract on suitable electrical stimulation, such asParamecium (Kamada & Kinosita, 1945; Pitelka, 1965). These findings are all sugges-tive of a contractile role for the M fibres, but there is not enough evidence yet to provethis beyond all doubt, nor to decide whether a sliding filament or contractile moleculetheory of contraction might be the more appropriate. Only one type of filament hasbeen found throughout the M fibres with any certainty, but the same is also true ofmetazoan smooth muscle which the M fibres of Stentor resemble (except for thepresence of dense bodies in smooth muscle).

The elongation of the M fibres into sinuous folds, observed after contraction,suggests that work is being done in relaxation either by an elastic system stretchedduring contraction, or perhaps by some type of reversal of the contractile processitself. No further evidence is at the moment available on this point.


The km fibres, in contrast to the M fibres, remain straight at all times, and shortenor lengthen with the changing length of the body. The functions of this system will bediscussed after considering its structure.

The arrangement of the kinetosomes and their associated microtubules reportedhere is in general agreement with the findings of Kennedy (1965). The main differenceis that we find each barren kinetosome to be attached not to 2 km microtubules, but to20 or 21 of them. Each barren kinetosome (with its attached ciliated companion)therefore forms the anterior termination of a vertical stack of about 21 microtubules.Since these stacks overlap one another, a cross-section of a km fibre contains severalstacks, each representing kinetosome pairs. Knowing the maximum number of stacks(66) which are found in cross-sections of km fibres in contracted animals, and also theminimum spacing of kinetosome pairs (0-3 fi), one may use Randall & Jackson's methodof calculating an approximate maximum length for a microtubule stack, which comesto about 20 ji (number of stacks per cross-section x distance between cilia). Of course,this is only an approximate figure, and assumes that all stacks are the same length in allparts of the body and in all animals. Within a stack, those microtubules which areclosest to the pellicle are the longest, since cross-sections of the aboral parts of stacks(i.e. those on the side of the km fibre away from the kinetosome row) show a pro-gressive loss of the inner microtubules.

Although animals which have been fixed in the extended position by the gelatinmethod cannot be regarded as truly relaxed, the state of their km fibres indicates thatindividual stacks of microtubules are able to slide upon one another as Stentor changesits length. If the calculated value of stack length of 20 /* is compared with the maximummeasured spacing of 25 ju, between cilia in extended animals, it would seem that thereis very little overlap between adjacent stacks (assuming of course that there is no grosschange in the length of the microtubules on fixation). This appears to confirm thesliding model of the km fibre first put forward by Randall & Jackson (1958). It isnoteworthy that the microtubules in the extended specimens are almost surroundedby the M fibres. The gelatin method of obtaining fixed extended specimens dependsupon the mechanical prevention of body contraction during the initial stages offixation. The contractile elements are undoubtedly stimulated to contract but areunable to shorten; that is, they undergo a virtually isometric contraction. Althoughthe main orientation of the M fibre filaments is parallel to the longitudinal axis of thefibre, this orientation is not strict and many filaments run at an angle with this axis.If the M fibres are contractile, one would expect a radial as well as a longitudinalcomponent in its contraction, and in an isometric contraction structures attached tothe edge of a fibre could be drawn towards its centre. Such a process probably under-lies the partial engulfing of the km fibre microtubules by the adjacent M fibre inextended specimens.

Turning to the detailed structure of the stacks of microtubules, the most remarkablefinding is the presence of cross-bridges between the microtubules of adjacent stacks.So far these have been found only in contracted specimens, presumably because thedetails of the microtubules in extended specimens are obscured by the surroundingM fibres. One of the participant microtubules is always microtubule 3, and the cross-


bridges are characteristically attached at the end farthest from the kinetosomal row.The other participant microtubule in such connexions varies in its position fromstack to stack. These findings indicate that these cross-bridges are a constant andintegral part of the structure of the km microtubules, and that in all cases they projectfrom the microtubules in the direction of the kinetosomal edge of the km fibre. Itwould also appear likely that these bridges can be disengaged at their kinetosomal endsunder certain conditions, and can link to whatever microtubule is adjacent to them; forinstance, when the regular array of stacks has become disrupted. This fits in well withthe sliding model of the km fibre stacks outlined above, since adjacent stacks must bedisconnected from each other to allow the sliding to take place, but must be reconnected,at least after body contraction, to give the appearance seen in contracted animals.

A mechanism of this type may be the cause of the delay in body extension seensubsequent to body contraction, associated with the sinuous folding of the relaxingM fibres. This could be brought about by locking together of adjacent microtubulestacks by means of cross-bridges. For elongation of the body to take place, thesecross-bridges must be broken and perhaps reformed, or alternatively they could slide.This phenomenon could be either passive or active, and if the latter, could be themotive force in the extension of Stentor, if it worked to slide the stacks apart, oralternatively could be the basis of local bending movements of the stalk if it acted inthe opposite direction. It is unlikely that such a system could be the main cause of therapid body contraction if one accepts that there is little overlap between adjacentstacks in the highly extended forms, since as in the Huxley-Hanson model of striatedmuscle contraction, contraction would depend upon interaction between overlappingelements.

These ideas are somewhat speculative, and await testing by other methods. It isinteresting though that ordered arrays of microtubules with cross-links between themhave also been described by Grimstone & Cleveland (1965) in the contractile axostylesof some flagellates, and these cross-links show a strong resemblance to those betweenadjacent microtubular stacks in Stentor. Although it is not known whether the axostylecontracts by a sliding mechanism or if the actual microtubules themselves are able toalter in length, a sequential breaking-and making of cross-bridges such as that suggestedfor Stentor could bring about the undulations observed in the axostyle. Similarcross-bridges have also been reported between the microtubules of the axopods ofActinosphaerium (Macdonald & Kitching, 1967), and these authors suggest thatthe streaming movements observed in the cytoplasm of the axopods may be broughtabout by a movement of the microtubules associated with the cross-bridges. In thecase of the peripheral microtubules of cilia, Satir (1965) has reported evidence favour-ing a sliding of adjacent microtubules rather than a contraction in microtubule length,and it is possible that the spokes connecting the central and peripheral cilium micro-tubules are analogous with the cross-bridges observed in Stentor. These suggestionsare obviously tentative and there is no evidence that the microtubules in all situationsare homologous (see Behnke & Forer, 1967). Nevertheless, it suggests that the study ofstructures associated with microtubules may be as important as studies of the micro-tubules themselves in understanding the function of these organelles.


The authors are indebted to Professor G. E. H. Foxon for his helpful advice and criticismof the manuscript, and to the Spastics Society for the use of the electron microscope situated atGuy's Hospital Medical School.

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(Received 23 August 1967)


ABBREVIATIONS USED ON PLATES

afbkccbckcsdbdffftkm

anterior fibre sheetbarren kinetosomeciliumcross-bridgeciliated kinetosomeclear stripedense body-dense fibrefilaments connecting M and km fibresaboral footkm fibre

ksktMmbmemsPpbpsst

kinetosomemicrotubules of km fibreM fibremembranemitochondrionstack of microtubulespelliclepigment bodypigment stripesubpellicular microtubules

Except where otherwise stated, micrographs are of material fixed in glutaraldehyde andpost-fixed with osmium tetroxide.


Figs. 3-10. Light micrographs of the surface regions of living specimens showing thefibre systems.

Fig. 3. Low-power view of the aboral half of an unrestrained Stentor which hasbegun to re-extend after contraction. Note the wrinkled longitudinal fibres.

Fig. 4. Contracted Stentor, showing broad km fibres with cross-striations due towrinkling of the pellicle. Pigment bodies lie in the folds of the pellicle in the pigmentstripe, giving it a ladder-like appearance.

Fig. 5. A km fibre showing pairs of kinetosomes along its margin.Fig. 6. A km fibre in a fully contracted specimen showing a row of kinetosomes

alongside, which appears to form a separate fibre.Figs. 7, 8. Fibres immediately after full contraction of body, taken in the same field

at different depths of focus. Asterisks mark corresponding positions. Fig. 7 shows thesuperficial km fibre with kinetosomes and adjacent pigment bodies. Fig. 8 shows thedeeper M fibre with adjacent mitochondria. Both km and M fibres are unwrinkled.

Figs. 9, 10. Fibres at onset of relaxation, again at different depths of focus in a singlefield. Asterisks mark corresponding positions. Fig. 9 shows the superficial km fibresto be unwrinkled. Fig. 10 shows the deeper M fibres to be thrown into lateral con-volutions.

Journal of Cell Science, Vol. 3, No. 2

« * 4

L. H. BANNISTER AND E. C. TATCHELL (Facing p. 308)

Fig. I I . Longitudinal section in stalk region of body showing prominent clusters ofdense bodies associated with M fibre (osmium fixation).Fig. 12. Transverse section through km and M fibres in stalk region, showing verticalmicrotubular stacks in km fibre, with cross-bridges between adjacent stacks, andfibrous connexions between M fibre and overlying structures.Fig. 13. Detail of M fibre showing a filament in longitudinal section (long arrow) andcross-sections of filaments with hollow centres (short arrows).


km

L. H. BANNISTER AND E. C. TATCHELL

Figs. 14, 15. Horizontal sections through surface of body showing relationshipbetween kinetosomes and adjacent structures.

Fig. 14. The km microtubules arise in close association with one of the triplets of abarren kinetosome.

Fig. 15. Subpellicular microtubules and anterior fibre sheet are in contact with theciliated kinetosome. Ribs are seen on one side of the anterior fibre sheet (arrows).Fig. 16. Dense prongs of fibrous material running between barren kinetosomes and kmmicrotubule stack.Fig. 17. Longitudinal section through km fibre stack where it joins the barren kineto-some, showing initial curvature in the course of microtubules. Fibrous attachmentsbetween M and km fibres are also shown.Fig. 18. Detail of Fig. 17, showing periodic cross-bridges between microtubuleswithin a stack (arrows).Fig. 19. Detail of Fig. 12, showing transverse section of part of km fibre. Cross-bridgesare seen between the microtubules of adjacent stacks, and projections are present onthe walls of some microtubules. The kinetosomal edge is toward the left. A tracingincluding this region is shown in Fig. 2 a.

Fig. 20. The regular sequence of stacks is disrupted in this transverse section of a kmfibre, and a secondary attachment is seen between two stacks in close proximity(arrow). The kinetosomal edge is towards the left.Fig. 21. Transverse section of km fibre at extreme adoral end of body, showing reduc-tion of the fibre to a single microtubular stack (arrow).Figs. 22, 23. Transverse sections through stalk region of specimen fixed in theextended position in gelatin.

Fig. 22. A single stack of microtubules representing the km fibre, almost sur-rounded by the M fibre.

Fig. 23. Detail of the stack of microtubules shown in Fig. 22.Fig. 24. Horizontal section through the surface region of the stalk in a specimen fixedin the relaxed state with previous cooling (osmium fixation). The km fibres andattendant row of cilia are straight but the M fibres show sinuous folding.

CONTRACTILITY AND THE FIBRE SYSTEMS OF STENTOR COERULEUS

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