Ann. Rev. Plant Physiol 1982. 33:651-98 Copyright @ 1982 by Annual Reviews Inc. All rights reserved

MICROTUBULES

Brian E. S. Gunning and Adrienne R. Hardham

Department of Developmental Biology, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra City, ACT 2601, Australia

CONTENTS

INTRODUCTION ...... ....... ............ . . ..... . . . . . ....... . . . ...... . . . . . . . . . ......... .......... ........ ........ . . .. .. . TUBULIN ..................................................................................................................... .

Isolation from Plant Material ................................................................................. .

Forms of Tubulin ..................................................................................................... .

Synthesis ................................................................................................................... .

Interactions with Other Molecules ........................................................................... .

Endogenous cofactors ....................•.......•............••...................................................

Drugs ................................................................................................................... . Microtubule-associated proteins ............................................................................... .

MICROTUBULES .... .. ......... .. .. ..... ..... .. . . ............................ . . . . . .... . . . . ............................. .

Structure, Assembly, and Polarity ........................................................................... .

Arrays in Wall-less Cells ......................................................................................... .

Movement of Organelles ........................................................................................... .

The Preprophase Band ............................................................................................. . Interphase Cortical Arrays in Walled Cells. .......................................................... ..

Nature and development ......................................................................................... .

Coalignment of microtubules and wall microjibrils .................................................... ..

Possible Orientation Mechanisms ............................................................................. . CONCLUDING COMMENTS ................................. .................................................. .

INTRODUCTION

651 652 653 654 655 655 656 657 660 662 662 666 667 670 672 672 676 680 683

Microtubules (MTs) have been considered twice before in this series. The first review, in 1969 (207), covered extensive but largely descriptive work stemming from the discovery that they are preserved in cells of higher plants by fixation with glutaraldehyde (167). Their major constituent pro­tein, tubulin, had been isolated, and by the time of the second review, in 1974 (121), the subject had entered a very rapid growth phase following publication of methods for polymerizing tubulin in vitro (17, 332). Since then many reviews have appeared, including several devoted to plants (64, 107, 118, 228, 264a). International symposia on MTs were held in 1974

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652 GUNNING & HARDHAM

(298), 1975 (16), and 1980 (47); books on MTs appeared in 1978 (56), 1979 (252), and 1981 (153); another volume in which plant MTs figure largely is in preparation (169).

Why do microscopic tubes composed of one major and some other asso­ciated proteins evoke such interest? The answer remains much as in 1969 and 1974. MTs are morphogenetic tools in eukaryotic cells. In plants they participate in regulating cell shaping and specifying the site and plane of cell division as well as spacing components within the cell. Knowledge of MTs and tubulin in plants has progressed relatively slowly; hence the present review, while in general botanically focused, includes material from the literature of cell biology that either is, or is likely to be, relevant to plant cells. Spindle MTs and their role in mitosis are not covered.

TUBULIN

The major structural unit of MTs is the protein tubulin, a dimer (MW 110,000) composed of a- and ,B-subunits of almost equal molecular weight (172). It accounts for about 3% of the total protein of fibroblasts [which, if all polymerized, would be equivalent to 16 mm of formed MT per cell (128)], up to 42% of the protein of brain (see 172), about 1 % of the protein of young Vigna epicotyls [extrapolation of data in (194)] and 0.2-0.4% of Aspergillus nidulans protein (46). In Chlamydomonas, tubulin accounts for about 5% of the total protein synthesis over much of the cell cycle and even more after deflagellation (329). Total lengths of MTs in higher plant cells range from 500-900 p.m [282] and 3-5 mm ( l00), to more than 10 cm (284).

The proportion of tubulin that is present as MTs varies from zero to nearly 90% (233). Also, the total tubulin in the cell may be partitioned among discrete pools (see 71) that are individually replenishable and used to produce different categories of MT array. Neither the subcellular locali­

zation of tubulin nor the means of separating pools is fully known. Proce­dures normally used for immunofluorescent localization of MTs almost certainly extract most of the free tubulin. Nevertheless, marked concentra­tions of nonfibrillar fluorescence have been seen at the nuclear surface in plant cells, especially at preprophase (335). Immunoelectron microscopy using ferritin- and peroxidase-labeled antibodies and fluorescence micros­copy using a fluorescent derivative of colchicine have revealed what is thought to be free tubulin close to developing MT arrays (2, 48, 336); these methods have not yet been applied to plant cells.

Tubulin can occur in cell membranes as well as in the cytosol; indeed membrane tubulin may account for observations of colchicine-binding by membrane fractions (12, 299). In'ciliary and flagellar membranes the tubu­lin is glycosylated (unlike that of the axoneme) and accounts for about

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MICROTUBULES 653

two-thirds of the intrinsic membrane protein (1,307). Other chemical differ­ences between ciliary and axonemal tubulins have been documented (307a). Membrane tubulin is in part accessible to non penetrating reagents and, when extracted, is capable of assembling into MTs (13). Tubulin can also assemble with phosopholipid into a membrane-like form (63). Membrane tubulin may provide links between membrane and cytoskeleton, links per­haps used during MT -mediated propUlsion of membranous surfaces or organelles (see later). It could also facilitate association between chromatin and nuclear envelope: a colchicine-binding protein not unlike tubulin is present in nuclear envelopes of Lilium microsporocytes, and treatment with colchicine induces release of a DNA-binding protein (131). Another possi­ble function for membrane tubulin is in nucleating growth of cytoplasmic MTs.

Isolation from Plant Material Initial work on isolation of tubulin from animal tissues was facilitated by the fact that colchicine, which binds to the protein, can be used to monitor the course of purification. Unfortunately, colchicine binding by plant tubu­lin is less stable (see 104), although it has been detected in a range of species and tissues (105, 106, 131, 173).

Gel electrophoresis is widely used as a preliminary means to identify tubulins in extracts (e.g. 262), but care must be taken with plant material because the large subunit of ribulose bisphosphate carboxylase migrates to the same position as tubulin (235,331) [this may account for reported high yields of tubulin from Chlamydomonas (61)]. To identify tubulin convinc­ingly in the absence of reliable binding by colchicine, it is desirable to demonstrate a capacity to assemble into MTs. Until very recently technical problems prevented such demonstrations. Inhibitors of polymerization oc­cur in algal (29,30,61,302), fungal (38), protist (258-260) and higher plant (68) material. Proteases attack both tubulin and tubulin-associated proteins; leupeptin (194, 258), lactalbumin hydrolysate (197), pepstatin A and p-methylphenylsulphonyl fluoride (154a) have been used to inhibit them. Inhibition by polyanions can be reduced by treatment with nucleases (38, 197, 258). GTPases in crude extracts can destroy the GTP that is needed for polymerization and can be combated by including a GTP-generating system (258). Polymerization also requires a critical concentration of tubu­lin; to attain this, brain tubulin can be added, or glycerol (258,302) or taxol (197) (see later) used to reduce the critical concentration. Given these additions, it has proved possible to co-polymerize brain tubulin with tubulin from carrot (297), tobacco (342), yeast (38, 1 54a, 328), Physarum (258), and Aspergillus (45, 290). It is notable that putative tubulin extracted from Chlamydomonas (29, 30, 61) and Paul's Scarlet rose tissue cultures (68)

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654 GUNNING & HARDHAM

inhibits assembly of brain tubulin; whereas cell-free translation products of mRNAs from the same sources is sufficiently free of inhibitors to co­polymerize (68,329). The alga Polytomella also yields tubulin together with inhibitors; the latter can be removed by phosphocellulose chromatography. Glycerol is still required for self-assembly, but in its absence, proteins derived from flagellar rootlets initiate assembly (302). Self-assembly of yeast tubulin has also been achieved ( l54a).

As far as higher plant material is concerned, tubulin has been obtained from fern sperm flagella ( l 68a, 1 73). It has also been isolated successfully from Vigna seedlings with affinity chromatography on ethyl N-phenylcar­bamate coupled to Sepharose 4B. The product shows labile colchicine bind­ing ( 194). Chromatography of extracts from tissue culture cells on DEAE-Sephadex A50 has yielded fractions which are electrophoretically similar to brain tubulins and which assemble into MTs in the presence of the drug taxol ( 197). These important advances should open the way to systematic study of plant tubulin.

Forms of Tubulin Tubulin is not one protein, but a class of proteins. Heterogeneity of both n- and ,B-tubulin has been detected both within ( 1 4) and between (see 1 72) organisms by using criteria of electrophoretic mobility, proteolytic cleavage patterns ( 168a), amino acid sequencing ( 157a, 236a), differences in interac­tions with drugs (see later), and specificity of antibody reactions (e.g. 163, 235). On the other hand, heteropolymers form when tubulin from one source is mixed with "seeds" from another (272), and wide spectrum cross­reactivity of antibodies occurs (see 170, 1 7 1 , 335), though not all antibody preparations raised against brain tubulin react with plant cells (S. M. Wick, personal communication). The heterogeneity therefore occurs against a background of extensive conservation of sequence and structure between tubulins of organisms as divergent as plants and animals.

It is not known whether variation of tubulins is related to specific func­tions. Thus in higher plants it is formally possible that there might be different tubulins in interphase cortical, preprophase band, spindle and phragmoplast arrays of MTs, and that they could each be tailored to suit particular roles. Different forms of tubulin occur at different times and locations in axolotl embryos ( 1 95), Drosophila ( 1 5 1 ), Naegleria ( 1 63), and sea urchins ( 1 4).

Analyses using cloned cDNA for n- and .B-tubulins have shown that the genes for both are in the form of families which are dispersed along and among the chromosomes (39, 40, 266), varying in number among organ­isms. Sequencing of amino acids and DNA (31 9) has revealed 40-55% homology between n- and ,B-tubulins, suggesting that the two may have

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MICROTUBULES 655

arisen from a common ancestral sequence. a- and ,8-tubulin genes are not found in tandem (39, 198); evidently linkage has not been favored during natural selection.

Post-translational modification of tubulin can give rise to further varia­tion by phosporylation, tyrosylation, and glycosylation. For example, a­tubulin has a C-terminal tyrosine when first produced, but later only 15-50% of molecules are tyrosylated (319). Detyrosylated a-tubulin can be retyrosylated by an ATP-requiring enzyme, tubulin-tyrosine ligase (172). Post-translational diversification of tubulin could participate in specifying particular functions or subcellular locations. For example, tubulin in mem­branes is not tyrosylated (see 299) but may be glycosylated (see above). The likely necessity for some such diversification is highlighted by the fact that Chlamydomonas probably has only two a-tubulin and two ,8-tubulin genes and a corresponding number of RNAs, yet many types of MT, each with a characteristic location, time of appearance, and (presumably) function, occur in the cells (192, 294).

Synthesis mRNAs for tubulins have been isolated for a variety of cells, including Chlamydomonas (192, 294, 329), and translated in cell-free systems. The messengers are apparently short-lived [fibroblast (9, 40a), Chlamydomonas (41)], although eggs can contain sufficient tubulin mRNA prior to fertiliza­tion to cater for early postfertilization mitoses in the embryo (see 71). The extent of tubulin production reflects the amount of mRNA (163, 192), and regulation is, at least partially, imposed at the level of mRNA induction. Defiagellation in Chlamydomonas leads to induction within a few minutes, before flagellar regeneration depletes the existing pool of tubulin. The na­ture of the inducing signal is therefore mysterious (330, 331). On the other hand, repression of further mRNA production is correlated with accumula­tion of tubulin, suggestive of a feedback inhibition (9, 331). Augmenting the pool of unpolymerized tubulin by treating cells with depolymerizing agents reduces transcription of tubulin mRNAs (40a). The herbicide amiprophos methyl (see also later) selectively and reversibly destroys tubulin mRNA in Chlamydomonas (41).

Interactions with Other Molecules At least three lines of research rest heavily upon investigations of interac­tions between tubulin and other substances. First, there is the study of interactions that modulate assembly-disassembly of MTs in vitro and possi­bly in vivo. Second, functions of MT arrays are often examined by observing effects of anti-MT drugs. Third, MTs have very diverse roles considering that they have a more or less standard substructure and are made of a highly

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656 GUNNING & HARDHAM

conserved protein; studies of associations entered into by MTs may reveal the molecular basis of some of this diversity.

ENDOGENOUS COFACTORS Interactions with calcium, which can in­duce disassembly, and magnesium, which is needed for assembly, are likely to be of regulatory importance in vivo (see 172, 245 for review). There is a high-affinity binding site for calcium in the tubulin dimer, together with about 16 low-affinity sites which influence assembly-disassembly. Up to 48 or so magnesium ions can bind to each dimer. Magnesium may be involved in interactions between tubulin, GTP, and MT-associated proteins, as well as in direct binding. It also potentiates CaH-induced depolymerization. Depending upon how the tubulin is prepared and the concentration of MgH, the effect of CaH is observed at micromolar (261) or millimolar (209, 214) concentrations. The alga Oocystis loses its MTs if exposed to 2 mM Ca2+ in the presence of the calcium ionophore A23187; tenfold less Ca2+, plus A23187, has no such effect. nor does it prevent cells from rebuilding their MT array after a disruptive treatment with colchicine (240).

Crude preparations of tubulin are likely to contain calmodulin, the calci­um-dependent regulator protein of cyclic nucleotide phosphodiesterase. If present in molar excess relative to tubulin. calmodulin makes brain MTs more sensitive to CaH by several orders of magnitude (158, 208) [but not so for sea urchin spindle MTs (247)]. Immunocytological work has revealed calmodulin in association with MTs in vivo (49); moreover, anticalmodulin drugs reduce CaH-induced disassembly of MTs (276). The activity of CaH may thus be modulated by calmodulin (known to occur in plants) and/or, as discussed in detal in relation to the mitotic spindle (122), by CaH -sequestering cisternae of endoplasmic reticulum. These mechanisms, together with localized CaH currents (142) and localized pH changes [which also affect assembly-disassembly kinetics (247a)], have the potential for producing a measure of spatial control at subcellular level.

Interactions of tubulin and two other naturally occurring small molecules -GTP and glycerol-have been much studied. There is one exchangeable and one nonexchangeable GTP-binding site per dimer. The latter is open in free tubulin but is masked in MTs and in vinblastine-induced crystals, so it may be at or close to sites of tubulin-tubulin interaction. Glycerol binds to tubulin (up to 5 molecules per dimer), stabilizes assembled MTs. and lowers the critical concentration for polymerization by displacing water molecules. These conclusions derive from in vitro experiments. However, with other polyols, glycerol is also a naturally occurring "compatible so­lute," present in quantity in many salt-tolerant plants, leading to the sugges­tion that the ability of certain flagellates to swim and maintain MT cytoskeletons at temperatures down to _4° in their natural saline Antarctic

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MICROTUBULES 657

environments may stem from protection by glycerol etc against low tem­peratute-induced depolymerization (H. J. Marchant, personal communica­tion).

DRUGS Biological effects of colchicine are commonly regarded as being indicative of the participation of MTs in whatever process is being investi­gated. Unfortunately, the situation is by no means clear cut, especially where plant material is concerned (104).

There is one colchicine binding site per dimer of brain (and other animal) tubulin (see 172). Tubulin from Chlamydomonas shows enhanced colchi­cine binding in the presence of dithiothreitol (65), while that from a higher plant binds colchicine only if dithiothreitol is present (194). This condition was not met in other attempts (7, 29, 105, 106, 213a) to detect colchicine binding in extracts of higher plants, perhaps accounting for the largely negative results. Sensitivity of higher plant cells to colchicine is less than that of animals by one to three orders of magnitude (reviewed in 104), and it may be that the binding ratio is also lower, as is the case with tubulin from colchicine-insensitive mutants of Chlamydomonas (66) and the lower euka­ryote Physarum, in which low sensitivity correlates with a 50-fold lower binding ratio (259). Low colchicine (or colcemid) binding activity has also been found in the fern Marsilea (173), the algae Chlamydomonas (65, 66, 173) and Caulerpa (106), yeast (94), and the fungus Saprolegnia (111). Cell division in Colchicum, which is the natural source of colchicine, seems especially resistant (267). As with other perturbations (e.g. 145), colchicine can affect different categories of plant MT differentially, even within one cell (see 99, 101, 285).

Responses of plants to colchicine vary, and, where present, may occur only at concentrations so high as to bring nonspecific effects. Use of the drug in identifying MT-mediated phenomena is therefore hazardous (104, 150). It has been considered that lumi-colchicine, produced by UV radiation of colchicine, retains nonspecific but lacks anti-MT effects, and hence can be used as a form of control treatment. Unfortunately, this too has hazards. In assays based on seedling growth, incomplete photoconversion introduced new effects (not shown by colchicine) and complete photoconversion abol­ished all activity (264).

Vinblastine, which probably has two high-affinity binding sites per dimer of brain tubulin (reviewed in 172), affects MTs in the fern Marsilea ( 173) and the algae Micrasterias (186) and Chlamydomonas (173), but not those in the fungus Sapro/egnia, though there is evidence that it enters the cells (110). It also affects aspects of cell division in onion stomata (225). In Physarum amoebae it precipitates tubulin but the aggregates so formed tend to be obscured by much more massive precipitation of actin (258). Its

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658 GUNNING & HARDHAM

source, Vinca, is considered to be resistant as compared with Lepidium, though the two genera, both flowering plants, are equally sensitive to colchi­cine (157). As in the case of brain tubulin, vinblastine stabilizes colchicine binding in extracts of Chlamydomonas and Marsilea (173), and carrot suspension cultures (213a). Another plant product, podophyllotoxin, inhib­its the colchicine binding of Marsilea and brain tubulin ( 173); the podophyl­lotoxin and colchicine sites are considered to overlap partially (see 172). Podophyllotoxin inhibits cell division in Marsilea microspores (173) and MT -mediated nuclear migration in Micrasterias (186) and removes MTs from another alga, Oocystis (255). It therefore seems likely that binding sites in plant tubulin for vinblastine (at least where effects do occur) and podo­phyllotoxin are similar to their counterparts in the protein from brain.

Studies of herbicides and fungicides have contributed several potent com­pounds to the experimentalist's armory of anti-MT agents. Trifluralin (N,­N-dipropyl-2,6 dinitro-4-trifluoromethylaniline) removes some or all MTs from Haemanthus endosperm (141), root tip cells (7, 126), and Oocystis (255). It is also active in Micrasterias (152, 186) and, in Chlamydomonas, induces flagellar shortening (and prevents outgrowth of flagella after ampu­tation) (125, 239). Interestingly, it is reported to have little or no effect on animal cells (reviewed in 127). Some of these effects are shared with the related dimethylaniline herbicide oryzalin (7), which mimics colchicine symptoms but is active at lOOO-fold lower concentrations (316, 317). Both herbicides inhibit polymerization and induce depolymerization of thrice­recycled brain tubulin (255). The latter is disputed (6, 7, 239), the discrep­ancy perhaps arising from differences in the purity of the tubulin preparations. Trifluralin binds to tubulin from Chlamydomonas flagella central tubules (127), but oryzalin does not bind to any plant protein in the size range for tubulin, though it does associate strongly with plant cell membranes (318).

Phenyl carbamates and benzimidazole carbamates both affect MT sys­tems. IPC (isopropyl N-phenyl carbamate, prophane) and CIPC (chloriso­propyl N-phenyl carbamate, chlorprophane) are antimicrotubular in algae (20, 43, 155a, 180, 186, 303), and IPC induces multipolar spindles and other abnormalities of cell division and differentiation in several higher plant (19, 120, 140, 225) and animal (174) cells. IPC (42, 255) and CIPC (255) do not bind to brain tubulin or affect its assembly-disassembly, or its assembly onto Poly tom ella MT organizing centers (MTOCs) in vitro (303). The ethyl ester, in the form of an affinity column, retards the passage of both plant and animal tubulin (194) and disrupts cortical MTs in a higher plant (293). Although there are indications that these carbamates act only at the level of MTOCs, it is not clear whether they can also cause breakdown of existing MTs in vivo-if they cannot do so, the implication is that their observed effects arise from turnover and disorderly development of MT arrays.

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MICROTUBULES 659

The benzimidazole 2-yl carbamate fungicides and anthelmintics benomyl (and its hydrolysis product MBC or carbendazim), parbendazole, nocoda­zole (= oncodazole), and others affect ascomycete but not oomycete or zygomycete fungi. Many examples of variations in tubulin binding and in the responses of fungal genera, species, and mutants to these compounds have been described (45, 46, 133, 134, 154a, 159, 160, 243, 290). Parben­dazole and nocodazole are more effective inhibitors of growth of Physarum amoebae than MBC, the response correlating with inhibition of assembly of Physarum tubulin (244). By contrast, brain tubulin is almost insensitive to MBC in vitro ( l54a, 244, 255) but is sensitive to nocodazole (see 45, 154a, 172) and parbendazole (107a), as are the MTs of many animal cells. In the algae, nocodazole is active in the anti-MT nuclear migration test in Micras­terias while MBC is ineffective (186); neither removes cortical MTs from Oocystis although MBC alters cell wall morphology (255) and has certain antimitotic effects in onion root tips (248).

CIPC, IPC, MBC, trifluralin, and oryzalin all release Ca2+ from plant mitochondria in vitro, the first being the most potent (123). Further, in Aspergillus, MBC treatment inhibits cAMP phosphodiesterase and raises cAMP levels, providing another possible route by which free Ca2+ levels in the cell could be elevated (161). However, if Ca2+-induced disassembly underlies the anti-MT effects of all of these drugs, it would be expected that they would have similar activities, and this is clearly not the case.

The organophosphorus herbicide amiprophos-methyl (APM or O-meth­yl-0- (4 - methyl -6 - nitrophenyl) -N-isopropyl - phosphorothioamidate) in­duces giant nuclei, spheroidal cell enlargement, and disruption of secondary wall deposition in seedlings (see 154). It blocks MT-mediated movement of the nucleus in Micrasterias at micromolar concentrations (154, 186), blocks movement of nuclei-also MT-mediated-in Acetabularia (155a), removes MTs from Oocystis (255) and Polytomella (303) cells, inhibits outgrowth of flagella in Chlamydomonas (41), and causes shortening of partially regenerated flagella (239). All of these effects are consistent with an anti-MT action, yet APM has little effect on polymerization of brain tubulin or depolymerization of brain MTs in vitro (255). It also inhibits CaH uptake by plant mitochondria (123) and selectively destroys mRNA for tubulin (41); however, only the latter effect occurs at micromolar concentrations. Tubulin synthesis does not persist for more than 10--15 min after applica­tion of 3 pM APM to Chlamydomonas (41). If such a response proved to be general, it might be possible to use APM to assess the turnover rate of MTs in the various types of array in plant cells.

One other compound deserves special mention because of its potential usefulness. Taxol, obtained from the western yew Taxus brevifolia, shifts the assembly-disassembly equilibrium toward assembly of tubulin into MTs, lowering the critical concentration for nucleation, enhancing the rate

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660 GUNNING & HARDHAM

of polymerization, and stabilizing MTs against the normally disruptive effects of low temperature and high Ca2+ concentrations both in vitro (274) and in vivo (275). As a consequence, it has marked effects, for instance, arresting dividing animal cells in G2 and M phases of the cell cycle (275) and inducing aster-like bundles of MTs and subsequent furrowing when it is injected into egg cells (116). It is not yet known how general its effects on plants might be, but, as does the animal hormone melatonin (140), it increases spindle birefringence in Haemanthus endosperm cells (339). It also enhances assembly of plant tubulin in vitro (197).

MICROTUBULE-ASSOCIATED PROTEINS MT-associated proteins (MAPs) almost certainly exist in plants. It is therefore appropriate to refer to major trends of research on these substances, despite the dearth of information on the plant versions. Specific points are referenced, but for general information see (245, 272).

The first proteins found to be associated with cytoplasmic MTs were those that co-polymerize with tubulin through successive rounds of assem­bly-disassembly, being separable from tubulin by chromatography on DEAE-Sephadex or phosphocellulose. Several classes have been distin­guished. The "tau-factor" is a group of proteins (55,000-62,000 mol wt) which, like others known as HMW or MAPz (280,000-300,000 mol wt in the case of brain extracts), bind to, co-purify with, and stimulate polymeri­zation of tubulin in vitro. The binding reaches a standard stoichiometry and promotes nucleation but not elongation of MTs. Ring structures which are formed as intermediates are of doubtful significance in vivo; indeed, MAP­free tubulin assembles into MTs, given high initial concentrations (26). Associations of MAPs and MTs do, however, occur in vivo, as shown by elegant immunocytological work [including the use of monoclonal antibod­ies (139)]. Diversity among the MTs of different arrays, cell types, and organisms can be achieved by varying MAPs as well as tubulin. Thus MAPs of HeLa cells (which differ antigenically and have different molecular weights from those of brain) are found in a variety of non-neuronal cell types in primates but not in similar cells in lower organisms (27); con­versely, certain antigenic determinants of MAP2 from pig brain are specific to differentiated neurons and are not found in spindles or cytoskeletons in non-neuronal tissue (139).

MAP2 of brain is visible on electron micrographs as regularly spaced projections in a helical superlattice (3). It consists of a nucleation-promot­ing domain (located on or in the MT wall) and the projection, which is tightly associated with protein kinase activity. Both parts of the molecule have phosphorylation sites (320), and cAMP-dependence of tubulin phos­phorylation has been observed (310). There is new evidence for the presence

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MICROTUBULES 661

of cAMP in plant cells ( 144), which, taken together with indications of regulation by cAMP of flagella growth in Chlamydomonas (263) and inhibi­tion of cAMP phosphodiesterase by MBC in a fungus (161), suggests that work in this area may well turn out to have significance in relation to plant as well as animal cells.

Other MAPs warrant mention because of their possible roles in assembly and function of MTs. The best known is dynein, found in flagella and cilia as two rows of angled projections every 24 nm along the A-subfibers of outer doublets. Here the tubulin is a framework, but not a stimulant, for en­zymatic activity in the MAP. Dynein has MgH ATPase activity, hydrolysis of its substrate leading to a transitory mechanochemical interaction with sites on the adjacent B-subfiber, and in turn to a sliding motion which drives the ciliary or flagellar beat [for recent reviews see (268, 268a, 326, 327)]. The A -subfibers also carry rows of periodic interdoublet links and radial spokes, the latter consisting (in Chlamydomonas) of some 12 polypeptides in a stalk region and 5 more at a bulbous head lying close to yet other projections extending from the central pair of MTs (234). The links and spokes may aid in transducing the mechano-chemical effects arising from dynein ATPase into beating movements. Additional molecules bridge the gap be­tween the outer doublets and intrinsic proteins of the flagellar membrane and are probably involved in flagellar surface motility (see later).

In the absence of MAPs, tubulin derived from outer doublet MTs reas­sembles in vitro in very much the same way as does brain tubulin, unlike the in vivo assembly process (62). Dynein from Chlamydomonas will bind linearly every 24 nm along MTs reassembled in vitro from brain tubulinj MAP2 (which has a different periodicity) prevents the otherwise readily accomplished reassembly of dynein onto dynein-depleted doublets (95). To some extent, then, properties of the MT-MAP complex are determined more by availability of particular MAPs during assembly than by heterogeneity of tubulins. This conclusion should not at present be ex­trapolated too far: spindle and interphase MTs in HeLa cells, despite their very different properties, apparently have similar amounts and types of MAPs (28).

Complexities of the MT-MAP arrangements in flagella serve to introduce the general concept that MAPs might link MTs into the rest of the cyto­skeleton. Very little is known about detailed architecture of those regions of plant cells-such as the cell cortex-that contain MTs, so what happens in animal cells is of especial interest. Major components of animal cell cytoskeletons include actin filaments, a range of intermediate filaments, a microtrabecular lattice, and, of course, MTs. Spatial associations between MTs and intermediate filaments have been observed by conventional elec­tron microscopy and by immunofluorescence (77, 80). In biochemical isola­tions, high molecular weight proteins that are very similar to MAPs (and

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662 GUNNING & HARDHAM

do in fact co"purify with tubulin and stimulate its assembly) are associated with intermediate filaments and may serve as linkers between them and MTs (80, 237, 334). Again, microtrabecular connections exist between MTs and other components of animal cells [summarized in (340)]. Finally, there are in vitro experiments which show that MTs without MAPs have little effect upon the viscosity of f-actin whereas MTs plus MAPs form a stable very high viscosity gel when mixed with filamentous actin (83, 84, 236, 268b). Myosin also associates with tubulin in vitro ( l08). It may be signifi­cant that the amino acid sequences of certain regions of the tubulin mole­cule resemble regions of muscle proteins, including actin, myosin head, and troponins (236a). These observations may or may not all be strictly relevant, but they set the scene for the more limited information on plant cells that is described later.

MICROTUBULES

Structure, Assembly, and Polarity

The molecular stqlcture of MTs has been reviewed thoroughly (3) and need not be detailed here. Cytoplasmic MTs in plants consist of 13 protofilaments (93, 145, 168) and presumably are basically similar to others. Each protofi­lament is a linear chain of alternating a- and ,B-tubulins. All 13 protofila­ments are aligned in the same way, slightly staggered so that the dimers lie in helices around the MT wall. It follows that MTs are intrinsically polar structures-a fact which relates to observable polar orientation of dynein sidearms (95, 268a); also there are differences in the rate constants for addition and deletion of tubulin at the two ends (see later). Moreover, the polarity could be of crucial importance to MT functioning (185).

MT assembly in vitro is a two-phase reaction sequence: condensation (nucleation) followed by polymerization. Formation of nuclei is strongly concentration-dependent. Polymerization is slow below a critical concen­tration (Cc), above which tubulin is rapidly added to initial nuclei, giving rise to MTs (312). The initial phase is bypassed if fragments of MTs are added as "seeds"; the kinetics of polymerization can then be studied in isolation.

Knowing that MTs have an intrinsic molecular polarity, an immediate possibility is that polymerization reactions might differ at the opposite ends. This in fact is the case. By using recognizable seeds (fragments of flagella or fragments of duplex or radioactive MTs), it has been shown that the association constants are different at opposite ends; at high concentrations of tubulin one end grows faster than the other, while at just above Cc growth can be essentially restricted to one end (10, 11, 44, 272). Similarly, using portions of Chlamydomonas flagella with MTs grown on at both ends,

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MICROTUBULES 663

sudden dilution of the tubulin induces depolymerization at both ends. Again the rates differ, the end with the larger association rate constant also having the larger rate of dissociation (10, 149). Other studies have shown that for net depolymerization induced by dilution of tubulin, by addition of Ca2+, or by lowering the temperature, dissociation is proportional to the number concentration of MTs but independent of their length, i.e. it occurs at the ends and not throughout the MTs (147, 148).

In vitro it is not the case that assembly occurs only at one end and disassembly only at the other (cf 183). Net growth or net loss is the sum of association and dissociation events. The data on Chlamydomonas flagella seeds indicate that at steady state a gain of one tubulin dimer at the fast­growing end, usually designated "+", is achieved by addition of 11 and loss of 10, while net loss of one at the opposite end, usually designated "-", involves addition of 3 and loss of 4. Tubulin added at one end will eventually be lost at the other, having passed along the MT, in this case at > 3 p.m h-i (10). This flux of subunits is described as "treadmilling" or "head-to-tail polymerization" and applies also to actin filaments (331a).

It has been pointed out that treadmilling would be theoretically impossi­ble if the "on" and "off' reactions at either end were strictly the reverse of one another (155, 331a). If they were, the two ends would have identical Ce values; both would grow or both would shrink depending upon the concentration of tubulin. The factor that introduces the disparity is believed to be coupling of assembly to hydrolysis of GTP at the exchangeable GTP site on tubulin. The "on" reaction involves tubulin-GTP while the "off' reaction involves tubulin-GDP. In agreement with this theory, stimulation of assembly by means of a nonhydrolyzable analog of GTP yields MTs in

which there is no treadmilling (44). Given hydrolysis of GTP, Ce, and hence assembly-disassembly kinetics,

can differ at the two ends. There is a steady state critical concentration (C:> at which net growth at one end is balanced by net loss at the other (as in the example specified above). At tubulin concentrations greater than C (slow end), both ends grow, while at concentrations below C+ e s e (fast end), both ends lose tubulin. Between C and C- growth at the fast

+ e s � end exceeds loss at the slow end; between C and C lOSS at the slow end e e exceeds growth at the fast end. It is suggested (155) that this spectrum of possibilities provides the cell

with a most valuable advantage-that if the concentration of tubulin is kept between CS and C+, then (a) there will be no spontaneous nucleation and growth of free Mrs; (b) if free MTs do arise by fragmentation (etc), they will disassemble; and (c) any MT whose slow end is rendered unavailable by attachment to an MTOC or nucleating site will have its growth domi­nated by the fast end and will grow (whereas if it were not "capped" at the

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664 GUNNING & HARDHAM

slow end, it would disappear). Here, then, are rationales for the role of GTP and the existence of organizing centers, with a kinetic background to the role of MTOCs in placing MTs in specified locations in the cell while at the same time avoiding indiscriminate development of other MTs. Such consid­erations are relevant to observations of apparently free short MTs in plants (see later).

The postulated role of "capping" at an MTOC is supported by data on growth of centrosomal MTs. Their in vitro growth rates match growth rates of the fast (+) end of fragments of flagellar outer doublets included in the reaction mixtures as internal controls. Upon dilution of the tubulin, the disassembly rate of the centrosomal MTs again matches that of the fast end of the flagellar system. The data for flagella are unequivocal and lead to the interpretation that in the centrosomal array the slow (-) end is "embedded in the organising material and . . . rendered unavailable for incorporation of subunits"; only the fast (+) end participates in growth, having been nucleated at the MTOC, and there is therefore no treadmilling (11).

This interpretation accords with predictions about the role of MTOCs based on kinetic theory [above and (155)], and with electron microscope visualizations of Chlamydomonas flagellar outer doublets, which when growing have distal ends opened out to sheets of protofilaments similar to those seen at growing ends of MTs in vitro (51). Also, MTs growing from spindle plaques of yeast nuclei have morphologically different ends: blunt and closed proximally, ,and open distally (32).

A method has been devised for labeling MTs so that their molecular polarity can be characterized and related to the orientation of the MTs and their +/- polarity (where that is known) (115, 117, 185). Note that the intrinsic molecular polarity has still not been identified in terms of the positions of the (1- and ,8-tubulins at the ends of the protofilaments. In a polymerization medium which contains dimethylsulphoxide, tubulin ex­tends preexisting MTs and also decorates both the seeds and the extensions with curved flanges. The curvature of the flanges is the indicator of molecu­lar polarity. For example, when basal bodies in Tetrahymena pellicles are used as seeds, MTs grow outward (at their + ends) and to a lesser extent inward into the cell body (at their - ends). Viewed from the + end toward the basal body, the great majority of flanges, which in end-on view appear as hooks, are curved in a clockwise direction. Inwardly growing MTs, viewed along their length from the basal body, are decorated in the same way. As evidenced by the curvature of the "hooks," the molecular polarity of the seed is propagated in both directions (117).

Thus far all MT arrays that have been investigated are remarkably homo­geneous in terms of molecular polarity (58, 59, 115, 117, 185). When viewed from outside the cell toward the basal body, both central tubules and outer

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MICROTUBULES 665

doublets show predominantly clockwise hooks. Looking from the distal end toward the cell bodies, predominantly clockwise hooks are seen in helio­zoan axonemes, neurotubules in axons, and the radiating processes of pigment-bearing cells in fish scales. The same applies to views from the distal end of mitotic aster MTs toward their MTOC, the centrosome. Bidi­rectional transport of cytoplasmic components in axonemes, axons, and pigment cells, and bidirectional surface motility on flagella (15) can evi­dently proceed in association with an array that is unidirectional with respect to molecular polarity of the constituent MTs-a conclusion to be remembered when considering the function of cortical MTs in plant cells. A second conclusion is that metaphase half spindles do not consist of interdigitating MTs of opposite polarity.

Relationships between molecular polarity (assessed by hook curvature), orientation with respect to MTOCs, and +/- growth polarity are known for outer doublets of flagella (see above) and can be expressed by a "right hand rule": when the fingers of the right hand are curled in the direction of hook curvature, the thumb points towards the - end of the MT (58). It would be very valuable if this could be applied as a general rule, thereby allowing sites of MT growth to be determined in the absence of direct data on kinetics (which would often be difficult or impossible to obtain).

Results for MTs in mitotic asters accord with the "rule" (58) and with generalizations concerning MTOCs (155), though this is to some extent inevitable in that their + ends were designated as such by extrapolation from the situation in flagella (11; see above). However, if the "rule" is applied to phragmoplast and midbody MTs, seen at telophase in plant and animal cells respectively, it becomes necessary to invoke addition of tubulin proximal to MTOCs.

The MTs of both phragmoplast and midbody consist of two sets which overlap slightly at the equator of the division figure and extend in opposite directions toward the poles. Molecular polarity changes at the overlap, where the + ends of each set (identified using the right-hand "rule") lie in dense matrix material (58). The two sets of midbody MTs may derive from the two asters of the whole spindle, which, by slight interdigitation in anaphase and telophase could produce the observed pattern (58). However, the phragmoplast probably has its own MTOC-capacity and is not merely a remnant of the spindle. It can form in the absence of any suitably oriented preceding spindle (5, 199, 251). It can extend in the plane of the equator, sometimes for long distances and in general beyond the diameter of the former spindle. If a damaged region is introduced into the array of phrag­moplast MTs array by a microbeam, the birefringence is restored provided that the cell plate at that part of the phragmoplast has not also been disrupted (136). This suggests that the MTOC for the MTs of phragmo-

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666 GUNNING & HARDHAM

plasts is at the level of the growing cell plate where, in terms of molecular polarity, ends corresponding to the + ends of flagellar outer doublets are located. However, this conclusion contravenes predictions ofMTOC behav­ior based on kinetic theory (155) and is at odds with one of the arguments used in designating + and - ends of centrosomal MTs, namely that if the end of an MT is embedded in MTOC material, then that end will be unavailable for incorporation of subunits (11 and above).

Unlike the outer doublets, the central pair of tubules in Chlamydomonas and Tetrahymena flagella are capped, and there is evidence that, in vivo, their growth is proximal to the basal body (51). However, in Tetrahymena, hook curvature shows that their molecular polarity matches that of the outer doublets, whose growth is distal (59, 185). If the caps are removed, growth in vitro, like that of the doublets, is primarily distal (51), so presum­ably capping at the + end (distal) constrains growth of these MTs to the end that is proximal to the basal body. There is a gap between the proximal ends and the basal body (e.g. 35) and it is not clear whether the basal body is the MTOC for the central pair. If it is, then the situation again contra­venes predictions concerning the properties of MTOCs. Note that the phragmoplast differs in that the + ends of its MTs are considered to be proximal to their MTOC.

The phragmoplast, which has been well described as "enigmatic" (58), and the central pair of MTs in flagella together caution that correlations between molecular polarity, +/- growth polarity, orientation with respect to MTOCs, and in vivo and in vitro growth characteristics are not uniform. Clearly it is necessary to look for "caps" on MTs as well as for MTOCs.

The above provides a background for a consideration of factors that might regulate MT development in vivo. Possible modes of regulation range from control of gene transcription and RNA survival and utilization to control of assembly-disassembly by promotors or inhibitors acting directly or indirectly on tubulin or MAPs. Not only nucleation and polymerization are controlled, but also length and position of MTs in the cell.

Arrays in Wall-less Cells Arrays of cytoplasmic MTs enable many wall-less plant and protistan cells to deviate from a spherical shape. Marginal bands in zoospores of the colonial green algae Sorastrum, Hydrodictyon. and Pediastrum support a discoidal shape without which correct aggregation and colony formation do not occur (175, 176, 181, 182, 191). Cytoskeletal arrays in the peripheral cytoplasm are seen in ovoid algal cells and zoospores [representative exam­ples are: Fritschiella (187), Poly tom ella (21), Chlorosarcinopsis (190)]. Vari­ous asymmetries may be initiated or supported by MTs, e.g. the horns of Pediastrum and Sorastrum, which have their own membrane-associated

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MICROTUBULES 667

MTOC (175, 176), the tail and platform of Ochromonas (18), and of course cilia and flagella, which may even have their basic cylindrical form modified by the presence of additional MTs [e.g. motile cells of Trentepoh/ia (81)]. The examples of Carteria crucifera and Chloromonas rosae provide a cau­tionary note; they develop very similar papillae but only in the former is the cytoplasmic extension supported by MTs, leading to the suggestion that there are other non-MT shape-determining elements of the cytoskeleton which become reinforced by MTs in only one of the two cases (51a).

There is a comprehensive literature on fibrous and microtubular "root­lets" which emanate from the vicinity of the basal body and have a range of functions in cell shaping, intracellular signaling, distributing the stress of ciliary/flagellar beating, and placement of organelles (reviewed in 44a, 113, 188, 189, 250). Some rootlets also serve as MTOCs for additional MTs, a role which has been investigated in vitro (303, 304) and by biochemical extractions of "tubule-inducing proteins" (302). Removal of the additional MTs from zoospores of Chlorosarcinopsis (they are selectively and revers­ibly sensitive to low temperature) changes the shape of the cell from elon­gate-ovoid to a shorter, tailed outline that is supported by the primary rootlet system (190). Alterations and recovery of cell shaping by the rootlet and additional MTs (nucleated on the "rhizoplast") of Ochromonas (18, 20) and Poly tom ella (303) have been thoroughly studied using physical and drug-induced perturbations. Spermatozoa, from algae to cycads, offer other spectacular examples of specific shapes, not just of the entire cell but also of nuclei and mitochondria, that are at least in part maintained by MTs (e.g. 53-55, 202-205, 315).

MT -mediated cell shaping also exists where cell walls are not formed in higher plants, as in sperm cells in pollen grains or pollen tubes (see 34). Cortical MTs remain in tapetal cells after wall dissolution (306) and, in "amoeboid" tapetum, are specifically clustered and oriented during cellular migration (220).

Movement of Organelles

MT -mediated movements in cells fall into several categories. In broad terms, there are movements involved in establishing and maintaining partic­ular polarities, including those related to preprophase bands and planes of division; there are movements of organelles (and the converse, anchoring of organelles); and there are specialized movements of cilia and flagella and those hypothesized to occur in the cell cortex during wall deposition. The cell cortex and preprophase bands are considered later, and reviews on ciliary and flagellar movements have already been cited. Even within the remaining classes of movement it is not to be expected that there is uniform-

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668 GUNNING & HARDHAM

ity of mechanism; indeed it is well known that there are other motility­generating systems not involving MTs (e.g. 146).

Nearly all of the evidence for participation of MTs in intracellular move­ments in plants comes from experiments with anti-MT drugs, coupled with electron microscope surveys of MT distribution. A notably rigorous excep­tion utilizes genetic approaches to show that ,g-tubulin is essential for migration of nuclei in hyphae (2 1 1). MTs radiate from spindle plaques or centrosomal regions in many fungi in which nuclear movement is sensitive to colchicine, vincristine, nocodazole, griseofulvin, or MBC (1 12, 1 13, 132-1 34, 165, 206, 2 1 1 , 246, 286, 31 1 ). The migration in several of these cases takes the form of maintaining a specified distance between the nucleus and the growing hyphal tip, as also seen in apical cells of filamentous gameto­phytes of the fern Adiantum (323, 324) and caulonema of the moss Funaria (277-280).

The position of the nucleus may change markedly betore or after mitosis, with concomitant changes in distribution of nucleus-associated MTs. In all of the following examples the nuclear envelope must be of great importance in attaching and/or nucleating MTs. In the desmid Closterium, postmitotic movement of the nucleus occurs along a groove between two recently divided chloroplasts. At the beginning of the process a "MT center" forms near the nucleus and migrates along the path to be followed later by the nucleus. MTs run back from the center toward the nucleus, which becomes highly deformed, one point becoming extended along the trail of MTs and eventually drawing the remaining bulk of the nucleus after it. The total journey is about 20 /Lm and is accomplished in 7-15 min (229, 230). Similar nuclear migration in Micrasterias (152, 3 13) has become the basis of a convenient bioassay for anti-MT activity (186). Postmeiotic movements of nuclei and polarity shifts in developing moss spores entail development of a spectacular "MT center" very like that of Closterium, with MTs appar­ently deforming the nucleus and attaching to its envelope (22, 23). In Uromyces (1 12) and Chytridium (31 1), the nuclei move from a larger compartment into a narrow tube along a funnel-shaped array of MTs [which in Uromyces are short, mostly < 2/Lm long ( 1 1 2)]. Branch forma­tion and tip growth in Funaria caulonema (278, 279) and asymmetric divisions in Azolla roots (91 ) and in Onoclea spores (8) also involve premi­totic movement of nuclei and again tracts of MTs appear to lay a path. Intra- and inter-cellular movement of nuclei in basidiomycete hyphae is also associated with MTs (245a, 246). In Schizophyllum commune the process is sensitive to griseofulvin (246), a drug which probably acts on MAPs (258a), and a mutant is known which shows continuous nuclear migration and possesses bundles of microfilaments and MTs passing from cell to cell through degraded septa (246a). Migration of secondary nuclei and "headed

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MICROTUBULES 669

streaming hands" in Acetabularia is reversibly sensitive to anti-MT drugs (155a). Nuclear cyclosis, at 2 /Lm sec-I, occurs throughout mitosis and interphase in Vaucheria, with prominent bands of MTs attached to the anterior end of the nuclei (i.e. the leading part in the motion) at persistent centrioles (218, 219). All of these motility phenomena deserve further inves­tigation to elucidate their molecular basis and to see whether the MTs participate in force generation or merely act as a framework along which movement can proceed. It has been pointed out that dynein binds to various types of MT, in addition to A-subfibers of flagella, and hence could be involved in intracellular MT -mediated movements (268a).

Anchoring of nuclei with the aid of MTs occurs in Acetahularia (341) and in moss caulonema subapical cells prior to prophase (278, 279). The shape of the nucleus (see 121, 286) and even the distribution of the pores in its envelope (54) may be markedly affected by the MTs. Anti-MT drugs release the nuclei and the same applies in certain cases where other or­ganelles are fixed in place (1 33; M. E. McCully, personal communication). Highly specific distributions of organelles, involving spectacular arrays of MTs, are seen in bryophyte sporocytes (22, 23, 23a). Close associations between MTs and mitochondria (253), an endosymbiont (217), virus parti­cles (33, 184), membranes (57), and vesicles (see 56) have also been ob­served. It is not always clear, however, whether such associations are transitory or whether they relate to anchoring or to saltations [which in some cases in both plants (113) and animals (135) are inhibited by anti-MT drugs]. With the exception of surface motility on flagellar membranes ( 15), there are no examples in plants of the spectacular bidirectional saltations exhibited by axopodia and chromatophore cells, where unipolar arrays of MTs evidently serve as a skeleton on which nonmicrotubular motility­generating systems can function. MTs are commonly found to be oriented axially in tip-growing filaments such as pollen tubes, fungal hyphae, and rhizoids, and roles in delivering vesicles to the "Spitzenkorper" are possible. Anti-MT drugs as well as cytochalasins disrupt both the MTs and the delivery of vesicles (133, 134). Participation of MTs in delivery of vesicles to growing cell plates and cell walls is another long-standing hypothesis (see 121, 135), but accumulation of vesicles in extending phragmoplasts and at tip-growing cell apices in moss caulonema has now been observed to be apparently independent of MTs (277, 280).

Finally, the "lipotubuloids" of Ornithogalum apparently have not been reinvestigated since the first descriptions of their ultrastructure and behav­ior (see 162�yet they are involved in one of the most extraordinary forms of intracellular motility, being aggregates of granules enmeshed in baskets of MTs, spinning at up to 3600 sec-I while moving by more conventional cyclosis around the cell.

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670 GUNNING & HARDHAM

The Preprophase Band

The most notable property of the preprophase band is that it predicts where cytokinesis takes place. Because movements of nuclei and cell plates are sometimes involved, it is convenient to deal with the topic after having considered other aspects of the movement of nuclei.

The preprophase band of MTs (231 , 232; see 9 1 for literature to 1978) is in general 2-3 J1m wide and girdles the cell cortex along the line where a cell plate, to be formed at telophase, will fuse with the parental cell walls. As with other cortical arrays, it consists of short overlapping MTs, cross­bridged in places (99). The total numbe! in the band ranges from more than 100, densely packed in any one cross section, to smaller examples contain­ing only 10-20 in a monolayer underlying the plasma membrane. There is evidence that the number of MTs in the band is related to the number found at interphase in the same cell ( 100, 102).

It is in fact useful to think in terms of a cortical site at which a variable number of preprophase band MTs may become organized. The fact that cytokinesis can occur under precise spatial control, but with no microtubu­lar component at the site, in situations such as Chara apices (229) and moss protonema (277) is an indication that other components, as yet invisible to the electron microscopist, are of fundamental importance. In the sole case in which a time sequence has been established, a phragmosome (which also predicts the future site and plane of division) precedes development of a preprophase band of MTs (322). Sporocytes in bryophytes, ferns, and gym­nosperms can specify sites and planes of division by making wall ingrowths before meiosis, and an equatorial band of organelles after the first meiotic division, all without benefit of a preprophase band of MTs (see 23a). Fur­thermore, preprophase band MTs disappear from the cell cortex as the nucleus enters prophase, but the site in the cell cortex remains, later in­fluencing the positioning of the cell plate. The point is especially evident in divisions which entail anaphase or telophase reorientation of spindle or cell plate into a predetermined axis. In Allium stomata, reorientation is achieved after the preprophase band MTs have disappeared (225); in moss protonema, reorientation occurs though there was no preceding prepro­phase band (277); in centrifugally displaced telophases in stamen hairs, the correct position is again restored after disappearance of the MTs from the preprophase band site (216).

Five years before preprophase band MTs were first described, it. was concluded that "a specially differentiated pattern is imprinted in the equatorial region of the cytoplasmic cortex, which is not moved by centrifu­gal forces of 3550 g or less, and a sort of pulling force is acting between the cortical pattern of the equator and the margin of cell plate" (2 16). Much

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MICROTUBULES 67 1

more recent experiments implicate actin in the "pulling force" by demon­strating sensitivity to cytochalasin B and phalloidin (222).

As mentioned above, the preprophase band of MTs is not ubiquitous. It has not been found in any fungus or alga (229). With one exception, it has only been found in divisions which involve fusion of a cell plate with a defined site in the parental wall [cf its absence during cellularization of endosperm, sperm division in pollen; see (91)]. The exception is that when Avena tapetum cells become binucleate, clustered MTs resembling prepro­phase bands appear before the primary nucleus divides (306). Nor is it found in disorganized tissue such as nodules of cells formed by regenerating protoplasts (69). It is absent from moss protonema (143, 277-280) but is present (given an appropriate light regime) in a filamentous protonema of a fern (323) and in uniseriate trichomes growing from three-dimensional tissues (3 1). Ordered two- and three-dimensional aggregates of cells in mosses, ferns, and flowering plants develop preprophase bands in cell divi­sions that occur in the normal course of development (see 31 , 9 1 for summa­ries) and in "unplanned" divisions that, although unplanned, are specifically placed in tissues undergoing various forms of regeneration or proliferation following wounding (102, 322).

The position of the preprophase band of MTs consistently predicts the site of fusion of cell plate and parental walls [despite certain suggestions to the contrary (295, 296; see (3 1 )]. Three categories of problem ensue. What determines the site? How does the band develop? What is its function and how does it function? Determination of the site and plane of division and its predictors, the phragmosome and preprophase band sites, is a major unsolved problem. Many environmental factors, internal genetic controls, and intercellular signaling all seem to feed "positional information" to dividing cells, the balance varying from situation to situation. The only indications about how the band forms come from observations on Azolla roots, where structures interpretable as nucleating sites arise in or close to regions where the future band intersects the edges of the cell. The impres­sion is gained that MTs grow out over the faces of the cell from these localized regions of the edges (87, 90, 92). Immunofluorescence microscopy of developing preprophase bands in onion root tips has so far not disclosed any such structures (S. M. Wick, personal communication).

In discussing functional aspects it is again useful to distinguish between functions of the preprophase band MTs and functions of the preprophase band site. The MTs have been suggested to be precursors for spindle MTs (228); they could also guide deposition of extra wall material at the site of the band (75, 76, 22 1). They might also participate in positioning the nucleus prior to mitosis, for which two partial processes can be envisaged -bringing the nucleus to a selected part of the cell [especially in asymmetri-

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672 GUNNING & HARDHAM

cal divisions (e.g. 91)] and orienting the nucleus in a particular direction. There is a question of whether a nucleus has a particular axis (228), but it may be that the distribution of spindle organizers at or near the nuclear surface, or of chromosome attachments to the nuclear envelope, could provide an axis for positioning the nucleus with respect to the preprophase band. In this context it is worth recalling that immunofluorescence micros­copy reveals concentrations of nonfibrillar tubulin and then MTs at the preprophase nuclear surface in plant cells (335) and that there is evidence of a colchicine-sensitive DNA-binding protein in plant nuclear envelope ( 1 3 1).

Two other possible functions concern the site rather than the MTs. The first, already referred to, is that of guiding the edges of the growing cell plate, which comes at least approximately to bisect the preprophase band site (3 1 , 87, 90, 9 1). The second is only a possibility and concerns develop­ment of interphase MT arrays in the daughter cells. In Azolla there is evidence that these are nucleated along selected edges of the cell, i.e. at bisected former preprophase band sites (87, 92). No evidence for such a distribution of nucleating sites has yet come from immunofluorescence microscopy of flowering plant material (335), but it remains possible that the preprophase band site could function as a form of MTOC long after the band itself has gone-a suggestion put forward in the previous review in this series ( 1 2 1 ). It is also possible that the edge nucleating sites derive from the phragmoplast MTOC system (87).

Interphase Cortical Arrays in Walled Cells.

It has long been held that MTs could exert an indirect effect on shaping walled cells by influencing the orientation of cellulose deposition and impos­ing directionality upon otherwise isotropic turgor forces acting to expand the cell. Recent confirmation is available (1 79). The concept will now be examined by reviewing the nature of the cortical arrays of MTs found in most walled cells at early stages of their development, presenting some case histories that illustrate the basic phenomena and examining mechanisms that might be involved.

NATURE AND DEVELOPMENT As described in earlier reviews (121, 207), cortical MTs lie close to and parallel to the plane of the plasma membrane. They may be dispersed over the entire waIls of a cell or they may be in localized groups. Serial sectioning of root meristem cells (86, 98, 99) and root hairs (289) has shown that the MTs are shorter than the dimen­sions of the face against which they lie-at least after glutaraldehyde fixa­tion. Short MTs have also been seen in protoplasts by negative staining, scanning electron microscopy, and immunofluorescence microscopy (52,

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MICROTUBULES 673

177, 178, 1 80, 321). Usually the ends of the MTs are scattered over the faces of the cell (99, 289), but many are near the edges where two faces meet; in mature arrays MTs may also curve around the cell comers from one face to another (100, 335).

Questions are immediately raised concerning the origin and life span of these short, apparently free MTs. Kinetic theory (above) predicts that they must be impermanent unless at least one end is "capped" or the local concentration of free tubulin equals or exceeds Cs• Yet they are consistently seen, with lengths in the general range 1-8 p.m (52, 98, 99, 289, 321). Steady state treadmilling occurs at several p.m h-1 (10, 44), and below C; free MTs would shorten faster than this. It is noteworthy that alkalinization, which may occur in the cell cortex as a consequence of proton secretion during growth, induces reversible disassembly of MTs in vitro to a new steady state in which the average MT length is shorter than at neutral pH (247a). There is no evidence to show whether the population of interphase cortical mi­crotubules is subject to turnover, though obviously it appears after cytokine­sis and disappears at preprophase in a dividing cell and may disappear or alter during cell expansion and differentiation. The ends of the constituent MTs may have "knobs," or densely staining particles (88), stated to occur at only one end in burst Mougeotia protoplasts ( 1 77, 1 78) but not seen in burst tobacco (321) or onion guard cell (52) protoplasts. Closed (rather than knobbed) ends also occur (e.g. 52, 88), as at the proximal end of spindle plaque MTs in yeast (32) and in many of the short free MTs of neurons (36). On the other hand, terminations of MTs can be splayed out (52) or project as C-shapes (99) or long narrOw protofilamentous beaks (88). As discussed in (99) there are no sure indications as to whether a C-shaped end is growing or depolymerizing, but when fixations were carried out in polymerization medium no such ends were found. Local shared polarity within small samples of interphase cortical arrays was detected through an asymmetric distribution of C-shaped ends [99].

MTs in the cell cortex might also be stabilized through bridging to each other or to the plasma membrane or to associated filamentous material. Bridges have frequently been reported [for recent examples see (88, 99, 321)], and temporal changes in the adherence of MTs to the plasma mem­brane of burst protoplasts have been attributed to progressive bridging (1 80). That cortical MTs in root tip cells often lie parallel to one another, with individuals sometimes leaving one group and passing to become paral­lel to another, is suggestive of interactions mediated by local bridging (98, 99). Bridges are, however, seldom seen in an obvious periodicity along plant cell cortical MTs [but see (99) for a sequence of regular bridges in a herringbone pattern.] Nevertheless, computer-assisted correlation of spatial patterns has revealed that the bridge-sites that are utilized fall into a series

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674 GUNNING & HARDHAM

of spacings closely similar to the 12-dimer superlattice proposed (3) for the association between brain MAPs and MTs (c. Jensen, personal communi­cation). In root hairs, by contrast, MTs seldom come within bridging dis­tance yet they have a predominant orientation and may therefore interact with other less obvious cytoskeletal components (289). Filaments associated with (though not always co-aligned with) cortical MTs have been seen (52, 86, 97, 114, 201, 289). Like those revealed at Oedogonium kinetochores by specialized fixation procedures (273), they may be connected to the MTs by fine strands, but can only be tracked through a few serial sections (86). Only in one case is there evidence that they are actin (289).

Individual MTs in cortical arrays may not be stable or static. Ori'a larger scale, there are many descriptions of qualitative and quantitative changes through the cell cycle and during differentiation. Also, expansion of meris­tematic cells in root meristems involves continual interpolation of MTs into the cell cortex. High densities are associated with newly formed longitudi­nal walls in Azolla roots (100; see also 281-283) and before the rapid elongation phase begins in liverwort setae (284). During initial phases of elongation, microtubule density declines slightly but subsequent interpola­tion is able to maintain the new level (100, 338). "Maintenance" in this sense is a complex process, interrupted as it is by mitotic intervals in which the whole cortical array is removed. The number of MTs JLm-1 of cell length is maintained while the cell expands during interphase; the same frequency is restored in the daughter cells after cytokinesis, and further maintenance by interpolation keeps pace with their expansion. Treatment of elongating cells with growth regulators and other substances can alter the rate of interpolation (284). Quantitative work on roots of Azolla (100) and four grass species (338) shows (a) that different cell types have their own charac­teristics when the same category of cell wall (e.g. tangential-longitudinal) is compared; and (b) a single cell may maintain different densities of MTs

on specific walls, as exemplified by endodermal cells in Azolla (100) and epidermal cells in wheat (338). In addition, it has been known since the first publication in the field (167) that different faces in one cell can have different MT orientations. Moreover, the interphase cortical MTs on a given cell face may or may not have the same orientation as later-formed preprophase band MTs.

The attributes described above have to be accounted for in considering how cortical arrays might develop. A minimum requirement is control of orientation and frequency at the level of the individual cell face, irrespective of whether the regulatory information is of internal origin, related to cell type, or is external, perhaps hormonal in nature.

The concept of the MTOC emerged from recognition that cells need to deploy MTs in particular places at particular times, and from electron

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MICROTUBULES 675

micrographs showing MTs focusing upon a specific structure or region of the cell, as if they had grown from it (227; see also 314). Such structures have now been found in the cortex of root primordium cells in Azalla. They are the best candidates so far for the role of MTOCs for cortical arrays though, in the absence of evidence concerning any capacity to "organize" the array, the less onerous term "nucleating site" has been preferred (87, 90, 92). The sites tend to lie in or close to the edges of the polyhedral cells, a strategic location for controlling arrays on individual cell faces. In other work on root tip cells (338) it has been found that rates of interpolation of MTs on longitudinal walls are proportional to the cell length but not to wall area or cell volume; the data are consistent with nucleation along a linear site such as an edge.

In Azalla these sites along cell edges stand out because the anatomy of the root favors detection of cells that are in the postcytokinesis phase of reinstating their cortical MTs, and because the density of ribosomes is fairly low in their vicinity. Other material is less favorable but regions at cell edges from which MTs fan out have now been seen in roots (88) and stomata (73, 75) of certain flowering plants. They have yet to show up in immunofluores­cence studies (335). "Focused" MTs have been seen in other parts of the cytoplasm, remote from cell edges (22, 23, 70, 74, 224).

The putative nucleating sites for cortical arrays in Azalia roots are most conspicuous at postcytokinesis and less obvious during interpolation phases. Preprophase band development and initiation of xylem thickenings also elicit the appearance of the sites, but at restricted portions of selected edges. Those for preprophase band development are least obvious when the orientation of the MTs in the band matches that of the preceding interphase array and most obvious when the band is at a new orientation. The capacity to carry the sites at a particular edge usually appears when the edge is newly formed by fusion of a cell plate with parental walls. Thereafter it can persist at the same edge through successive cell cycles (87, 88). There is a tendency for the emerging MTs to assume an orientation roughly normal to the edge bearing their nucleating sites, but sometimes they fan out from particular vertices. Overlapping MTs on one face can on occasions be tracked back to opposite edges; however, thus far there are no indications of molecular or + / - kinetic polarity regarding the sites. It is possible that short free MTs found on cell faces detach and migrate from nucleating sites at cell edges. Obviously it is impossible to rule out nucleation and polymerization of individual tubules on the faces, but such a process is difficult to reconcile with theoretical considerations presented earlier.

The above system cannot account for all subtleties of MT -orientation in cortical arrays (even if the putative nucleating sites do turn out to be widespread). Problems include cell faces that do not have edges normal to

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676 GUNNING & HARDHAM

the orientation of their MT arrays, e.g. cylindrical cells in trichomes, and arrays which are localized and contain very specifically oriented MTs, sometimes remote from cell edges. Clearly there are pattern-determining systems which, although they may act in part by promoting MT-nucleation at selected edges or selected parts of selected edges, also have "fine tuning" capabilities for placing MTs. Nevertheless, the idea that cell edges are endowed with some morphogenetic regulatory properties has received sup­port from an entirely different form of analysis, showing that shape change in growing tissues can be geometrically described in terms of primary events at cell edges, which perforce dominate the shape and expansion of the circumscribed cell faces (156).

COALIGNMENT OF MICROTUBULES AND WALL MICROFIBRILS The initial suggestion (167) that cortical MTs are responsible for regulating the orientation in which wall microfibrils are deposited has general support from numerous further observations of MT lmicrofibril coalignment and colchicine-induced abnormalities. Some striking examples of coalignment are included in (22, 23, 60, 90, 97, 333, 337, 343) and many others are cited in (257). Uncertainties relating to a role for MTs in wall deposition (see 207, 212) stemmed largely from observations of apparent noncongruence, but results on systems that are changing through time show that these can be interpreted as MTs that have reoriented prior to the deposition of microfi­brils in a corresponding new orientation. Summaries of selected case histo­ries follow to illustrate coalignment during primary or secondary wall development or both.

The mature secondary wall of Oocystis, an ellipsoidal unicellular green alga, consists of about 30 layers of large microfibrils of cellulose. Within each layer the roicrofibrils lie parallel to each other, but are approximately perpendicular to those in adjacent layers. Cortical MTs can be seen to be cross-bridged to the plasma membrane and almost always lie parallel to the innermost layer of wall microfibrils, at an average of2.S microfibrils per MT (254, 265).

Oocystis has been used in extensive and revealing studies of relationships between MTs and microfibrils during production of its secondary wall (24, 85, 196, 238, 240-242, 254-256, 265). MTs are not necessary for production of cellulose, nor indeed for supporting the continued growth of microfibrils in parallel arrays once parallelism has been established. Cellulose produc­tion can be disrupted with or without loss of the cortical MTs. A major conclusion is that without MTs the cell loses its ability to change the angle of cellulose deposition. If they are destroyed without disrupting the cel­lulose synthesizing complexes, layers of microfibrils continue to be depos­ited, but in register; if they are restored, alternation of orientation in successive layers resumes every 30 min or so.

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MICROTUBULES 677

The desmid Closterium displays MT -microfibril coalignment during pri­mary wall formation, but, unlike Oocystis, not during secondary wall for­mation. Mter mitosis the symmetry of the crescent-shaped cells is restored by localized expansion at one end of the daughter cell [1 30, 230]. Microfi­brils are initially deposited at the septum in circular configurations, MTs in the underlying cell cortex being similarly disposed. For about 2.5 h after septum formation, as expansion (mainly elongation) proceeds, both MT and microfibril alignments are transverse (1 30). Colchicine treatment in this period leads to loss of MTs and random deposition of microfibrils and isodiametric expansion of new semicells (129). Transition to secondary wall formation and cessation of growth, about 1 . 5 h later, is not markedly delayed by colchicine. As in untreated controls, the secondary wall consists of bundles of parallel microfibrils lying in various orientations, covering the primary wall and produced independently of MTs, presumably by mem­brane-particle complexes comparable to those seen in Micrasterias during secondary wall production (79).

MTs in root hair cells, which figured in earlier reviews (121 , 207, 2 1 2), have recently been reinvestigated. In the zone of secondary wall formation they are short and lie at various angular deviations from the longitudinal axis of the hair, similar to the deviation pattern of the wall microfibrils (289). In cotton hairs the orientation of the primary and secondary wall microfibrils changes from transverse to nearly longitudinal, but MTs are generally coaligned at all stages (333, 337), even following reversals in microfibril orientation (343).

Some of the clearest examples of parallelism between MTs and microfi­brils are seen at secondary wall thickenings in xylem (see 19, 100, 101, 1 19, 12 1), stomatal guard cells (see 52, 72, 76, 1 2 1 , 223, 226), and other less widespread cell types (210, 282, 283, 288a). In kidney-shaped guard: cells of Allium, Vigna, and during early development of guard cells in grasses, MTs cluster in the middle region of the edge where the ventral wall meets the outer periclinal wall and radiate outward congruent with microfibrils that form the pore-thickening. In grasses developmental changes leading to net axial alignment and attainment of dumbell-shaped guard cells are again characterized by predominant parallelism with MTs. Both forms of guard cells in Cyperus, (either kidney- or dumbell-shaped depending upon the conditions under which the plants are grown) show congruence (193).

Treatment of developing guard cells and xylem elements with anti-MT drugs can disrupt both the localization of thickenings and the orientation of their microfibrils (e.g. 101, 223). The pathway of differentiation may not be totally blocked in that an irregularly pitted type of secondary wall, still recognizably "xylem," can develop (101, 103, 1 2 1). Recovery of spacing and localization mechanism following pulse treatment with colchicine has been followed in Vigna stomata and Azolla root xylem. In the latter,

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678 GUNNING & HARDHAM

nucleating sites for MTs form at the normal 2 p.m intervals in newly differentiating xylem elements (88, 1 0 1). Vigna stomata have provided the striking result that during recovery certain wall thickenings can form at their normal locations in what should be paired guard cells, even though the wall that normally segregates them was not formed in the guard mother cell as a result of prior treatment with colchicine (72).

The above examples describe changes-either single or repeated-in wall architecture of individual cells during development. Coordinated reorga­nization of MTs and cellulose deposition have also been documented on a larger scale-across hundreds of cells-during the formation of leaf pri­mordia on a plant meristem (97). Removal of the leaf in the succulent Graptopetalum stimulates the residual meristem at the leaf base to produce leaf primordia (82). During early development the local pattern of trans­verse microfibrils in the outer wall of the epidermis (the only wall analyzed in detail) and the adjacent MTs changes congruently to yield circumferen­tial arrangements around each emerging, roughly cylindrically shaped, leaf primordium, thereby facilitating its expansion as a radial organ from a tract of cells with uniform apico-basal polarity.

Two modes of analysis revealed close correlation between the alignment of microfibrils and MTs. In one, alignment of wall microfibrils in outer walls of epidermal cells was determined using polarized light microscopy to view dissected sheets of cells. The MT arrays, adjacent to these same walls, were then examined using serial sectioning. There was asynchrony among cells which were changing their polarity. Birefringence patterns at an early stage indicated that some cells had shifted their microfibril orientation through 90° while others had not. Nevertheless, the MT arrays paralleled the mi­crofibrils. During reorganization of wall deposition, Mrs with different alignments coexisted within single cells, but the newly deposited wall mi­crofibrils in any given locality were again consistently parallel to the nearby MTs. Cells which were changing their polarity had the greatest frequency of MTs.

Without claiming that MTs participate in all types of cell shaping (it is clear that they do not), the above case histories suffice to illustrate that they may do so from unicells all the way up the morphological scale to develop­ments that demand integrated behavior across large tracts of cells [see (8 1 a, 8 1 b) for models of large-scale morphogenesis based on coordinated cortical control of wall biophysics and plane of division]. Some such growth phe­nomena are subject to regulation by growth regulators, posing the question: do events in the cell cortex (production and orientation of arrays of inter­phase cortical MTs) mediate in coordinating responses to morphogenetic signals (hormonal or environmental) which affect the mode of growth of cells, tissues and organs? Available information is given in the following section. Further details are reviewed in (96).

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MICROTUBULES 679

Auxin treatments usually do not lead to change in polarity unless incuba­tion times are prolonged (29 1 , 292). In accord with this, MTs have been reported to be unaffected by IAA (292) or 2,4-D ( 1 70). Although IAA has marked effects on extension of Pellia sporangiophores (287), the cells ap­pear to maintain approximately the same frequency of transverse MTs per unit of cell length as controls (284). The effects of colchicine on auxin­stimulated growth vary according to the experimental system, in some cases having no inhibiting effect, even when MTs are present (27 1 , 292), in others being inhibitory and removing microtubules (4, 287), and in the classic coleoptile straight growth test, inhibiting (as does oryzalin) (1 66, 3 1 6) even though the cells contain exceedingly few MTs except in the vascular paren­chyma (2 1 3). Were it not for the existence of membrane tubulin, the latter result might be taken to mean that growth inhibition by colchicine is not caused by its property of binding to tubulin. However, in counteracting responses to gibberellic acid (GA3), colchicine does at least have effects (see below) that are not shown by lumi-colchicine ( 1 50).

Growth regulators which alter axes of cell expansion have rather more interesting interactions with MTs. Shoots of azuki beans, peas, and lettuce have been much used. Their cortical and epidermal cells deposit cross­polylamellate walls, and the effect of the growth regulators seems to be to alter the amount of time that is spent depositing the successive orientations. This is observed as a change in the thickness of the layers within the cell walls. For example, GA3 enhances stem elongation with minimal lateral expansion (269, 270, 29 1 , 292, 308, 309) and gives rise to a predominance of transversely oriented MTs and newly deposited microfibrils in the walls of epidermal cells in azuki bean (308, 309) and of cortical cells in lettuce (269, 270). Auxin-induced elongation of azuki bean epicotyl segments, while promoted by GA3, is inhibited by kinetin and benzimidazole (292). Coapplication of either of these with auxin results in stem thickening. Longitudinal microfibril deposition at tangential walls of the epidermal cells occurs after treatment with benzimidazole. A change from transverse to longitudinal alignment of MTs was noted after kinetin treatment. In gen­eral, MTs are oriented normal to the major axis of expansion (292, 293), just as in Azolla roots, where longitudinally expanding walls have trans­verse orientation of MTs while radially expanding transverse walls have tangential orientations (88, 89).

Like kinetin and benzimidazole, ethylene causes stem thickening and reduced elongation in pea shoots (4, 249). Before treatment the polylamel­late walls of the cortical cells contain predominantly transversely oriented microfibril layers, and transverse MTs are six times more frequent than those longitudinally aligned. After treatment with ethylene, longitudinal deposition of microfibrils predominates in cortical and epidermal cells and longitudinal MTs become eight times more frequent than transverse MTs

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680 GUNNING & HARDHAM

in cortical cells (164). Microfibrils are also switched to a predominantly longitudinal orientation in Zea root cortex cells (201).

That MTs mediate hormone-induced changes in axes of expansion is supported by results of experiments in which the cortical arrays are de­stroyed. Colchicine (1O-2M) inhibits GA3-induced elongation (269, 29 1) and reverses kinetin or benzimidazole inhibition of growth of azuki beans (292). Colchicine and low-temperature treatments reverse the ethylene effect on the growth of pea epicotyls (305). In azuki beans ethyl N-phenyl­carbamate also reverses the hormone effects and, moreover, does so by disrupting MT orientations without inhibiting cellulose synthesis (293). Colchicine must be present either as a pretreatment or during the first hours of GA3 treatment to be effective. Its inhibition of growth decreases progres­sively at later application times, being lost completely 6 h after GA3 applica­tion in bean (291) [36 h in lettuce (269)]. Unless growth is stimulated by GA3, colchicine application on its own does not lead to any significant lateral swelling (269).

Colchicine [but not lumi-colchicine (150)] destroys MTs in hypocotyl cells within about 4 h. Nevertheless, the microfibrils in the inner wall layers do not become deposited randomly. Instead they show partial ordering, with localized parallel arrangements which may be different within a single cell or even along the same wall (301) [this also applies to Zea root cells (201) and cotton hairs (343)]. In bean a slight predominance oflongitudinal microfibril arrangements has been observed after the colchicine treatment, and the epidermal cell walls assume a uniform rather than cross-polylamel­late structure (309). These results resemble the effect of anti-MT agents on Oocystis and are important in considering how the orientation with which microfibrils are deposited might be regulated by MTs.

Possible Orientation Mechanisms Facts that help in interpreting the coalignment of cortical MTs and wall microfibrils may be summarized as follows:

1 . Certain walls can develop and certain cells can be shaped to a high degree of specificity without benefit of cortical MTs [e.g. Micrasterias pri­mary and secondary walls (3 13), Closterium secondary walls (130), and cells with tip growth]. Similarly, wall microfibrils can be deposited in very highly ordered configurations without participation of MTs, most notably in algal "scales" (e.g. 44a).

2. Cell shaping may continue even though the cells have diminished or eliminated their population of cortical MTs. Thus there are very few MTs in a coleoptile segment but it continues to show "straight growth." The same probably applies during the major phase of elongation in roots ( 100, 338). MTs were not seen along walls undergoing specificially directed ex-

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pansion in the genesis of new apical cells for lateral roots in Ceratopteris (unpublished observations).

3. Artificial removal of MTs does not block cellulose synthesis (85, 293) and may not totally abolish oriented deposition of microfibrils. The contin­ued parallelism of microfibrils seen in Oocystis after selective removal of MTs by colchicine, APM, trifiuralin, and oryzalin (255) has its counterpart in higher plants, though perhaps only in local areas of cell wall (1 19, 1 37, 1 38, 201 , 223, 343). In other cases colchicine is more disruptive, eliciting random deposition and nonspecific shaping (78, 1 30, 180). Known cases of survival of specific shaping after colchicine treatment-formation of "pri­mordiomorphs" by cell expansion in the absence of mitosis (37. 67. 215)­deserve to be subjected to ultrastructural analysis. Like examples in the preceding parapraph, they could be accounted for if a prepattern mediated by MTs had developed early in development and was later expressed even though the MTs had disappeared either naturally or through drug treat­ment.

4. The highest densities of MTs occur in three situations: (a) when microfibril deposition is initiated, for example at newly formed walls in Azolla (100) and Sphagnum (282, 283); (b) in cells which increase their rate of wall deposition during differentiation [e.g. cotton hairs (343) and xylem and sieve elements (100)]; (c) during reorientation of the direction of cel­lulose deposition, for example during formation of a lateral axis (97). In only one case has a 1 : 1 relationship between MTs and microfibrils been found [at the growing edge of the lorica in Poterioochromonas (288)], though frequencies as high as 20 MTs p.m-1 of wall length have been seen (343).

5. Cortical MTs are short relative to the dimensions of the cell wall and most are shorter than the length of the "average" cellulose microfibril (see 1 14, 257); they may also be transitory, mobile. and subject to treadmilling. Their kinetic and molecular polarities have not been determined.

6. Unlike chitin synthetase, cellulose synthetase has not been isolated and made to function in vitro. However, there are plausible identifications of "terminal complexes" on microfibrils in several recent studies of plasma membranes by freeze-fracturing of turgid cells in the absence of cryo­protectants. The terminal complexes lie within the membrane and take various forms: a single globular particle, suggested to fit into the center of a rosette of other particles (see 200, 201); single rosettes (325); large aggre­gates of rosettes (79); or elongated triple rows of particles (24, 196). Apart from their location, the best evidence that these terminal complexes are involved in producing cellulose concerns Micrasterias cells that are under­going secondary wall formation; the more rosettes there are in line at the terminus of a microfibril, the wider the microfibril (79). Directionality of

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microfibril production in secondary walls of Micrasterias, where the large rafts of rosettes are found, is insensitive to colchicine, whereas the smaller terminal complexes, which might be expected to be more mobile, are in colchicine-sensitive cells or cells with no predominant orientation of mi­crofibrils. In only one case has the orientation of MTs and that of microfi­brils with attached terminal complexes been visualized in the same preparations: they are coaligned even though there may be heterogeneity of orientation within the sample (20 1).

The above considerations limit both the generality and the nature of any mechanism purporting to show how MTs might provide spatial guidance for cellulose deposition. Several proposals have been made, the first dating from 1971 ( 1 1 9). All involve speCUlation and all may contain elements of truth. Plants are sufficiently diverse to have adopted a variety of mecha­nisms to control something that is of such basic morphogenetic significance. A detailed critique of the proposals is available ( 1 14).

All proposals recognize that cellulose synthetases must move in the plane of the plasma membrane, trailing their cellulose behind them. Some propos­als see cortical MTs merely as guidance elements, the motive force coming from crystallization of cellulose, which effectively drives translational mo­tion of the synthetases along channels delimited by MTs that are bridged to the plasma membrane (25, 1 24). It has been pointed out that the kinetic force released by crystallizing cellulose chains into microfibrils is sufficient to propel an Acetobacter cell and should therefore be able to move a synthesizing complex in the plane of a fluid membrane ( 1 24). If adjacent microfibrils adhere to each other by hydrogen bonding. then cohorts of synthesizing complexes, themselves not tightly aggregated, might be moved in unison along preferred channels, thus accounting for the observed pro­duction of bundles of microfibrils and, through the inertia of the whole system, for survival of bundles that maintain, at least partially, their original orientations following removal of cortical MTs (25, 1 37, 1 38). The mi­crotubules can be viewed as providing fences beside or between which an unspecified number of microfibrils extend. The proposal also accounts for the importance and abundance of MTs at times when the orientation of deposition is being altered (see above). An earlier proposal can also be invoked. It was suggested that inter-MT bridging and sliding contracts the array in the long axis of the MTs so that, given fixed MT-plasma membrane links, the plasma membrane pulls away from the wall, against turgor forces, and thereby creates a local oriented channel for wall deposition (283). While this may not apply to uniform deposition over the face of a cell (see 1 14), it seems quite appropriate in cases where MTs congregate in high density -indeed micrographs that portray something very like this in developing xylem have appeared (88). It is not known whether the constituent MTs

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would have to be of different polarities,' or have differing properties (as in A and B subfibers in flagella), in order to interact with one another so as to produce contractility. Images of inter-MT bridges in "herringbone" arrangements are not inconsistent with inter-MT sliding and recent por­trayals of the mechanochemical cycle of dynein arms (268a). As discussed previously, the polarity of interphase cortical MTs is conjectural.

Another variation on MTs as guidance elements is that "oriented mem­brane flow," powered by actomyosin, drives cellulase synthetases along channels delimited by MTs [although the observed microfilaments were not coaligned with the MTs (20 1)]. In other proposals, cortical MTs participate in motility generation as well as guidance. Synthesizing complexes could be coupled directly to cortical MTs by bridge molecules (109, 289), presum­ably akin to those now known to link the outer doublets of flagella to tubulin in the flagellar membrane (15, 50). Externally applied particles or endoge­nous membrane components can be propelled at high rates along flagella, as if the underlying MTs were being used as guide tracks. This is what was envisaged in one of the proposals (109); the other takes into account the observation that cortical MTs are often too short to be effective guide tracks and suggests that whole MTs, with synthesizing complexes attached, move in the cell cortex, guided and powered by other cytoskeletal elements in­cluding actin microfilaments (289). Treadmilling, should it occur, might also generate movement. Finally, the original proposal (1 19, 1 2 1) differs in that the ends of the microfibrils (nowadays = sites of terminal complexes) are not directly linked to the MTs. Rather, the MTs are the seat of an unspecified form of force generation which creates a shear in the plasma membrane and so aligns nearby growing microfibrils.

Little concrete evidence is available on which to assess proposals that invoke force generation over and above that produced by crystallization of cellulose itself, but cytochalasin and phalloidin, both of which affect actin, do not disrupt oriented cellulose deposition in Allium stomata, though both penetrate into the cells (222). Methods for visualizing and measuring mem­brane flow and fluidity do now exist. It is to be hoped that they will soon be used on plant cell surfaces, for the architecture of cell walls is immensely important in the morphogenesis and general biology of plants. Additional information is badly needed to help elucidate mode(s) of microfibril deposi­tion and its regulation.

CONCLUDING COMMENTS

Tubulin is a highly conserved protein. The order that is embodied in its molecule finds expression in the specificity and variety of its interactions with cofactors and MAPs and above all in its capacity to generate a struc-

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684 GUNNING & HARDHAM

ture of higher order, the microtubule. Microtubules in turn emerge as mUltipurpose tools, used in the cell as skeletal and active elements. They do not function as individuals so much as in the form of the next level in the hierarchy of ordering that is founded upon tubulin, namely arrays of microtubules. Following the principle that order, once established, can be used to generate still higher levels of order, microtubule arrays either di­rectly or indirectly participate in ordering the shapes of cells and, as de­scribed above, of tissues and organs.

Of all the steps in this hierarchy, that of accurate deployment of MTs seems the most problematical. This review has been able to report substan­tial progress and healthy speculation in other areas, but there do seem to be spatial control systems about which very little is known. How does a cell establish an array ofMTs with a particular orientation? How does it switch to a new orientation? How does a cell that has been caused to become spherical, having lost its ordered transverse deposition of microfibrils through treatment with colchicine, recover its initial shaping system (78)? How has the memory been preserved? There are parallel problems in ani­mals cells [300]. The next review on plant microtubules in this series can be expected to contain much new knowledge on mechanisms involving microtubules, but the deeper problems of development may need profound conceptual advances.

ACKNOWLEDGMENTS

We thank all those who generously provided preprints of their recent work, also P. Hepler, R. McIntosh, D. Sabnis, E. Schnepf, and S. Wick for their helpful comments on the manuscript, which was written while A. R. H. was in receipt of an Australian Commonwealth Government Queen Elizabeth II Fellowship in the School of Botany, University of Melbourne.

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