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y. Ceii sd. so, 259-280 (1981) 259Printed in Great Britain © Company of Biologists Limited 1981

MICROTUBULE-ORGANIZING CENTRESAND ASSEMBLY OF THE DOUBLE-SPIRALMICROTUBULE PATTERN IN CERTAINHELIOZOAN AXONEMES

J. C. R. JONES AND J. B. TUCKERDepartment of Zoology, The University, St Andrews,Fife KY16 gTS, Scotland

SUMMARYThe double-spiral microtubule pattern is established by a self-linkage procedure when

axopodial axonemes reassemble after cold treatment in multinucleate Echinosphaerium nucleo-filum and mononucleate Actinophrys sol. Nuclei are related spatially to axoneme morpho-genesis in both organisms but in rather different ways.

Microtubules grow out in all directions from discrete clumps of dense material situatedclose to nuclei in E. nucleofilum as axonemal assembly begins. Each clump acts as a microtubule-organizing centre (MTOC) in so far as it is associated spatially with the assembly of micro-tubules for a single axoneme. The dense material spreads along the sides of a developingaxoneme for several (im, where it probably promotes further microtubule assembly as thedouble-spiral pattern is established. Pattern is generated as microtubules that are randomlyoriented to begin with become more closely juxtaposed and aligned with each other. Thereare indications that juxtaposition is brought about by the contractile action of a filamentousmeshwork that interconnects the microtubules. Final positioning and alignment appears tobe accomplished by a ' zippering' together of adjacent portions of microtubules that proceedsin both directions along the lengths of developing axonemes as self-linkage is effected.

Considerable numbers of more or less radially oriented microtubules remain and projectfrom the surface membrane of the single central nucleus during cold treatment of A. sol.Additional tubules assemble and become associated similarly with the nuclear envelopeimmediately after cold treatment. Initially these microtubules are not arranged in a double-spiral pattern, which is subsequently generated by procedures similar to those outlined abovefor E. nucleofilum. It is suggested that the surface of the nuclear envelope may act as anMTOC.

INTRODUCTIONThere are two basic ways in which microtubule patterns are established during

the assembly of highly ordered microtubule bundles (Tucker, 1977, 1979)- Patternmay be established by a highly ordered array of interconnected microtubule-nucleatingelements that acts as a microtubule-nucleating template so that microtubules aresituated in a specific pattern as their assembly begins. This is the case during assemblyof centrioles and basal bodies (see Tamm & Tamm, 1980), and certain cytopharyngealmicrotubule bundles (Tucker, Dunn & Pattisson, 1975; Pearson & Tucker, 1977).Alternatively, pattern may be generated by a self-linkage procedure in which micro-tubules are not positioned precisely when they start to assemble. Pattern is establishedas intertubule links join microtubules together.

26o J. C. R. Jones andj. B. Tucker

The axopodial axonemes of actinophryidian heliozoans exhibit one of the mostcomplex microtubule patterns so far described (MacDonald & Kitching, 1967;Tilney & Byers, 1969; Roth, Pihlaja & Shigenaka, 1970; Ockleford & Tucker, 1973;Cachon & Cachon, 1974). The 12-sectored double-spiral pattern is considerablymore elaborate than the cylindrical arrangement of centriole microtubules or thehexagonal microtubule lattice in cytopharyngeal bundles. The double-spiral latticeof microtubules and links represents a non-equivalent pattern (Tucker, 1977) ofmicrotubule packing and linkage. Microtubules at certain loci in the pattern are linkedand positioned with respect to their immediate neighbours in a different way fromthose situated elsewhere in the pattern. Nevertheless, examination of axonemesreassembling after microtubule breakdown that has been induced by cold treatmentindicates that the double-spiral pattern is generated by self-linkage (Tilney & Byers,1969). However, serial cross-sectioning of the first-formed basal portions of develop-ing axonemes has never been undertaken. The central portion of the double-spiralis the most complex part of the pattern and is constructed before the more peripheralregions. The possibility that it is established by a pre-existing template that 'starts-off' the double-spiral pattern has not been excluded. Other important questions alsoremain unanswered. Is assembly of axonemal microtubules initiated in discrete cyto-plasmic localities by microtubule-organizing centres (MTOCs) so that microtubulesare in fairly close proximity to begin with ? There is no evidence for such MTOCsin these particular organisms to date. How do an axoneme's microtubules becomeclosely juxtaposed alongside each other so that self-linkage can be effected if a tem-plate is not employed? These issues are dealt with in this spatio-temporal analysis ofaxonemal assembly for 2 species of actinophryidian heliozoans.

MATERIALS AND METHODS

Culture

Echinosphaerium nucleofilum and Actinophrys sol were obtained from the Culture Centre ofAlgae and Protozoa, 36 Storey's Way, Cambridge CB3 oDT, England (reference nos. 1507/1 and1502/2, respectively). They were cultured at room temperature (20 °C) as described byOckleford & Tucker (1973), except that 1 ml of an aqueous solution of disodium EDTA(0-29 g I"1) was added to each litre of culture medium for E. nucleofilum. All organismsexamined were taken from cultures approximately 6 days after subculturing so that noneof the organisms was packed with freshly formed food vacuoles (this occurs shortly aftersubculturing) or had been subjected to prolonged starvation.

Light microscopy

Living organisms were examined using a Zeiss (Oberkochen Ltd) Stereomicroscope IIIfitted with a transilluminator or a Universal Microscope fitted with differential interferencecontrast optics. For high-resolution examinations a coverslip was supported by a ring ofsilicone grease that surrounded a drop of culture medium and organisms. This drop formed acolumn between slide and slip, and its sides did not contact the grease ring. Organisms survivein a healthy state for at least 24 h in these preparations, which are referred to as ring-preparationsin the account that follows. The lengths of axopodia and diameters of cell bodies were assessedusing an eyepiece micrometer.

Axonemal assembly 261

Electron microscopy

The procedure devised by Roth, Pihlaja & Shigenaka (1970) was used during preparationof organisms for electron microscopy. Fixatives were all at 20 °C prior to addition to culturemedium and organisms (even if the latter had been cooled), because this provided betterpreservation than when the temperature of the fixative was lowered to that of the culturemedium. When cold-treated organisms were fixed, a duplicate control preparation (of organismsfrom the same culture as those destined for fixation and contained in identical watchglasses)was always cooled simultaneously and examined as it warmed at room temperature, shortlyafter the other organisms were fixed, to ascertain that more than 90 % of the organisms in thecontrol preparation had survived the cold treatment and retained the ability to form new axo-podia. This was undertaken to minimize the possibility that organisms were in a moribundstate and/or potentially unable to generate new axonemes and axopodia when fixed.

Cold treatment

Organisms and culture medium contained in watchglasses or ring-preparations were cooledby placing them on ice-chips in a polystyrene container or by using a cooled incubator (setat — 2 CC). The rise in temperature of the culture medium after removal from the ice-containeror incubator to room temperature was monitored using a thermocouple as described byOckleford & Tucker (1973).

E. nucleofilum was cooled to o °C for 6 h. The organisms did not usually survive cooling tolower temperatures or being kept for more than 6 h at o °C. Hence the o °C/6 h regime wasthe most severe form of low-temperature treatment available. Over 90 % of the organismssurvive this treatment. For A. sol the most severe cold treatment giving at least 90 % survivalwas also used; organisms were cooled to — 2 °C for 17 h.

RESULTS

E. nucleofilum

Three untreated organisms, 3 organisms fixed immediately after 6 h cold treatmentat o °C, and 3 organisms fixed 7 min after termination of this treatment when thetemperature had risen to 10 °C were examined ultrastructurally.

Untreated organisms. The spherical multinucleate cell body is highly vacuolatedand consists of 2 distinct cytoplasmic regions (Fig. 1, arrows). Most of the vacuolesin the centrally situated endoplasm are much smaller than those in the peripheralectoplasm. Nuclei are distributed more or less evenly in a single layer around theecto-endoplasmic border. Axonemes terminate proximally at this level. In somecases the base of an axoneme appears to make contact with the surface of a nucleus(Tilney & Porter, 1965; Roth & Shigenaka, 1970); most bases do not but are usuallysituated within 1 -5 fim of the nearest nucleus.

Sections of axonemes close to their basal extremities (within 1-2 /tm) reveal thepresence of small patches of apparently amorphous dense material positioned alongthe sides of axonemes. This material appears to make contact with the walls of someof the microtubules at the peripheries of axonemal cross-sectional profiles (Fig. 3,arrows). Whether these patches are spatially discrete entities or, alternatively, rep-resent interconnected portions of a loose anastomosing network has not been ascer-tained. Such patches are also apparent in a micrograph (Fig. 9, Roth & Shigenaka,

262 J. C. R. Jones andj. B. Tucker


1970) but did not attract comment. They are distinct from the less substantialdense material clustered at the basal tips of axonemal microtubules (Tilney & Porter,1965; Roth & Shigenaka, 1970).

The cross-sectional profile of a mature axoneme consists of 2 main interlockingmicrotubule spirals (Fig. 3). In some instances cross-sectional profiles of axonemeson opposite sides of an individual were examined because Tilney & Porter (1965)suggested that such axonemes might have opposite spiral handedness and that thismight be related 10 axopodial activity during locomotion. All spirals followed anti-clockwise courses (progressing from centre to periphery of a cross-sectional profile)for profiles viewed distally (looking from tip to base of an axoneme). This is alsothe case for mature axonemes in A. sol and for developing axonemes shortly aftercold treatment in both organisms. There are no micrographs published showingaxonemes of opposite hand in the cell bodies of these organisms. However, Tilney& Porter (1965) have shown for E. nucleofilum that a few axopodia contain a pair ofoppositely spiralling axonemes. Such axopodia might have bent over on themselvesand portions proximal and distal to a bend might have fused together (during orjust prior to initial fixation) so that the same axoneme passes through a section twiceand with opposite polarities.

Organisms fixed immediately after cold treatment. Axopodia retract completelyduring cold treatment (Tilney & Porter, 1967) but numerous short pointed pro-tuberances that are not present usually project from the surfaces of cell bodies(Fig. 2). The ectoplasm contains considerable numbers of macrotubules with diametersof about 40 nm (Tilney & Porter, 1967; Toyohara, Shigenaka & Mohri, 1978). Mostof these appear to be arranged randomly but inside the protuberances the majorityare oriented along the lengths of the protuberances. Hence, these macrotubules mayhelp to maintain the elongated (about 10 /tm long) shapes of the protuberances.

Organisms still contain a few double-spiral microtubule arrays near the endo-ectoplasmic border. This is in contrast to the apparent lack of any double-spiralarrays after cooling to o °C for 3 h, as reported previously (Tilney & Byers, 1969).These axonemal remnants are very much shorter, far less numerous, and includeconsiderably fewer microtubules (only the central portion of the double-spiral

Figs. 1,2. These 2 micrographs each show one half of a living organism {E. nucleofilum)and have been closely juxtaposed to show changes in appearance induced by coldtreatment. Two different organisms with cell bodies of slightly different sizea areshown. Magnifications have been adjusted so that their cell surfaces are in registerto facilitate comparison.

Fig. 1. Numerous axopodia radiate from the surface of an untreated organism. Theposition of the ecto-endoplasmic border is indicated by the arrows. Interferencecontrast, x 690.

Fig. 2. Relatively short pointed protuberances project from the surface of anorganism that had been cooled to o °C for 6 h. Interference contrast, x 475.Fig. 3. Cross-section of an axoneme cut close to its base in an untreated E. nucleofilumshowing the 2 main microtubule spirals of the double-spiral microtubule array andpatches of dense material (arrows) attached to the periphery of the array, x 116000.


pattern) than the mature axonemes of untreated organisms (compare Figs. 3, 5).Dense material is concentrated around their sides at certain points along theirlengths. This material is similar in appearance to that of the dense patches at thebases of mature axonemes and to that which forms small, approximately spherical,clumps (o'2-o-4/im in diameter) that are not associated with double-spiral micro-tubule arrays immediately after cold treatment. Each of these clumps is situatedusually within 1-2 fim of a nucleus and clumps are encountered with about thesame frequency in sections near the ecto-endoplasmic border as the bases of matureaxonemes in untreated organisms. The spacing of clumps (and axonemal bases inuntreated organisms) corresponds closely to that of nuclei. Microtubules projectat an apparently random variety of orientations from the surfaces of the clumps(Fig. 6). Apart from these microtubules and the axonemal remnants no other groupingsof microtubules were detected. A few microtubules are sparsely distributed amongthe macrotubules in the ectoplasm.

Axopodial outgrowth after cold treatment. Small axopodia (5-10 /im long) are firstdetectable at the surfaces of organisms contained in ring-preparations examinedusing high-resolution interference contrast optics about 8 min after removal fromthe cold, when preparations have warmed to 20 °C. Prior to this point it is difficultto distinguish projections that may represent axopodia from the pointed protuberancesthat are still present. However, the fixation procedure for electron microscopy requiresthat organisms are cooled in 5 ml of culture medium contained in watchglasses.Warming after removal to room temperature proceeds more slowly than it does forring-preparations, which reach 20 °C after 4 min, and recovery of axopodia has tobe monitored with a dissecting microscope that provides less resolution than inter-ference contrast optics. Under these conditions, axopodia (which probably havelengths of 15-40/im) are first detectable 12 min after removal from the cold, whenwatchglasses and their contents have warmed to 13-5 °C. Organisms were fixed forelectron microscopy 7 min after removal from the cold, when the temperature hadrisen to 10 °C. These organisms contain a range of stages in the assembly of newaxonemes.

Organisms fixed ) min after cold treatment. The frequency with which double-spiral axonemal microtubule arrays were encountered in sections cut through theectoplasm was considerably greater than for organisms fixed immediately after coldtreatment. They all include fewer microtubules than those of untreated organismsand exhibit the patterns found at the centres of mature axonemes. Most of them aremore or less radially oriented with respect to the spherical cell body. No indications

Fig. 4. Cross-section through a surface protuberance of an organism fixed 7 minafter the termination of cold treatment. Macrotubules surround an array of micro-tubules (arrows) that has a double-spiral-like arrangement. E. vucleofilum. x 96000.Fig. 5. Cross-section of a small axonemal remnant in an organism fixed immediatelyafter cold treatment. E. nucleofilum. x 119000.Fig. 6. Microtubules radiating from the surface of an MTOC clump in an organismfixed immediately after cold treatment. E. nucleofilum. x 107000.

266 J. C. R. Jones and J. B. Tucker

were obtained of the way in which this orientation is accomplished. A few of thedeveloping axonemes project into the macrotubule-containing surface protuberances(Fig. 4). Examination of ring-preparations reveals that new axopodia often extendfrom the tips of protuberances during recovery from cold treatment.

Clumps of dense material are still present near nuclei. They are considerablylarger, more irregularly shaped, and are associated with more microtubules thanthose in organisms fixed immediately after cold treatment (compare Figs. 6, 7).

Fig. 7, Microtubules radiating from the surface of an MTOC clump in an organismfixed 7 min after cold treatment. E. nucleofilum. x 83000.

Serial sectioning revealed 2 main types of clumps. Some only have microtubulesradiating in apparently random directions from their surfaces (Fig. 7). The densematerial of the others, in addition to exhibiting this type of microtubule association,extends around and along the sides of small groupings of well-aligned microtubules(Fig. 8), which when cross-sectioned exhibit a packing pattern identical with thatat the centre of a double-spiral pattern (Fig. 9). Whether the clumps associatedwith an individual developing axoneme are all interconnected by thinner strips andstrands of dense material has not been ascertained. The clumped material extendsfor up to 5 fim along the sides of the basal portions of these developing axonemes.


8

Fig. 8. Longitudinal section through a portion of a microtubular axoneme duringits morphogenesis showing other microtubules projecting from MTOC clumpssituated along the sides of the axoneme in an organism fixed 7 min after cold treat-ment. E. nucleofilum. x 63000.


11


The tips of irregularly arranged microtubules that surround each axoneme areembedded in the clumps. These microtubules splay out from the clumps at a varietyof orientations with respect to the longitudinal axis of a developing axoneme (Fig. 8).

Individual developing axonemes can occasionally be recognized and followed inserial sections for distances of up to 25 fim along their lengths. They can be distin-guished because of the extent to which assembly of the double-spiral pattern hasprogressed. The organization of 3 axonemes in 2 organisms was examined in thisway. They showed that the developing double-spiral array includes more microtubulesclose to its base than it does at more distal levels (Figs. 10-12). However, some lociin the pattern that are occupied by microtubules at distal levels are not so occupiedat more basal levels (Figs. 10-12, short arrows). Thus portions of tubules are addedto the pattern at both distal and basal levels, although the former is more common.Such addition might be achieved by microtubule elongation as microtubule assemblyprogresses or, alternatively, because portions of tubules splay out from the patternas indicated in Fig. 13 and are subsequently drawn into the pattern. Some of themicrotubule profiles that are grouped around and well-aligned with developingdouble-spiral arrays may represent such portions (Figs. 8, 11), but in no case wasit possible to demonstrate unequivocally that tubules providing such profiles joinedthe pattern. It is difficult to follow these tubules in sequential sections because theyare not positioned very regularly and oriented with respect to each other and thedouble-spiral array. Because such tubule portions are much less numerous in thevicinity of mature axonemes, and because it is difficult to see how they could becomeprecisely positioned simultaneously at all points along their lengths, it is reasonableto suppose that microtubules are incorporated progressively into the pattern in bothdistal and proximal directions (Fig. 13). If this is not the case then these tubulesmust be disassembled at some point and can have little direct relevance to axonemalassembly. This possibility seems most unlikely, bearing in mind the large numbersof microtubules involved (Fig. 8). Fine strands of dense material that are situated

Fig. 9. Cross-section of a microtubular axoneme during its morphogenesis showingother microtubules radiating out from MTOC clumps positioned around the sidesof the axonemal array in an organism fixed 7 min after cold treatment. E. nucleofilum.x 74000.Figs. 10-12. A sequence of cross-sections at progressively more distal levels throughan axoneme fixed during its morphogenesis in an organism 7 min after cold treat-ment. The row of microtubules situated between the short arrows includes moremicrotubules at more distal levels than it does at more proximal levels, but the rowsituated between the long arrows near the base of the axoneme is not present at moredistal levels. E. nucleofilum. All x 189000.

Fig. 10. Cross-section cut close to the base of the axoneme near the ecto-endoplasmicborder about 40 fim below the surface of the cell body. Fine dense strands (s) aresituated between some of the microtubules positioned around the axonemal array.

Fig. 11. Cross-section at a level 20 fim distal to that of Fig. 10.Fig. 12. Cross-section 5 /'m distal to that of Fig. 11.


between these tubules and those in double-spiral arrays (Fig. 10, s) may connectmicrotubules together (Fig. 13).

Although the central portion of the double-spiral is constructed before its peri-phery, the 2 spirals are not only produced by addition of microtubules one-by-oneto the most peripheral end of each spiral. Portions of spirals can be built up at(apparently) any region around the edge of a developing double-spiral array (Fig. 10).

Fig. 13. Schematic diagram showing the probable sequence of events in E. nucleofilumas axonemes start to assemble after cold treatment. Shortly after cold treatmentmicrotubules begin to grow out from dense MTOC clumps in increasing numbers.These tubules become interconnected by fine strands (wavy lines) that may facilitatetubule alignment and juxtaposition. As axonemes assemble tubules are incorporatedinto the double-spiral pattern at several points along its length. Such tubules maysplay out from the pattern and be zipped-up in both proximal and distal directionsas self-linkage proceeds. A supply of microtubules appears to be maintained forsome time as the assembly of new ones is nucleated by MTOC clumps that spreadalong the sides of the basal portions of developing axonemes.

A. sol

Three untreated organisms, 3 organisms fixed immediately after 17 h cold treat-ment at — 2 °C, and several organisms fixed at intervals after this treatment (3 organ-isms at 2 min, 12 min and 16 min; 2 organisms at 5, 6-5 and 10 min; 1 organism at15 min) were examined ultrastructurally.

Untreated organisms. Axonemes radiate from the outer surface of the singlecentral nucleus. The basal portions of their microtubules terminate in lateral registerwith one another where they contact the nuclear envelope (Fig. 14). The double-


spiral pattern is apparently identical with that of E. nucleofilum but the largestaxonemes in the cell body and proximal portions of axopodia include fewer micro-tubules than those of E. nucleofilum (Ockleford & Tucker, 1973; Patterson, 1979).No patches of dense material were detected at axonemal bases.

Organisms fixed immediately after cold treatment. A few short axopodia remainafter cold treatment. They contain microtubules aligned parallel to their longitudinalaxes but these are not arranged in a double-spiral pattern. A few sparsely distributedmacrotubules with diameters of about 40 run are also present in the ectoplasm nearthe surface of the cell body. Numerous microtubules project from the outer surfaceof the nuclear envelope. Unlike the microtubules of mature axonemes, these micro-tubules are not well-aligned with their neighbours, nor are they grouped in compactarrays and confined to certain surface regions of the envelope (compare Figs. 14, 15).They are distributed more or less evenly over the entire surface of the envelope.None of these microtubules are arranged in a double-spiral pattern. The proximalends of most (perhaps all) of them are attached to the nuclear envelope.

Organisms fixed 12 min after cold treatment. Examination of ring-preparationsreveals that new axopodia start to extend from cell bodies 12 min after removalfrom the cold. These preparations reach room temperature (20 °C) 4 min after suchremoval but organisms cooled in watchglasses for fixation have only warmed to10 °C after 12 min.

Greater numbers of microtubules radiate from the surface of the nuclear envelope,have a more precise radial orientation, are more exactly aligned with their neighbours,and are less evenly distributed where they contact the envelope than the juxtanuclearmicrotubules, in organisms fixed immediately after cold treatment (compare Figs. 15,16). Many of them are concentrated together to form fairly compact bundles. Withinbundles they are not packed in well-defined patterns except within 2-3 fim of thenuclear envelope where some of them are arranged as short slightly curved rows oftubules. Some of these rows are grouped in pairs in configurations closely approxi-mating to those at the centre of the double-spiral axonemal pattern (Fig. 17). Serialsectioning did not reveal any configurations that were exactly identical with thecentral portion of a mature double-spiral; presumably they represent stages in theself-linkage of microtubules to form axonemal centres. They were not detectedin organisms fixed 2, 5, 6-5 or 10 min after cold treatment. Fine strands of densematerial that may interconnect tubules are sometimes situated within these double-spiral groupings (Fig. 17).

Organisms fixed 16 min after cold treatment. Numerous groupings of radiallyoriented microtubules are present in the vicinity of the nucleus in these organisms.Many of these groupings include the central portions of the double-spiral arrays ofdeveloping axonemes (Figs. 18, 19). The spatial organization of the basal portionsof 4 developing axonemes that could be followed in sequential sections through2 organisms was examined. More microtubules were found at basal levels than atmore distal levels and this provided one instance in which a row of microtubulessplays out distally from an axonemal centre (compare Figs. 18, 19, arrows). Noexamples were found of the absence of microtubules from loci at basal levels in


these arrays that are occupied by microtubules at more distal levels. It is perhapsto be expected that this feature would be less common than it is in E. nucleofilumbecause the microtubules are initially most closely juxtaposed at a single level (thesurface of the nuclear envelope), so that pattern generation is most likely to beginat tubule bases. The spatio-temporal sequence of events as axonemes start to assembleis summarized in Fig. 20. No clumps of dense material were detected at the bases ofdeveloping axonemes.

DISCUSSION

Nuclei, MTOCs and microtubule nucleation

The double-spiral pattern is rapidly established after cold treatment, within 7 and16 min for E. nucleofilum and A. sol, respectively. In both organisms this appears tobe facilitated by nucleation of microtubule assembly in highly localized juxtanuclearcytoplasmic regions.

In E. nucleofilum clumps of dense material act as MTOCs. They apparentlynucleate microtubule assembly, because microtubules grow out from them. Con-tinuation of a highly localized supply of microtubules as axonemes assemble seemsto be achieved because clumps spread along the sides of the first-formed portionsof axonemes. The clumps appear to do little in the way of ' organizing' apart fromensuring that microtubules are concentrated in a number of discrete cytoplasmicregions. The dense patches at the base of each mature axoneme of untreatedorganisms probably represent remnants of an MTOC clump. They may providethe material basis for maintenance of axoneme number and initiation of an activeMTOC following situations such as exposure to low temperatures in which mostaxonemes are completely disassembled.

MTOC clumps are situated close to nuclei although direct structural connectionbetween them was not detected. This raises the question of whether there is a precisenumerical correlation between nuclei, MTOCs, axonemes and axopodia. Possibly,cell size, nuclear number and the number of axopodia 9tay in register during inter-fission growth because each nucleus is involved in the maintenance of one axonemal

Fig. 14. Longitudinal section through the base of an axoneme where it makescontact with the nuclear envelope in an untreated organism. A. sol. x 51000.Fig. 15. More or less radially oriented tubules close to the outer surface of thespherical nucleus in an organism fixed immediately after cold treatment. The basalends of some of the tubules make contact with the envelope. A. sol. x 51000.Fig. 16. Numerous radially oriented microtubules contact the surface of most portionsof the nuclear envelope in an organism fixed 12 min after cold treatment. A. sol.x 51000.

Fig. 17. Cross-section of radially oriented microtubules in an organism fixed 12 minafter cold treatment. A group of tubules is packed in an arrangement that closelyapproximates to that at the centre of an axonemal double-spiral array. A strand ofdense material is situated within this grouping. A. sol. x 330000.


19


Fig. 20. Schematic diagram showing the probable sequence of events in A. sol asaxonemes start to assemble after cold treatment. Microtubules assemble near thenuclear envelope (towards the bottom of the figure) and are interconnected by finestrands (wavy lines) that may facilitate more exact radial arrangement and juxtapositionof tubules so that self-linkage into a double-spiral array can proceed. This mainlybegins near the bases of tubules so that the more distal portions of such tubulesmay splay out from the pattern as indicated until they are 'zipped-up' into thepattern as self-linkage proceeds distally to interconnect tubules along their entirelengths.

Fig. 18. Cross-section of radially oriented microtubules cut at a level approximatelyi /tm distal to that at which their bases make contact with the nuclear envelope inan organism fixed 16 min after cold treatment. Many of the tubules have beenincluded in the central portions of double-spiral axonemal arrays. The curved rowof tubules situated between the arrows includes more tubules than it does at moredistal levels (compare Fig. 19, arrows). A. sol. x 125000.Fig. 19. Axonemal arrays included in Fig. 18 cut in cross-section at a more (3 /tm)distal level. They all include fewer microtubules than they do at the more proximallevel. The row of tubules situated between the arrows splays out distally from thecentre of a double-spiral array (compare Fig. 18, arrows), x 125000.


MTOC. Under the culture conditions employed in this study the basal portionsof all axonemes of untreated organisms exhibited cross-sectional profiles of similarsize, included similar numbers of microtubules, and their spacing correspondedclosely to that of the nuclei. The majority (perhaps all) of these axonemes extendedinto axopodia and each axopodium included a single axoneme. However, this situationmay not be typical. It is clear from the micrographs of Tilney (1968), Allison et al.(1970), Roth & Shigenaka (1970) and Roth, Pihlaja & Shigenaka (1970) that, some-times, under both normal and experimental conditions considerably more than 2axonemal bases are positioned near each nucleus. In these instances axonemes varymarkedly in cross-sectional size and it is possible that, as in A. sol (Ockleford &Tucker, 1973), many of the smaller axonemes are confined to the cell body anddo not extend into axopodia.

E. nucleofilum apparently stores much of its tubulin in the form of macrotubulartubulin aggregates during cold treatment. It has been suggested that macrotubulesmay convert directly to microtubules by rearrangement of protofilaments duringrecovery from cold treatment (Tilney & Porter, 1967; Toyohara, Shigenaka & Mohri,1978) rather than dissociating completely into soluble tubulins, which then reassembleto form microtubules. There is direct evidence for such conversions (Suzaki, Toyo-hara, Watanabe, Shigenaka & Sakai, 1980). The concentration of microtubules atthe surfaces of MTOC clumps while macrotubules remain scattered throughout theectoplasm indicates that, at least initially, axonemal microtubules assemble fromsoluble tubulin.

The outer surface of the nuclear envelope of A. sol may act as an MTOC. Aftercold treatment microtubules assemble in its immediate vicinity. Microtubule-nucleating elements might be firmly bound to, and oriented at, the surface of theenvelope so that microtubules are radially oriented alongside each other as theystart to assemble. This would facilitate subsequent production of the parallel, moreclosely packed, array of axonemal microtubules in axonemes. However, the possi-bility that assembly is initiated close to, but not in contact with, the envelope and isfollowed by attachment of tubule tips to the envelope has not been eliminated inthis study. In either case the nuclear surface clearly plays some part in organizingthe microtubules. If attachment and/or nucleation of tubule tips is related to tubulepolarity, the association will define the polarity of the axoneme as well as its radialorientation. Tip attachment to the outer membrane of the envelope presumablymust be such that tips can move in the plane of the membrane. If this were notthe case most of the tubules could not be included in the compact double-spiralpattern that is finally established right down to the level at which tubules contactthe membrane.

In A. sol there does not seem to be any nucleus-mediated specification of axonemalnumber. Large numbers of axonemes start to assemble near the nucleus. They arefinally of very variable cross-sectional size (consisting of correspondingly variablenumbers of microtubules). Only the largest project into axonemes (Ockleford &Tucker, 1973). Thus it appears that production of central axonemal portions is notunder strict numerical control, that they may 'compete' for 'free' microtubules on


a 'first come, first served' basis in which the 'large get larger' because they havemore peripheral sites available for self-linking free microtubules, and that only thoseabove a certain critical size continue to elongate and promote outgrowth of axopodia.

Starting the double-spiral pattern

The central portion of the double-spiral pattern is more complex in terms ofmicrotubule packing and linkage than its periphery (MacDonald & Kitching, 1967;Tilney & Byers, 1969; Harris, 1970; Cachon & Cachon, 1974). Establishing thecentral portion may require more detailed spatial instructions than are needed toextend the 2 spirals once the central portion has been constructed. Bearing in mindthe speed with which central portions are established (7-16 min) and their intricacy,it is not unreasonable to consider whether the double-spiral pattern is initiated bya pre-existing microtubule-nucleating template. It has been suggested that a templateis not involved in E. nucleofilum because sections of developing axonemes revealmicrotubule configurations that appear to represent stages in the self-linkage andpositioning of microtubules to form the central portions of double-spiral arrays(Tilney & Byers, 1969). However, the present study (see below) and that conductedon Raphidiophrys (Tilney, 1971) show that in developing axonemes pattern can beestablished at one level in the microtubule bundle prior to its appearance at another.Patterning is apparently achieved as adjacent microtubules are 'zipped' together bya self-linkage procedure at progressively more distant levels from those at whichpattern is first established. Hence, even if stages in pattern establishment are detectedat a particular level in an axoneme they may not represent the initial establishmentof pattern. Examination of cross-sectioned axonemes at all levels during patternestablishment is needed to eliminate the possibility of template specification ofmicrotubule positioning.

Such examinations in the present study show unequivocally that the positions ofmicrotubules at the periphery of the double-spiral pattern are not template-specifiedbecause these tubules join the patterned array at several levels as axonemes assemble,and in E, nucleofilum most microtubules in the vicinity of the axonemal MTOCclumps are not precisely positioned when they start to assemble. Stages in theinitial positioning of microtubules at the centre of a double-spiral were not foundin E. nucleofilum but may have been obscured by the dense MTOC material. Cross-sectional profiles that appear to represent such stages were obtained for A. sol. Thesemicrotubule groupings did not exhibit perfect double-spiral microtubule positioningat any points along their lengths. Thus, the entire double-spiral pattern is probablyestablished without the exploitation of a microtubule-nucleating template. Lack ofprecise positioning when microtubules start to assemble also eliminates 'linker-nucleation' (Bardele, 1977) as the mechanism for generating pattern.

The 'template controversy' (Tilney, 1971) includes the centrohelidian Raphidiophryswhere the axonemal microtubule pattern is less complex than it is in an actino-phryidian double-spiral. In Raphidiophrys a single dense body called the centroplastnucleates axonemal microtubule assembly, but pattern is established initially at


some distance from its surface and so, presumably, it does not act as a template andself-linkage generates pattern (Tilney, 1971). However, Rieder (1979) demonstratedintertubular rod-like components where axonemes emanate from the centroplast andsuggested that they form part of a template that establishes axonemal microtubulepattern.

Self-linkage

Neighbouring microtubules need to be aligned and situated within a ' link's length'of each other before self-linkage can proceed. Neither of these conditions initiallyobtain to any great extent as microtubules start to assemble in E. nucleofilum aftercold treatment. In A. sol some of the microtubules are fairly well-aligned. High-resolution light microscopy does not reveal any detectable Brownian movement inthe cytoplasm (excluding the vacuoles) of these 2 organisms. The degree of cyto-plasmic gelation is probably such that microtubules need to approach each otherwith the assistance of some agency other than random diffusion if self-linkage andpattern formation are to be effected as rapidly as observed. Fine strands are situatedbetween microtubules during pattern formation. These might form part of a con-tractile meshwork that draws microtubules together to facilitate self-linkage. Thefilaments in such a meshwork would not necessarily need to have a very well organizedspatial arrangement to do this. Provided some portions of some microtubules aredrawn to within a link's length of each other, self-linkage of those that are alignedcould begin. It may not be necessary to align and position microtubules preciselyalong their entire lengths before self-linkage can start. Once short portions of 2adjacent microtubules are connected by links, regions immediately 'above and below'these portions would be more suitably positioned for self-linkage. Thus self-linkagecould proceed progressively along tubules moving away from the initially linkedportions and by a 'zipper-like action' draw the flexible microtubules (Ockleford &Tucker, 1973) together along their entire lengths eventually. The evidence that someof the microtubules splay out from the patterned array as axonemes assemble is com-patible with such a procedure, which is similar in some respects to that envisaged in the'zipper hypothesis' for microtubule repositioning in certain spindles (Bajer, 1973).

Serial sectioning reveals that microtubules are incorporated into the double-spiral pattern at various levels, proceeding both proximally and distally alongdeveloping axonemes in E. nucleofilum. Hence it is difficult to account for patterngeneration in the entire axoneme unless individual tubules can be zipped togetherin both directions. It is not known if all tubules in an axoneme have the samepolarity. The distal migration of bends (produced by manipulation with a micro-needle) during axonemal repair in A. sol (Ockleford & Tucker, 1973) suggests thatall the microtubules do have the same polarity and may be an indication that self-linkage proceeds more rapidly in a distal than in a proximal direction.

The ways in which various microtubule/link interactions might establish the12-sectored double-spiral pattern during self-linkage have been considered in detailby previous investigators (Tilney & Byers, 1969; Roth, Pihlaja & Shigenaka, 1970;


Cachon, Cachon, Febvre-Chevalier & Febvre, 1973; Cachon & Cachon, 1974;Bardele, 1977).

J.C. R.J. held a Science Research Council (U.K.) Studentship during this study and grantsupport from the S.R.C. to J.B.T. is gratefully acknowledged.

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{Received 20 November 1980)

Note added in proofIt has recently been shown that most of the microtubules (more than 98%) in the

axonemes of E. nucleofilum have the same polarity.

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microtubule-organizing centres and assembly of the … · survive this treatment. fo solr a. the...

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