J. Cell Set. 47, 167-185 (1981)

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ARRANGEMENT OF SUBUNITS INMICRORIBBONS FROM GIARDIA

D. V. HOLBERTONDepartment of Zoology, The University of Hull, Hull HU6 7RX, U.K.

SUMMARY

Ultrasound has been used to disperse the cytoplasm of Giardia muris and Giardia duodenalistrophozoites, releasing disk cytoskeletons for negative staining and study by electron micro-scopy. Sonication also breaks down the cross-bridges uniting microribbons in disks. Individualribbons, and small bundles of these structures, are found in these preparations and have beenimaged both from their edges and in flat face view. The outer layers of ribbons are 2 sheets ofregularly arranged globular subunits, held apart by a fibrous inner core. The axial repeat ofthe microribbon is 15 nm, which is also the distance separating cross-bridge sites along ribbons.Pronounced striping at this interval is a feature of ribbon faces where they are joined in bundles.

Subunits in the outer layer are arranged in vertical protofilaments that are set orthogonally tothe long axis of the ribbon. Protofilaments bind tannic acid and are seen clearly in sectionedribbons. Three protofilaments fit into the 15-nm longitudinal spacing. Optical diffractionpatterns from ribbon images are dominated by orders of the 15-nm periodicity, including thethird-order reflexions expected from protofilament spacings. Fourth-order reflexions indicatethat the ribbon core may also be structured. Ribbon face images give rise to a strong 4-nm layerline, corresponding to the vertical spacing of subunits in protofilaments. Neighbouring proto-filaments are staggered by about 0-67 nm.

The lattices found in ribbons are consistent with studies of cytoskeleton composition.

INTRODUCTION

The accompanying paper (Holberton & Ward, 1980) described the disk cytoskeletonof Giardia and showed how a useful preparation of this structure can be obtained byTriton extraction of cells harvested from culture. A disk is largely composed ofmicroribbons: insoluble ordered structures which have huge dimensions and appear tospring from the walls of microtubules. Efforts to purify microribbons were hamperedby the very strong bond between microribbons and microtubules, and by the con-siderable numbers of axonemes that were also Triton-insoluble.

The protein composition of disks included much tubulin, and quantities of asecond smaller protein with a polypeptide molecular weight of about 30 000.Becausemicroribbons are much larger than microtubules, to account for the predominance oftubulin in gel patterns it was thought likely that tubulin is also one of the micro-ribbon proteins.

After conventional fixing and sectioning, microribbons appeared at moderatemagnifications to be trilaminar in cross-section (Holberton, 1973; Holberton & Ward,1980). Their densely staining faces carry large numbers of bridges by means of whichthey are rigidly linked in situ. In many sections the more translucent central core of theribbon appeared to have continuity with a microtubule wall.

168 D. V. Holberton

In this paper the structure of some isolated ribbons is examined at higher magnifi-cations, and is analysed by optical diffraction from these images. Their faces are foundto consist of 2 sheets of regularly packed subunits that have the dimensions of tubulinprotomers. The sheets do not appear to touch the microtubule with which they areassociated. They enclose a filamentous matrix of different electron-staining properties,through which this bond is apparently expressed.

MATERIALS AND METHODSThe micrographs reproduced in this paper are of G. muris and G. duodeiialis. The preceding

paper (Holberton & Ward, 1980) gave details of how these 2 organisms were obtained respec-tively from mouse gut and from monoxenic cultures. Disks were isolated by the sonicationmethod rather than by Triton extraction, since structures can be collected on electron-micro-scope grids more rapidly this way. It was thought that microribbons might be better preservedif chemical extraction were avoided.

To obtain clear images of disk microribbons in face view it was necessary to disrupt the linksbetween these elements. Flagellates were disintegrated by exposure to ultrasound (mean output100 W) for 30-60 8. In some cases cells were first fixed in 2-5 % glutaraldehyde in o-i M phos-phate buffer at pH 7-2. The material collected by a low-speed centrifugation (5 min at 1200 g)was membrane-free, and consisted of fragments of disks, and some axonemes. Single drops ofsuspended material were negatively stained on grids of 400-mesh overlayed with thin carbonfilms; various stains were used (see figure legends).

For studies of ribbon cross-sections, washed flagellates were fixed in 2 % glutaraldehyde,8 % tannic acid (GTA), according to the method of Mizuhira & Futaesaku (1971), as used byTilney et al. (1973). Pellets of fixed cells were postfixed and prepared for sectioning as before(Holberton & Ward, 1980).

Specimens were examined at 80 kV in a JEOL 100C electron microscope equipped with a± 6o° single tilt-rotating stage. To prevent radiation damage, microribbons were usuallyphotographed at a low beam current and at nominal magnifications of x 33000 and x 50000.Micrographs of crystalline bovine liver catalase (Agar Aids) taken on the same occasion underthe same conditions were used to calibrate spacings.

Diffraction studies were carried out on a 3-m double rail bench, with optical componentsfollowing the construction suggested by Markham (1968).

RESULTS

Preparing microribbons for electron microscopy

Disks were stable in Triton because many of the cross-bridges between micro-ribbons resist extraction. Even when disks were violently swirled in extracting solutionsit was difficult to separate ribbons. On the other hand, when disks were preparedfrom cell suspensions by ultrasonic agitation they disintegrated rapidly. Amongst the

Figs. 1, 2. Electron micrographs of microribbons found amongst unfixed disk frag-ments from G. muris. Washed flagellates were broken open by ultrasonicating for1 min. Fragments pelleting after 5 min at 1200 g were embedded in films of 05 %uranyl oxalate with 50 fig ml"1 bacitracin at pH 6-5.

Fig. 1. Separated ribbons emerging from the posterior notch region at top left ofthis micrograph. Single ribbons are very faintly striated, x 40500.

Fig. 2. A flat bundle of ribbons showing strong vertical striping at 15-nm intervals,x 146600.

Subunits in Giardia microribbons 169

500 nm

- • > • .

Subunits in Giardia microribbons 171

fragments were found some free ribbons and many bundles where ribbons were stilljoined side by side.

Isolated ribbons generally lay flat and straight like pieces of stiff tape. Where atintervals they were twisted, the folds also were flattened so that first one face and thenthe other was uppermost. Negatively stained, these preparations gave finely detailedimages that included information from a number of levels of ribbon structure, andfrom which it was possible to resolve subunits by optical diffraction. Often ribbonsremained attached at one or two points but elsewhere were splayed apart (Fig. 1). Insome cases large frayed bundles were tied tightly together only at the posterior notchwhere transverse linkages were intact. Where bundles of ribbons lay flat they weresometimes seen to be regularly striped at the cross-bridge interval of 15 nm (Fig. 2).

Microribbons in bundles occasionally sat vertically on grids so that their edgeswere presented to the electron beam. In this orientation the clearest images werefound near the ends of ribbons where they are low in height (probably less than 50 nm)and can stand upright without folding or buckling.

Sections of disks fixed in GTA helped with the interpretation of the images ofisolated ribbons. Tannic acid was crosslinked to ribbon layers by glutaraldehyde,producing a dense contrasting matrix around their subunits. These could be imagedfairly easily by tilting thin sections until rows of subunits came into alignment withthe microscope axis. Comparable alignments were rarely achieved for isolated ribbonsstanding on edge in negative stains, except over very short distances. Single ribbonstended to lean or fold over; those still held by cross-bridges in bundles generally stoodtoo high off the surface of the grid and trapped excessive amounts of stain.

Ribbon edges

Negative stains penetrated ribbons and in some cases outlined a number of struc-tural layers when ribbons were viewed from their edges. Most clearly visible in these

Figs. 3-7. Electron micrographs comparing ribbons in edge view in various prepara-tions. Figs. 3-5: G. duodenalis ribbons. Figs. 6, 7: G. muris ribbons.

Fig. 3. A fixed isolated ribbon, negatively stained with 1 % ammonium molybdateat pH 5-0, to show ribbon layers. The two superficial layers are 4-5 nm thick. Theyenclose a core of indeterminate structure which in places appears to be made up of2 interlocking layers (arrows). Periodic structure along the ribbon is poorly preserved.The cross-bridges have been dissolved away, leaving short projections at some sites,x 420000.

Fig. 4. Part of an intact fixed disk in which cross-bridges between ribbons arepreserved and ribbons are nearly parallel to the beam. Ribbons are negatively stainedwith 0-5 % uranyl formate at pH 4-4. Periodic substructure is apparent in ribbon layers.The diffraction patterns from the framed area, and from the inset micrograph (Fig. 5)are shown in Fig. 8 c and Fig. 8D respectively, x 163300.

Fig. 6. Longitudinally sectioned disk fixed in GTA. Microribbons and cross-bridges are densely stained by tannic acid. The diffraction pattern of Fig. 8B istaken from the 4 ribbons on the right of this micrograph, x 226700.

Fig. 7. Sectioned ribbon at high magnification. The black lines indicate subunits,or protofilaments, that can be distinguished in ribbon-face layers after tannic acidstaining. Compare this micrograph with the interpretative diagram of Fig. 13.x5933OO.

i72 D. V. Holberton

micrographs are the 2 similar layers at the faces of a ribbon, which are of uniformthickness and give rise to cross-bridges at regular intervals. They enclose a broadercore which is less distinctly organized; stains infiltrate this layer irregularly. Fig. 3shows a region close to one end of an isolated ribbon that has lost its cross-bridges. Inplaces the core is divided lengthways, so that for short distances 4 layers can becounted across the width of the ribbon.

After fixing in GTA, ribbons in cross-section showed the same arrangement oflayers; also some features that were not apparent in negatively stained preparations.The outer layers were again of constant thickness, between 4 and 5 nm, but whensectioned horizontally were seen to be composed of subunits, which must be arrangedin vertical columns, or protofilaments. Figs. 6 and 7 show that cross-bridges are verydensely stained by tannic acid. Cross-bridges are precisely spaced along a ribbon, andin horizontal sections it was possible to count 3 protofilaments in the interval separat-ing 2 cross-bridges.

Elements of the ribbon core also bound tannic acid but, in general, were imperfectlyaligned however sections were tilted. Consequently they were not well defined inany of the electron micrographs. In places the core appeared to comprise rather smallsubunits or filaments, much smaller than the distance between the 2 outer layers. Insome regions there seemed to be two rows of subunits running side by side, or theymight be arranged across the centre of the ribbon in zig-zag fashion (Fig. 7). Wherethese were not visible, tannic acid outlined larger triangular or square blocks whichmight be clusters of the smaller structures.

In order to clarify the arrangement of components along the ribbon, micrographsof negatively stained ribbons, and of sectioned ribbons, were examined by opticaldiffraction. Ribbon images lend themselves to this sort of analysis because the struc-tures run a nearly straight course for 10-20 cross-bridge intervals. Detail in the outerlayers and the core seemed to be periodic at shorter range than the cross-bridgeinterval, so this distance included a large number of repeats. When tilting sections itwas difficult to be sure when ribbon layers were exactly parallel to the beam, especiallyat low beam intensities. As a result, most of the recorded images had a slightly obliqueviewpoint which meant that diffraction patterns differed in the relative intensities ofsome reflexions. When single ribbons were masked off, the most consistent informa-tion from optical transforms was the layer line values from features that are seriallyrepeated along the ribbon.

As Fig. 8 illustrates, patterns from both sets of images are dominated by a 15-nmsystem of layer lines. This is the cross-bridge spacing and might be expected to relateto the longitudinal repeat of subunits in the face layers, but it appears that someperiodicities from the core also fit into this interval. In transforms, longitudinalspacings can be calibrated internally from the cross-bridge spacings to allow for sectiondistortion, where this occurs. Apart from the strong 15-nm layer line, reflexions arealso present on the second-order (7-5 nm), third-order (5 nm), and fourth-order(3-75 nm) layer lines. The prominent third-order reflexions are due to the spacing ofprotofilaments in the outer layers. The spots on this line are always off the meridianbut asymmetrically positioned to either side of it. This 'pseudo-helical' pattern

Subunits in Giardia microribbons 173

nm

3 - 7 5 -5-0 -7-5 -

15-0 -

• •:? • •

8A 8B

nm

8c \ I 8D4-5 4-4 3-85 nm

Fig. 8. Optical diffraction patterns from microribbon edge views. Axial periodicitiesin microribbons from both G. duodenalit and G. muris give rise to a system of layer linesbased on the 15-nm interval. The strong 15-nm reflexions come from the cross-bridge spacing along a ribbon. The higher orders show that this interval is integrallysubdivided by periodic arrangements of subunits in ribbon face and core layers (seetext and Fig. 13). A. Diffraction pattern from the image of a single sectionedribbon shown in Fig. 7. B. Diffraction pattern from 4 ribbons in Fig. 6. In thisimage the cross-bridges diffract strongly. Their reflexions on the 15-nm layer lineare streaked because the angle they make with ribbons varies between 650 and 1350.C, D. Diffraction patterns from the images in Figs. 4 and 5 where ribbon layers werestrongly contrasted by uranyl formate staining. Central spot attenuated photo-graphically. Higher-order reflexions on the equator indicate the dimensions of somestructures across the width of the ribbon: in the face layers (4-4 nm), and in theribbon core (3-85 nm).

174 D. V. Holberton

indicates that the 2 outer layers are probably not in register across the ribbons, but thepositions of their subunits are offset to a small extent (see Fig. 13, p. 179).

Structures in the ribbon core gave rise to weak layer lines once face layers weremasked out; 375-nm and 5-nm spacings were detected in different images and some-times both layer lines were given by a single masked micrograph. Occasionally a weak15-nm layer line was also present. However, since the viewpoint or section planethrough a ribbon was often slightly oblique, it was difficult to mask core structuresreliably. Probably some periodicities of face layers coming from some level in thesection remained in the masked image. Where visible, structures in the core seemedsmaller than face subunits, suggesting that the 375-nm repeat may be a genuineperiodicity of core layers.

Where ribbons are transversely linked in bundles, the spacing between them isextremely uniform and acts as a strong grating if the diffraction mask is widened toinclude a number of ribbons. In addition to the layer lines, diffraction patterns nowfeature on the equator a large number of orders of the ribbon separation (Fig. 8).Ribbon layers were contrasted more by negative stains than in thin sections, so whenthese images are used for diffraction the equatorial orders are modulated by higher-angle diffraction from ribbon layers. Most consistently present are a pair of peakreflexions corresponding to 4-4 nm, which may be attributed to the thickness of theouter layers. In Fig. 8 c these reflexions are nearly coincident with the seventh orderof the ribbon separation, which was 30-31 nm in these images from the posteriornotch region of the disk (Figs. 4, 5). In addition, this series of transforms showedbroad maxima over the fourth orders (7-7 nm), or intense eighth-order reflexions(3-8 nm, see Fig. 8D) where there seemed to be 2 layers in the ribbon core.

Ribbons in transverse section

Ribbon layers show particularly strongly in transverse sections after tannic acidstaining (Fig. 9). More obviously in this orientation than in longitudinal section thecore of the ribbon appears divided down its centre. However, the 2 sublayers also fuseat intervals, suggesting that they are not independently continuous sheets. Ratherthey appear as beaded columns of subunits that at different levels in the section crossover in some way.

Over short distances subunits are packed sufficiently regularly in the outer layersto be brought into alignment at certain angles of tilt. The orientations that show themmost favourably do not usually coincide exactly with an axial view of the microtubuleto which the ribbon is attached, but are found when the section is tilted about io° ineither direction from this position. The reason for this is evident from face views ofisolated ribbons (see below); subunits fall into rows that are not horizontal butinclined at a shallow angle to the long axis of the ribbon. Fig. 9A shows that thecentre-to-centre spacing of subunits in outer protofilaments is about 4 nm. Althoughcore filaments are not straight, where they could be followed for short distancesattempts were made to measure their bead-like subunits against this scale. Usually6 core 'beads' were counted alongside 5 outer subunits over a distance of some 20 nm.

Subunits in Giardia microribbons 175

9AFig. 9. Electron micrographs of transverse sections of microribbons and micro-tubules in intact G. duodenalis cells fixed in the presence of 8 % tannic acid. 13 proto-filaments are outlined in microtubule walls. The black dashes indicate that in theface layers of microribbons subunits are spaced at 4-nm intervals. Structures in theribbon core appear in this orientation to be arranged in 2 sheets or sets of filaments(arrows). The way in which core filaments seem to intertwine at fairly regular intervalscan best be seen by looking at the page from the bottom (A, central ribbon) or fromthe top (B, both ribbons), x 500000.

I 76 D. V. Holberton

Subunits in Giardia microribbons 177

This measurement averages the size of beads. In fact they are not very uniform inmicrographs, largely because of the irregular way in which they are superimposed.

Fig. 9 shows that disk microtubules have the normal 13 protofilaments. The widthof a ribbon is broad enough (16-17 nm) to span 3 microtubule protofilaments wherethese 2 structures join. In fact tannic acid staining shows that 5 or 6 microtubuleprotofilaments may be involved in this junction. On the side facing outward (towardthe rim of the disk), a densely-staining sheet emerges from the bottom of the ribbonand partly wraps over the microtubule wall. The sheet is continuous with the ribboncore and is seemingly the means by which the structures are bonded together, sinceneither of the ribbon protofilament layers appears to make direct contact with themicrotubule. Its presence explains why microtubule protofilaments in this region arestable against degradation by Triton (Holberton & Ward, 1980).

Ribbon faces

Flat ribbons allowed the imaging of ribbon faces over considerable distances. Theribbon bundle in Fig. 10, for example, was followed for almost 20 /on. It was foldedover at 3 places, but stretches between the folds were straight for up to 300 cross-bridgeintervals. Because ribbons are also broad structures, areas equivalent to about o-i fim2

of ribbon surface could be masked off in micrographs used for diffraction. This meantthat diffraction patterns averaged information from between 3000 and 5000 unit cellsof the ribbon face lattice. Nonetheless, reflexions were sharp and not streaked,indicating that the packing of subunits was undistorted over large areas. Ultra-sonication removed cross-bridges from ribbons, in many places so completely thatribbon faces were notably smooth, and lacked projections or heavily sculpturedfeatures of any kind. The protofilaments seen in horizontal sections were not obviouslydelineated by negative stains. Possibly this is because contrast was reduced in themultilayered structure, the core material partly filling the grooves between protofila-ments. Rather, the image seen most often was more like an undivided sheet weaklyetched by fine striations running both along and across the ribbon. The transverselines were narrowly spaced and exactly perpendicular to the ribbon axis. Those at thecross-bridge interval sometimes stained more heavily in patches along the ribbon,although all of these lines were interrupted in places as if the stain lay in rather shallowgrooves. Ribbons stacked in flat bundles also generally appeared featureless, likesingle ribbons (Fig. 11), but where they were still joined by cross-bridges they becamestrongly striped at the 15-nm spacing. Striping was characteristic of the posterior

Fig. 10. Part of a bundle of 5 microribbons lying flat on the carbon support film,negatively stained in 0-5 % uranyl oxalate at pH 6-5. The junctions of ribbons withmicrotubules are toward the bottom of the micrograph. Fine vertical striationsindicate the orientation of face layer protofilaments. 15-nm striping is particularlyclear on the left, x 114700.Fig. 11. An enlargement from Fig. 10. The low contrast of ribbon subunits gives riseto a smooth appearance. The white lines trace lateral alignments of subunits, whichare best seen by viewing the page from the side, x 390000.

i 7 8 D. V. Holberton

notch region of isolated bundles where ribbons are more closely stuck together thanelsewhere.

The diffraction patterns from flat ribbons varied very little in their principal featuresfrom image to image, including images of flat bundles of ribbons. In the latter case,micrographs recorded similar periodic information from a number of different levelsin the bundle. Compared to single ribbons, bundles held more stain and gave imagesof higher contrast that were more easily focused at low beam intensities. For thisreason micrographs of bundles generally showed structures that were better preserved,and there was less noise in the optical transforms. Images were selected where ribbons

nm

4-0 ft *

*•••"•*•• • >JT

• " * ' . ' ' • •

•' ' * v .4:1^ .=•

12A 12BFig. 12. Optical diffraction patterns from the framed areas in Fig. io. The 4-nm layerline comes from the regular arrangement of subunits at ribbon faces. Reflexions on thelayer line and on the equator are orders of 15 nm, which is the fundamental axialperiodicity of face and core structures (see Figs. 13, 14). In Fig. 12B the pattern isfrom a part of the image (area 2) that includes some 15-nm striping associated withcross-bridges. In Fig. 12A, from a low-contrast image (area i), the 15-nm equatorialreflexions are weak, suggesting that cross-bridges are lost or disordered. The strongestspots on the equator correspond to the 5-nm axial repeat of face-layer protofilaments.

were not noticeably splayed. There was little evidence from their diffraction patternsthat ribbons in a bundle were rotated with respect to one another.

Ribbon face transforms are illustrated in Fig. 12. Reflexions on the single layer lineand on the equator conform to the same 15-nm orders as governed axial spacings in theribbon edge array. The equatorial reflexions indicate that the structures diffracting atthese spacings are set at right angles to the long axis of the ribbon. According to howmicrographs were masked, the different orders varied in relative intensity, just asribbons varied locally in the extent to which they were striated. As shown in Fig. 12B,sometimes the cross-bridge spacing is stressed. More typically patterns were like thatin Fig. 12 A, in which the shorter 5-nm repeat from protofilaments contributes the

Subunits in Giardia microribbons 179

7-5 nm

4-5 nm

50 nm 50 nm

Fig. 13. Periodic structures in microribbons, from optical transforms of ribbon edgeand face images, A. Structures repeating axially in microribbon layers. Unit cells ofsubunits in the face layers are marked to explain the positions of prominent spots onthe 5-nm layer line of the diffraction pattern from 1 sectioned ribbon (Fig. 8A).B. Lattices of subunits at ribbon faces. Protofilaments are at right angles to themicrotubule axis (X axis). Left: Subunits at the near face. Cross-bridges (solidtriangles) are arranged at top and bottom in 2 possible superlattices (parallel andantiparallel) which have the same unit cell dimensions and are related by mirrorsymmetry. Right: Subunits at the far face, as seen through the ribbon. Cross-bridgesites marked by solid circles.

D. V. Holberton

Subunits in Giardia microribbons 181

strongest reflexions. The 7-5- and 375-nm reflexions are usually weaker than theother orders. The 375-nm periodicity may be explained by assuming that staininfiltrates ribbons to an extent that differentiates some core elements.

The sharp 4-nm layer line is in agreement with the vertical spacing of the subunitsseen in ribbon face protofilaments in transverse sections. In the absence of anotherlayer line, it must be supposed that core structures are not strongly periodic in thisdirection, unless they are spaced more closely than the useful resolution of theseimages (about 2-5 nm). The beaded structures outlined by tannic acid in sections arenot resolved by face diffraction.

A surprising result was that no reflexions were found in diffraction patterns closerto the equator than the 4-nm layer line. From sections one might have expected orderat longer range, either from the association of core structure with face protofilaments,or from the vertical spacing of cross-bridges. Where cross-bridges can be brought intoalignment by tilting transverse sections of ribbons, their separation suggests that theyare sited further apart on protofilaments than the subunit repeat, and probably at adistance of 8-10 nm, or a multiple of that dimension. However, these periodicities donot appear in face images, even in those images of bundles where the 15-nm spacing ofcross-bridges along ribbons gives strong reflexions on the equator. Perhaps wherethey remain attached, cross-bridges collapse in a disordered manner against theribbon face but still stain in broad bands at the 15-nm interval.

The fact that 15-nm equatorial reflexions also appear in diffraction patterns ofribbons that are apparently free from projections suggests that protofilaments tend toanticipate the cross-bridge repeat by being in some other way grouped in threes.

The arrangement of subunits at the faces of a ribbon is not orthogonal since thereflexions on the 4-nm layer line were all off the meridian. However, as Fig. 12 shows,they were symmetrical in their positions about the meridian, indicating that a viewthrough a bundle of ribbons includes both left-handed and right-handed patterns.The pair of reflexions nearest the meridian were often not of equal intensity. One wasconsistently more intense than the other reflexions on the layer line. These 2 reflexionscorrespond to the 2 (01) reflexions from the opposite-handed images of the 2 facelattices, one seen from in front and the other from behind (through the ribbon). Theirpositions, which did not vary at all from pattern to pattern, indicate that subunits fallinto rows that are sloped at a gentle angle (7-5°) to the long axis of the ribbon. Theresulting face lattice is drawn in Fig. 14, and is found to have special properties ofsymmetry that are discussed below.

When micrographs are examined closely, fine lines can be seen running obliquely

Fig. 14. A. An optically filtered image of the microribbon in Fig. 11, reconstructedfrom the equatorial and 4-nm layer line reflexions of a diffraction pattern. Periodicfeatures from separate ribbon layers are superimposed to varying extents in differentparts of the image. Near the top, sets of 3 protofilaments are marked in a region wherethe reconstructed lattice is the image of one of the face layers. B. When superim-posed, simple diagrams of the front and back ribbon lattices generate moire' patternsthat are reasonable models of the filtered image in areas where optical interactionbetween ribbon layers is evident (framed in Fig. 14A, above).

182 D. V. Holberton

across ribbon faces at the lattice angle, but usually in both directions simultaneously(Fig. 11). The near-meridional reflexion that is stressed most often in transforms is theone corresponding to a left-handed pattern in the micrograph. Micrographs areprinted as if the observer is viewing a specimen from underneath the carbon supportfilm. For flat structures like ribbons both surfaces are stained to some extent, otherwisethere would be no ambiguity of handedness in images. However, it is perhaps likelythat the surface in contact with the support film is more completely embedded instain and is contrasted more strongly. This argues, somewhat inconclusively, for aribbon model that is essentially a back-to-back arrangement of two similar layers thatappear left handed when viewed from outside.

DISCUSSION

A model of ribbon structure

Figs. 13 and 14 illustrate lattices appropriate to the face structures that will explainthe optical diffraction patterns. The chief features of ribbon construction that thesesuggest are interpreted more schematically in Fig. 15.

The face subunits are arranged in vertical columns, which I have called protofila-ments; they are also arranged in nearly horizontal rows, with an included angle of82*5° between these 2 alignments. An important feature of this lattice is that there isleft-right symmetry about protofilaments for subunit positions in every third proto-filament. Consequently, at orders of 15 nm (3 protofilaments), the reflexions from aback-to-back double layer, in which both left and right-handed lattices are projectedin the image, will superimpose on the layer line corresponding to the subunit dimen-sions (4 nm). For this reason, when a number of orders appear on the layer line (suchas in Fig. 12A and B), it is not possible to determine from position symmetry alonewhether both front and back lattices are present in ribbons.

Fifteen nanometres is also the spacing of cross-bridges along ribbons. The patternmade by cross-bridges at ribbon faces can be seen in fixed disks sectioned as nearly aspossible parallel to ribbons. A detailed analysis of these images by optical diffractionwill be the subject of a later paper. The results show that cross-bridges are in align-ment vertically, and that the vertical repeat is fairly regular. Once foreshortening inimages due to ribbon tilt is allowed for, the spacing seems to be about 8 nm; thiscoincides with every other face subunit. Cross-bridges are arranged in patterns thathave the same lattice angle as the arrangement of face subunits, but may be left orright-handed at different locations on the same ribbon. Because of the mirror sym-metry of ribbon subunits at 15-nm intervals, both lattices superimpose on ribbonsubunits in a straightforward way. In other words, cross-bridge sites can be specifiedby the positions of ribbon face subunits, whether these are arranged in lattices that areright-handed or left-handed. This makes it possible for a set of parallel bridges to linktogether 2 similar ribbons whose opposing faces will be sloped in opposite directions.

Ribbon face subunits and microtubule subunits are about the same overall size.However, the setting of ribbon protofilaments orthogonally to the axis of their

Subunits in Giardia microribbons 183

companion microtubulc is bound to make the seam discontinuous. In this orientationit is improbable that subunits of the 2 structures will bond directly because they willnot be aligned. This is because the true longitudinal repeat at the junction is 60 nm, adistance that encompasses an even number of ribbon protofilaments and cross-bridgeintervals, but an odd number of microtubule subunits. By the same argument,

QDOFig. 15. Perspective drawing of the subunit arrangement in microribbon faces, showingthe discontinuous seam with a disk microtubule. Ribbon core structures are drawncrudely from their appearance in transverse sections, like those reproduced in Fig. 9.

structures that are periodic in the ribbon core cannot be equivalently positioned withrespect both to ribbon face layers and to microtubule subunits. It is possible that corefine structure is differently organized along the bottom edge of a ribbon where itwraps over microtubule protofilaments. Core molecules may have separate sitescapable of making 2 sets of fairly specific interactions: with ribbon surface layers onthe one hand, and with microtubules on the other. Alternatively, the mechanism ofcore adhesion to microtubules could be a quite general one.

184 D. V. Holberton

In the accompanying paper the composition of disks was found to include moretubulin than was expected from the number of disk and axonemal microtubules,giving grounds for proposing that microribbons might also contain tubulin. The factthat diffraction analysis of ribbon faces has resolved tubulin-sized subunits supportsthis suggestion.

In ribbon faces, protofilaments are staggered by 0-67 nm, a much smaller shift thanbetween microtubule protofilaments (~o-o, nm), according to the surface latticessuggested by Amos & Klug (1974), Erickson (1974, 1975) and Cohen et al. (1975).However, when precipitated by Zn2+, sheets of brain tubulin have a changed packingof subunits. In these aggregates, columns and rows of subunits are angled at 24-5°,which is equivalent to a stagger between protofilaments of 2-1 nm (Crepeau, McEwen,Dykes & Edelstein, 1977).

Recently, Burton & Himes (1978) have shown that bovine brain tubulin purified byphosphocellulose chromatography can be assembled in 10% dimethyl sulphoxideinto variously shaped structures, including branched and corrugated ribbons, andmicrotubules of different numbers of protofilaments. At higher pH values (pH 7-5-77), almost 90% of protofilaments were in microtubules, predominantly of 14 proto-filaments, but also of 13, 15 or 16 protofilaments. This result, confirming earlierstudies of protofilament variations in the cross-section of microtubules (Pierson,Burton & Himes, 1978), suggests some plasticity in the way protofilaments can packlaterally, expecially under conditions that exclude the structuring influence of micro-tubule-associated proteins.

Conceivably, in Giardia also the packing arrangement of ribbon face subunits is notdue solely to intrinsic bonding geometry, but is governed by their association withstructural proteins in the ribbon core.

The positioning of cross-bridges at intervals along ribbon protofilaments implies thedifferentiation of bonds at a spacing of about 8 nm, but as discussed above there wereno 8-nm reflexions in the diffraction patterns from isolated ribbons. There is thereforeno direct evidence that subunits are paired in ribbon protofilaments in a way thatcompares with the heterodimeric composition of tubulin protofilaments. However, thedimer spacing in tubulin structures cannot always be detected by optical diffractionfrom electron micrographs. In hoop aggregates of brain tubulin a weak 8-nm line inthe diffraction pattern identifies the dimer (Mandelkow, Mandelkow, Unwin &Cohen, 1977), but this periodicity is not usually apparent when the same protein formstubules (Amos, 1977), although it is present in flagellar tubules (Amos & Klug, 1974).

In microribbons, the structural basis for bonding bridges at 15-nm intervals may bethe special status associated with groups of 3 protofilaments. Fig. 14 is a filtered imageof a microribbon reconstructed optically from all of the visible reflexions on the equa-tor and layer line of a ribbon face transform. In some regions only 1 of the 2 facesappears imaged, and here protofilaments can be seen clearly grouped in threes. Evenwhen isolated ribbons were naked of projections the 15-nm orders dominated thediffraction patterns, possibly because this is the fundamental repeat from combiningface and core lattices. It is interesing that Mandelkow et al. (1977) reported that whenprotofilaments of brain tubulin form double-layered sheet and hoop aggregates, they

Subunits in Giardia microribbons 185

also have a tendency to triple. These authors have suggested that cytoplasmic tubulinin general is disposed to bond this way.

The model of the Giardia microribbon presented in this section is one that canaccount for almost all of the features of diffraction patterns at the current level ofresolution, but leaves open an interpretation of ribbon core structures. The reason forthis uncertainty relates to the overall thickness of ribbons and the difficulty of stainingcore structures in a discriminating way. In comparison with the diffracting power offace layers and of cross-bridge lattices, the less regularly organized core contributespoorly to face view images. A better understanding may come indirectly from studyingthe structural forms adopted by ribbon proteins in vitro, once these are available inpurified preparations from improved techniques.

This work was supported by a grant from the Science Research Council.

REFERENCES

AMOS, L. A. (1977). Arrangement of high molecular weight associated proteins on purifiedmammalian brain microtubules..7. CellBiol. yz, 642-654.

AMOS, L. A. & KLUG, A. (1974). Arrangement of subunits in flagellar microtubules. J. Cell Sci.14, 523-549-

BURTON, P. R. & HIMES, R. H. (1978). Electron microscope studies of pH effects on assemblyof tubulin free of associated proteins. Delineation of substructure by tannic acid staining.J. CellBiol. 77, 120-133.

COHEN, C , DEROSIER, D., HARRISON, S., STEPHENS, R. E. & THOMAS, J. (1975). X-Ray patternsfrom microtubules. Aim. N. Y. Acad. Sci. 253, 53-59.

CREPEAU, R. H., MCEWEN, B., DYKES, G. & EDELSTEIN, S. J. (1977). Structural studies onporcine brain tubulin in extended sheets. J. molec. Biol. 116, 301-315.

ERICKSON, H. P. (1974). Microtubule surface lattice and subunit structure and observations onreassembly.7- Cell Biol. 60, 153-167.

ERICKSON, H. P. (1975). The structure and assembly of microtubules. Ann. N.Y. Acad. Sci.

253.6I-77-HOLBERTON, D. V. (1973). Fine structure of the ventral disk apparatus and the mechanism of

attachment in the flagellate Giardia muris.J. Cell Sci. 13, 11-41.HOLBERTON, D. V. & WARD, A. P. (1980). Isolation of the cytoskeleton from Giardia. Tubulin

and a low molecular weight protein associated with microribbon structures. J. Cell Sci. 47,139-166.

MANDELKOW, E.-M., MANDELKOW, E., UNWIN, N. & COHEN, C. (1977). Tubulin hoops.Nature, Lond. 265, 655-657.

MARKHAM, R. (1968). The optical diffractometer. Meth. Virol. 4, 503-529.MIZUHIRA, V. & FUTAESAKU, Y. (1971). On the new approach of tannic acid and digitonin to the

biological fixatives. 29th A. Proc. Electron Microsc. Soc. America, Boston, Mass., pp. 494-495. Baton Rouge, La: Claitor's Publishing Division.

PIERSON, G. B., BURTON, P. R. & HIMES, R. H . (1978). Alterations in number of protofilamentsin microtubules assembled in vitro.jf. Cell Biol. 76, 223-228.

TILNEV, L. G., BRYAN, J., BUSH, D. J., FUJIWARA, K., MOOSEKEB, M. S., MURPHY, D. B. &

SNYDER, D. H. (1973). Microtubules: evidence for 13 protofilaments. J . CellBiol. 59,267-275.

(Received 21 December 1979 - Revised 25 June 1980)