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Microtubule-organizing centres in plants
reviews
Microtubule-organizing centres in plants , o o The organization of mierotubules into ordered arrays is essential for cell division and differentiation. Centrosomes have long been recognized as major components of this process in metazoans. Most plant cells, however, do not have such morphologically distinct organelles, although they do have functionally equivalent microtubuleorganizing centres. Recent discoveries have shed new light on these mysterious organelles. A key element is ~/-tubulin, which is ubiquitous in eukaryotes, including plants. Multiprotein complexes with ~-tubulin - '~/-somes' - probaby serve as templates for microtubule initiation. Microtubules can also self-organize into ordered arrays in the presence of motor proteins, providing evidence that the organization of microtubule arrays involves both nucleation and polymer translocation. These exciting findings pave the way for elucidating the identity of the diffuse microtubuleorganizing centres in plants and the principles of microtubule organization without centrosomes. icrotubules, which are dynamic polymers of a- and ~-tubulin heterodimers, operate in key developmental events, including cell division, growth and differentiation 1. Like dancers in a cytoplasmic ballet, they rearrange into new arrays according to each phase of the plant cell cycle. During G2, the interphase microtubule array is replaced by a preprophase band, which is soon replaced by two nuclear polar caps with microtubules oriented at right angles to the band. During prophase, the polar caps appear to give rise to the mitotic spindle. After anaphase, the spindle is replaced by the phragmoplast and, finally, a new cortical array is reinstated during the telophase-interphase transition. In cells that depart the division cycle (Go phase) and enter-the final act - differentiation - microtubules form cortical arrays with distinct geometry, including transversely oriented arrays in elongating plant tissues, banded patterns in epidermal cells and tracheary elements 1, or radial arrays in stomatal guard cells 2. Even in differentiating cells, microtubules still undergo dynamic interconversions 3. In mosses, ferns and zooidogamous gymnosperms, microtubules form spectacular, geometrically precise arrays during the differentiation of the motile sperm 4. How do microtubules generate such a multitude of patterns? Several basic principles have been established. First, microtubules have intrinsic polarity: a slow-growing (or slow-shrinking) minus end, and a fast-growing (or fastshrinking) plus end. Elongation is accomplished by the addition of heterodimers, with the ~-tubulin subunit containing an exchangeable GTP-binding site, at the growing end. Shrinkage, or loss of heterodimers, involves GTP hydrolysis and the loss of a GTP cap. Second, microtubules are highly dynamic. Stabilization of microtubules and organization into arrays is mediated by interactions with various partner molecules, termed microtubule-associated proteins 5. These may be structural, modulating microtubule dynamics and facilitating mechanical cross-linking with other organelles and cytoskeletal elements. Alternatively, they may be ATPpowered motor proteins ~, either travelling along the microtubules in a plus end or minus end direction and delivering specific cargo to specific locations, or driving intermicrotubular sliding. Finally, microtubule arrangements are facilitated by microtubule-organizing centres 7 (MTOCs),
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© 1997 Elsevier Science Ltd
entities that nucleate microtubules and influence their number, structure, polarity and spatial positioning s.
Morphological diversity of the microtubule-organizing centres The centrosome has long been regarded as the major microtubule organizer in metazoan cells s'9. Electron micrographs show that microtubules radiate from it in a star ('aster') formation. Following drug- or cold-induced depolymerization, new microtubules again originate from the centrosome. There is evidence that centrosomes nucleate the minus end of microtubules and orient the fast-growing plus ends distally. Morphologically and structurally diverse metazoan centrosomes share a common feature - a pair of centrioles surrounded by amorphous pericentriolar material. It is from this pericentriolar material that microtubules are nucleated 8. The centrosome, which remains in close association with the nucleus, replicates itself faithfully before each cell division, suggesting that it is an autonomous organelle. Plant cells do not have centrosomes, but they make flawless mitotic spindles and generate ordered interphase arrays. Do they contain organelles functionally homologous to the centrosome? Distinct microtubule-organizing centres are seen in algae and lower land plants in association with the nucleus. Two basal bodies function as templates for the assembly of fiage]lar axonemes in Chlamydomonas; remarkably, the axonemes are resorbed before mitosis, and the basal bodies convert into a pair of centrioles located at the poles of the mitotic spindle 1°. Another microtubuleorganizing centre, a multilayered structure, is involved in spermatogenesis in Characean algae and bryophytes. It generates a ribbon of microtubules, which is inserted at right angles to the lamellae of the Characean multilayered structure, but at a 45 ° angle in the bryophytes 1°, and it also carries two basal bodies. A distinct, spherical microtubuleorganizing centre - the blepharoplast - emerges during spermatogenous divisions in zooidogamous vascular plants (ferns, cycads and Ginkgo). It gives rise to a multilayered structure with a large ribbon of microtubules and cohorts of basal bodies4 (Fig. 1). Gigantic blepharoplasts, up to about 500 ~m in diameter and giving rise to many thousands of flagella, are typical for spermatogenesis in cycads. However, despite the suggestive location of blepharoplasts at the PII S1360-1385(97)01042-X
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reviews spindle poles of spermatogenous divisions, because they arise de novo they may be mere passengers rather than true spindle organizers. Clusters of small microtubule-organizing centres are involved in monoplastidic divisions in bryophytes and some primitive ferns 11.The nucleation and organization of spindle microtubules is associated entirely with the single plastid. Microtubules clearly emanate from close to the plastid envelope and, in plastids recovering from drug-induced depolymerization, microtubules again reassemble from numerous foci on the envelope. These microtubule-organizing centres are therefore particulate and dispersed, unlike the compact basal bodies, blepharoplasts and multilayered structures, although they still associate with a membranebound organelle. Conifers and flowering plants, along with acentriolar animal cells, such as early mouse embryos or Xenopus eggs8 (and including vegetative cells from the Charophytes upward to angiosperms), lack centrosomes or similarly distinct microtubule-organizing centres. Again, this has no apparent effect on cell division, raising a conceptual dilemma regarding the exact role of the centriolar centrosome. This dilemma was rationalized by Mazia 12in his illuminating concept of a 'flexible centrosome', where the nucleation of microtubules is accomplished by the amorphous pericentriolar material itself, regardless of the degree of its condensation or dispersion, or the presence of centrioles. Indeed, microtubule nucleation in plants is generally dispersed within the cell, possibly in relation to the evolution of rigid cell walls containing aligned microfibrils and the underlying, ordered cortical cytoskeleton1. Fittingly, the lack of a centriolar centrosome is in line not only with the absence of a centrally focused radial array of interphase microtubules, but also with a loosely distributed Golgi apparatus, which is in contrast to the microtubule motor-driven perinuclear location of Golgi in animal cells ~a. It is not surprising that the blepharoplasts, multilayered structures and basal bodies, which are all involved in the construction of a motile apparatus, have been lost, because a pollen tube that delivers the sperm to the egg has evolved in higher plants.
Fig. 1. Immunofluorescent images of developmental stages of spermatogenesis in the fern Pteridium, obtained with antibodies raised against [~-tubulin. (a) Spherical blepharoplasts (arrowheads) emerge at the spindle poles of a spermatogenous mitosis (left, prometaphase; right, metaphase). (b) A microtubule ribbon (R) extends from a multilayered structure (MLS) and carries basal bodies (arrow). (c) A microtubular ribbon with basal bodies (arrow) in the process of assembling flagellar axonemes (F). Bar represents 10 tzm. Reproduced, with permission, from Ref. 4.
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~,-Tubulin, the microtubule nucleator Many proteins have been implicated in microtubule nucleation. For metazoans, proteins that associate with centrosomes include microtubule-associated proteins, microtubule motors, other cytoskeletal components, pericentrin, elongation factor-l~, and nuclear matrix proteins s'9'14. Various regulatory proteins may also briefly associate with centrosomes, including epitopes that react with monoclonal antibody MPM-2, which recognizes phosphorylated proteins associated with centrosomes and other microtubuleorganizing centres ~5, and the cell division cycle regulators p34cdc2,p13sue1and cyclins 8. For plants, proteins that colocalize with perceived microtubule-organizing centres include: phosphoproteins that react with the antibody MPM-2 (Ref. 15); epitopes that react with autoimmune serum 5051, which recognizes pericentriolar material in animal cells (but see Ref. 16); and a 180-kDa protein detected with monoclonal antibody 6C6 that has been raised against isolated calf centrosomes 17. Centrin, a calcium-binding phosphoprotein, also colocalizes with certain plant microtubuleorganizing centres ~s'~9. However, centrin is absent from taxol-induced microtubule asters in metazoans 2°, and has
reviews been localized to the cytokinetic cell plate, but not the phragmoplast microtubules 21. Generally, the exact place, if any, for these proteins in the microtubule-organizing centre jigsaw puzzle remains to be established. Recent consensus has emerged that ~-tubulin, a unique member of the tubulin superfamily originally discovered as a suppressor of a ~-tubulin mutation in Aspergillus 2~'23, is essential for microtubule nucleation. It is now almost certain that ~-tubulin is the long sought, universal microtubule nucleator in morphologically diverse microtubuleorganizing centres: • Disruptions of the ~-tubulin gene in Aspergillus and yeast eliminate mitotic spindles and are lethal. • Xenopus sperm centrioles alone cannot nucleate astral microtubules, but are activated by recruiting ~-tubulin from egg extracts; conversely, immunodepletion of ~-tubulin inhibits the process2°. • ~-Tubulin localizes to 'classical' microtubule-organizing centres, such as centrosomes, centrioles, spindle poles and spindle-pole bodies23. • ~-Tubulin genes have been identified in various organisms, from yeast to mammals, indicating that it is widespread throughout the eukaryotes. • Nucleotide sequence analysis shows that ~-tubulin genes are highly conserved; indeed, a human ~-tubulin can rescue a yeast ~-tubulin mutant. • Experiments with labelled ~-tubulin confirm that it localizes to microtubule minus ends 23, which also fits neatly with the expected microtubule-organizing centre model. The exciting breakthrough in determining ~-tubulin's role in microtubule nucleation soon spread into the plant field. A ~-tubulin homologue was identified in various plant protein extracts using a peptide antibody raised against a conserved sequence of the ~-tubulin gene 24, although the immunoreactive polypeptides were unexpectedly-large - 58 kDa instead of the 48-50 kDa found in other eukaryotes. Immunofluorescence microscopy revealed an extensive, punctate staining pattern in association with microtubule arrays throughout the cell cycle24, with stronger labelling towards the expected microtubule minus ends ls'24'25. Some ~-tubulin signal may be located along microtubule bundles, presumably reflecting staggered microtubule minus ends; a secondary localization along individual microtubules may be transport or storage forms of ~-tubulin 25rather than incorporation into the ~ heterodimer lattice. An antibody, Rb27, raised to a ~tubulin peptide, specifically cross-reacts with ~-tubulin in algal basal bodies and mammalian centrioles 27. Sequencing of the first plant ~-tubulin gene from the fern Anemia revealed that it is approximately 20 amino acids longer than its fungal and metazoan counterparts 25. Subsequently, the genes TubG1 (encoding ~l-tubulin) and TubG2 (encoding 72tubulin) were isolated from Arabidopsis, each encoding a protein of approximately 53 kDa (see Ref. 25). A ~-tubulin gene has also been isolated from maize 23. Therefore, as in other eukaryotes, ~-tubulin is widespread throughout the plant kingdom 2s, Plant ~]-tubulin genes are highly conserved, sharing about 90% identity in amino acid sequence, and with only 60-70% identity to fungal, algal and metazoan ~-tubulins.
Dispersed plant microtubule-organizing centres: subcellular locations during the cell cycle The recognition of ~-tubulin as a universal microtubule nucleator has brought a fresh approach to the study of plant microtubule-organizing centres. Despite the absence of
Fig. 2. An immunofluorescent image of a wheat root-tip cell at prophase, stained with an antibody raised against ~tubulin. This is a polar view of a wide cell, surrounded by a preprophase band of microtubules and showing a polar cap with irregular margins around the central gap. Bar represents 10 t~m.Reproduced, with permission, from Ref 48.
visible morphological markers such as centrioles, it is now possible to locate microtubule-organizing centres within cells using antibodies raised against ~-tubulin. A consensus picture of microtubule-organizing centres in the various arrays has emerged, taking into account: ~-tubulin immunoreactions; loci of converging microtubules with vesicles and dense material, seen by electron microscopy; and results based on functional markers, such as points of microtubule reassembly after depolymerization. The mitotic apparatus During prophase, the nuclear envelope first develops uniform ~-tubulin staining, which progressively concentrates towards the opposite poles and eventually forms polar caps. Interestingly, each cap often has a central gap with an irregular margin (Fig. 2), which coincides with intense ~-tubulin staining 24. This gap may be a stage in a microtubule shift towards the pole, because the cap can also extend beyond the nucleus into a sharply focused pole29. Following breakdown of the nuclear envelope, the polar caps give rise to a broad ('anastral') prophase spindle, with most prominent staining accumulating along the kinetochore trunks and only a faint signal along the polar fibres tra= versing the equator u. As the kinetochore trunks shorten during anaphase, ~-tubulin staining also retreats towards the poles (Fig. 3a). Thus, microtubule-organizing centres are likely to redistribute along with the microtubules. The division site: the preprophase band and the phragmoplast The preprophase band has a pioneering role by marking out the cell division site. ~-Tubulin colocalizes with the band 24, and labelled tubulin incorporates into it 3°, suggesting that new microtubules are nucleated and assembled onsite. Nonetheless, the import of intact microtubules capped with ~-tubulin, from the cortex or from the nucleus, may contribute to the process. The cytokinetic phragmoplast facilitates the construction of the new cell wall. It consists of two sets of interdigitating June 1997, Vol. 2, No. 6
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reviews Furthermore, a phragmoplast can also form between non-sister nuclei 33, reinforcing the idea of its de novo origin. Nucleation of microtubules in interphase cells: perinuclear or cortical? The origin of cortical microtubules in interphase cells has been debated for some time. In the perinuclear model, microtubules initiate on the nuclear surface and then migrate to the cortex, where they are ordered into appropriate geometries by a separate mechanism 17'84'~5. Clearly, the nuclear envelope is a powerful microtubule nucleator and shows strong ~/-tubulin staining - particularly during preFig. 3. Onion root-tip cells stained with peptide antibodies raised against a conserved ~-tubulin sequence - EDFATQGGDRKDVFFY(J. Marc and T. Dibbayawan, prophase and prophase ~4'25. Isolated unpublished). (a) A cell at late anaphase, showing prominent accumulation of maize or tobacco nuclei have been immunoreactive signal at the spindle poles. (b) A cell at cytokinesis, showing two shown to initiate microtubules in vitro, rows of a punctate signal along the phragmoplast. Bar represents 10 ~m. although microinjected, fluorescently labelled tubulin failed to incorporate at the interphase nuclear envelope 1. antiparallel microtubules, their plus ends facing the cell Therefore, it remains to be established how many nuclei in plate, along with accessory motor proteins and other intact cells initiate microtubules during the G1 or Go cytoskeletal elements ~1. The origin of the phragmoplast is phases, when perinuclear ~-tubulin staining is weak and a unclear. Its initial, rod-shaped stage may arise from rem- new ~- and ~-tubulin pool is synthesized. nants of the spindle, although the subsequent ring-shaped In the cortical model, interphase microtubules are nuclestage is probably generated de novo by active microtubule- ated and ordered at the cell cortex 1. Foci of converging organizing centres. The phragmoplast ring grows centrifu- microtubules have been found along cell edges3~; microgally at about 0.2 ~m min 1, with a rapid microtubule tubules reassembling during recovery after depolymerizturnover (half-time of about 1 min; see Ref. 31). Again, the ation initiate from dispersed cortical sitesl; and microlocalization of ~/-tubulin (Fig. 3b) is redistributed along with injected, fluorescently labelled tubulin incorporates in the the microtubules of the expanding phragmoplast ring 24. cortex~7. Moreover, punctate ~/-tubulin staining is clearly also located in the cell cortex, including spots at the ends of microtubule bundles 2'~.Again, further analysis is needed to evaluate the generality of the cortical model for various tissues. A special case of cortical microtubule organization is seen in Alliurn stomatal guard cells2. Following division of the guard mother cell, new microtubules originate exclusively from a unique cortical domain along the new, longitudinal wall, and then elongate while spreading out into a radial array. In cells recovering from microtubule depolymerization, microtubules again regenerate from this domain, even if the nucleus has been displaced by centrifugation 2 (Fig. 4). The domain also reacts with antibodies against ~-tubulin 25, confirming that it represents an aggregation of microtubule-nucleating sites. Because it has a dual function - nucleation and spatial organization - the domain represents a true microtubule-organizing centre. Fig. 4. A pair of developingguard cells fromAllium cotyledons stained with an antibody raised against ~-tubulin [(b): differential interference contrast view of (a)]. The cotyledon was A ~,-tubulin ring complex: a template for microtubule incubated for 1 h with 200 ~M colchicineto depolymerizethe assembly microtubules completely,and then briefly centrifuged to sediHow does ~-tubutin nucleate microtubules? A ~/-tubulin ment the nuclei (n) to the bottom, along with small organelles ring model involving the nucleation of 13 protofilaments (vertical bar). Although only diffuse cytoplasmic staining was was proposed several years ago22, based on existing genetic, seen at first, after 10 min of ultraviolet irradiation (which biochemical and immunochemical evidence. Supporting evideactivates colchicine) a prominent layer of tubulin (arrowdence quickly followed. It was shown that Drosophila ~/heads) emerged on either side of the separating wall. The laytubulin binds to a tubulin affinity column as part of a proers are positioned centrally, even though the nuclei were distein complex8 and, similarly, Xenopus ~-tubulin binds to placed by the centrifugation. Bar represents 10 ~m. microtubules and to centrosomes as a large, 25S complex, or Reproduced, with permission, from Ref. 2. '~-some '2°. Recently, two papers have provided ultrastructural and biochemical evidence that ~/-tubulin does indeed 226
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reviews form a multiprotein ring complex (~/-TuRC). Moritz and colleagues "s used immune-electron microscopic tomography of Drosophila centrosomes to identify hundreds of microtubule-nucleating sites within the pericentriolar material. These were mostly ring-shaped, 25-30 nm in diameter and contained multiple copies of ~/-tubulin. Using Xenopus eggs, Zheng and colleagues ~ purified a 2000-kDa complex that consists of seven different proteins, including ~- and ~-tubulin and 10-13 molecules of ~-tubulin. The proposed ~/-TuRC model is a helical template that nucleates a three-start helix with a B-surface microtubule lattice and caps the microtubule minus end. Given the highly conserved nature of the tubulin superfamily, it is probable that other eukaryotes will also have followed this pattern 4°. A simple working model of ~-some assembly is shown in Box 1; assembly of microtubule-organizing centres and microtubule arrays is shown in Box 2. This outstanding breakthrough has elevated ~-tubulin to the status of the anticipated minus-end nucleator of microtubule assembly. Nonetheless, as a testimony to the rapid development of this area, the ~-TuRC model has already been challenged 4~. The counter-argument is that the microtubule minus end terminates with ~- instead of B-tubulin, and that ring structures are also formed by a- and B-tubulins, and by a bacterial relative of eukaryotic tubulins, FtsZ (Ref. 41). In the proposed alternative model, ~-tubulin forms a straight protofilament onto which a[~-tubulin heterodimers assemble laterally, forming a sheet. Because the lattice naturally curls, the edges of the laterally expanding sheet eventually touch and seal into a tubule 41. One could counter, however, that a straight, longitudinal ~-tubulin protofilament could not easily explain the specific number of ~ protofilaments in the tubule. Self-organization of microtubules into ordered arrays Ironically, while ~/-somes have clearly substantiated the function of microtubule-organizing centres in microtubule nucleation, another recent discovery has shaken the concept of the centrosome as a master microtubule organizer. In an experiment employing a mixture of plasmid DNA-coated beads, cytoplasmic extract of meiotic frog eggs and fluorescently labelled tubulin or polarity-marked microtubules, Heald and colleagues4~'4~showed that microtubules can selforganize into a bipolar spindle. Initially, microtubules assembled at random orientations on the surface of the beads. The microtubules then gradually coalesced, presumably by crosslinking with microtubule-associated proteins, into bundles of mixed parallel and antiparallel polarity. In the presence of the minus end-directed motor, cytoplasmic dynein, the mixed microtubules segregated by sliding with their minus ends in two opposite directions; these eventually formed a bipolar spindle, each half spindle containing microtubules of uniform polarity. This was achieved without centrosomes. This result is particularly gratifying for plant cell biologists. It is certainly consistent with the dispersed plant microtubule-organizing centres, but is also consistent with several types of microtubule behaviour that are arguably best explained by self-organization. First, various rearrangements ofmicrotubules are known to precede mitesis ~, including: transformation of three or more polar caps into two poles in some endosperm cells; extension of annular polar caps away from the nuclear envelope, drawing out into a tight focus; and invasion of microtubules into the nucleoplasm following breakdown of the nuclear envelope.
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Second, microtubuIes in Haemanthus endosperm and other plant cells can self-organize into 'fir-tree' configurations, with their presumed minus ends forming 'converging centres' that point towards the spindle poles33. Third, there are examples of ATP-driven sliding of antiparallel microtubules in the spindle and in isolated phragmoplasts, presumably involving the action of plus end- and minus enddirected motors 6. In addition to mitosis, 'fir trees' also appear during interphase in Haemanthus 3~ and, similarly, V- or Y-shaped structures or entire branching clusters appear in the cortex of Nitella 37, suggesting that selforganization operates throughout the cell cycle. Various reorientations of interphase cortical microtubules are also commonTM. It is therefore likely that microtubule selforganization does occur in plants, although relatively little is known about the partner proteins involved. June 1997, Voi. 2, No. 6
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reviews connections to anchoring points within the cell. In addition, the activity of microtubule-organizing centres may be modulated, possibly by reacting with proteins that act as a scaffold, such as (D Assembly of e - o r g a n i z ~ centres pericentrin, which is essential for the assembly of centrosomes 2°. Components of microtubule-organizing centres are also regulated by phosphorylations. Recruitment of ~/-tubulin to centrosomes requires ATP, presumably for the phosphorylation of centrosomal proteins 2°. Phosphoprotein epiMembrane Firm support Matrix topes that react with the MPM-2 A microtubule-organizing centre is envisaged as a cluster of several to many antibody have been localized to centrohundreds of v-somes. The ~/-somes(circled) within the cluster are presumably crosssomes, and also to the nuclear envelope, linked inone of three ways: by flexible or contractile fibrous proteins; by a complex spindle and phragmoplast in plants 15, matrix; or by being attached to membranes. A rigid attachment of ~-somes to a in a pattern that resembles the distrifirm support would facilitate precise ordering of microtubutes along the multibution of ~/-tubulin. The highly conlayered structure of bryophytes and ferns. Because of their large mass, isolated served regulators of the cell cycle, microtubule-organizing centres may sediment by a mild centrifugation (e.g. cyclin-dependent kinase p34 ode2and its 10000g for 10 min). tn immunofluorescence microscopy using antibodies raised regulatory subunit cyclin B, associate against ~/-tubulin, microtubule-organizmg centres would appear as dots or spots~.2~; immuno-electron microscopy would show large aggregates of gold both with centrosomes 9 and with particles. mitotic arrays in plants 45. Interestingly, microinjection of Chlamydomonas (2) 0 ation of mierotubules into arrays p34CdC2/cyclin B-like kinase into Tradescantia staminal hair cells induced a rapid degradation of the preprophase band 3~, although it is unknown whether the treatment Microtubule Membrane affected the nucleation of microtubules or their stability. MicrotubNe arrays generated by microtubule-organizing centres reflect the geomet~ of these centres. Individual microtubules may be released from microtubuteThe net product of nucleation is con°rganizing centresS, however, presumably with their minus ends still capped by founded by microtubule dynamics. The ~/-somes (circled~. Such mierat-ubutes may interact with structural microtubule~/-tubulin molecule may nucleate either assoeiated proteins (NAPs, shaded boxes), facilitating mntuat cross-linking or dynamically unstable or intrinsically a~achments to membranes'.5 Interactions with motor proteins 6 (cireles surrounded stable microtubules according to its by shaded boxes) facilitate intermicrotubule sliding or sliding over a solid supporL alternative conformational states, driving the self-organization ofmicrotubules into new arrays. which are regulated by the phosphorylation of a centrosomal protein 26. Microtubule dynamics in metazoans is also modulated by a variety of cytoplasmic and centrosomal Regulation of microtubule organization Microtubule organization involves their nucleation, microtubule-associated proteins 9, some of which involve cell dynamics, association with structural and motor proteins, cycle-dependent phosphorylation5. Microtubule-associated and interactions with other cytoskeletal elements, proteins have been described in plants, and currently organelles and membranes. How are all these processes include a 76 kDa protein from carrot, a 65 kDa protein from choreographed? This area is largely not understood, but tobacco, 50 and 100 kDa proteins from maize, elongation factor-l~ (Refs 1 and 46) and a 90 kDa protein recently isosome basic principles are emerging. Nucleation, the key step, is facilitated by the location and lated from tobacco membranes 47. The mechanism of action activity of the disperse microtubule-organizing centres and of these proteins remains to be established. possibly also free ~/-somes2°. Their essential component, soluble ~/-tubulin, is presumably the net product of its syn- Future directions Recent work has brought remarkable discoveries. We thesis, post-translational modifications and chaperoninassisted folding26'2s. In cultured animal cells, about half of now know that ~-tubulin is a key element of microtubulethe cellular ~-tubulin is distributed in the cytoplasm in a organizing centres, that it functions as a complex of several soluble forms'2°. In plants, the succession of distinct micro- proteins in stoichiometric proportions, that the complex is tubule arrays and corresponding rearrangements of ~/-tubu- probably ring-shaped and that it nucleates microtubule lin staining during the cell cycle mean that ~/-somes or assembly and caps the minus end. Microtubules can initiate microtubule-organizing centres in various stages of their from microtubule-organizing centres that are dispersed in construction (see Boxes 1 and 2) are relocated within the the cell, and assembled microtubules can self-organize into cell. It can be speculated that free ~/-somes may migrate a bipolar spindle, without a centriolar centrosome. These within the cytoplasm, ~/-somes still attached to assembled exciting findings open doors for further exploration. The microtubules may travel together during self-organization, basic challenge will be to identify plant ~/-somes and deterand whole microtubule-organizing centres may be pulled by mine their molecular composition. Given the disperse 228
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reviews nature of plant microtubule-organizing centres, it will be important to elucidate how ~-somes become organized into larger assemblies - microtubule-organizing centres - and how these are targeted to specific locations in the cell. We also need to know how the activity of microtubuleorganizing centres is regulated, and how dynamic microtubules become stabilized. Also, because microtubules can self-organize into arrays by interacting with partner proteins, we need to know who the partners are and what the necessary conditions are for initiating the interaction. The recent molecular advances put this research onto solid ground and set the stage for making further progress and new discoveries.
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Dedication Dedicated to the memory of Daniel Mazia.
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Acknowledgements
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Only a small proportion of the vast literature on microtubule-organizing centres is cited here because of space limitations; my apologies to all authors whose work could not be directly cited. I thank Dr John Harper for providing valuable suggestions on the manuscript, Dr Teresa Dibbayawan for preparing Fig. 3 and Dr Richard Cyr for valuable comments and suggestions on the manuscript, and for expert preparation of electronic versions of the figures. I also thank three anonymous reviewers for their suggestions. Financial support was provided by the Australian Research Council.
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endosperm cells of higher plant Haemanthus, Cell Motil. Cytoskeleton 31, 34-44 Harper, J.D.I. et al. (1989) Does the autoimmune serum 5051 specifically recognise microtubule organising centres in plant cells? Cell Biol. Int. 13, 471-483 Lambert, A-M. (1995) Microtubule-organizing centers in higher plants evolving concepts, Bot. Acta 108, 535-537 Hoffraan, J.C., Vaugtm, K.C. and Joshi, H.C. (1994) Structural and irmmmocytochemical characterization of microtubute orgmlizing centers in pteridephyte spermatogenous cells, Protoplasma 179, 46-60 Salisbury, J.L. (1995) Centrin, centrosomes, and mitotic spindle poles, Curr. Opin. Cell Biol. 7, 39-45 Stearns, T. and Kirschner, M. (1994) In vitro reconstruction of centrosome assembly and function: the central role of y-tubulin, Cell 76, 623-637 Del Vecchio, A.J. et al. Centrin in higher plants associates prominently with the developing cell plate, Protoplasma (in press) Oaldey, B.R. (1995) ~-Tubulin and the fungal microtubule cytoskeleton, Can. J. Bot. 73 (Suppl. 1), $352-$358 Joshi, H.C. and Palevitz, B.A. (1996) ~-Tubulin and microtubule organization in plants, Trends Cell Biol. 6, 41-44 Lin, B. et al. (1993) A ~-tubulin related protein associated with the microtubule arrays of higher plant cells in a cell cycle dependent manner, J. Cell Sci. 104, 1217-1228 Liu, B., Joshi, H.C. and Palevitz, B.A. (1995) Experimental manipulation of ~-tubulin distribution in Arabidopsis using anti-microtubule drugs, Cell Motil. Cytoskeleton 31, 113-129 Burns, R.G. (1995) Analysis of the ~/-tubulin sequences: implications for the functional properties of ~-tubulin, J. Cell Sci. 108, 2123-2130 Dibbayawan, T.P. et al. (1995) A ~-tubulin that associates specifically with centrioles in HeLa cells and the basal body complex in Chlamydomonas, Cell Biol. Int. 19, 559-567 Vassilev, A. et al. (1995) Identification of intrinsic dimer and overexpressed monomeric forms of ~-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas ~-tubufin sequence, J. Cell Sci. 108, 1083-1092 Baskin, T.I. and Cande, W.Z. (1990) The structm'e and function of the mitotic spindle in flowering plants, Annu. Rev. Plant Physiol. Plant Mol. Biol. 41, 277-315 Cleary, A.L. et al. (1992) Microtubules and F-actin dynamics at the division site in living Tradescantia stamen hair cells, J. Cell Sei. 103, 977-988 Staehelin, L.A. and Hepler, P.K. (1996) Cytokinesis in higher plants, Cell 84, 821-824 Hush, J. et al. (1996) Plant mitosis promoting factor disassembles the microtubule preprophase band and accelerates prophase progression in Tradescantia, Cell Biol. Int. 20, 275-287 Smirnova, E.A. and Bajer, A.S. (1994) Microtubule converging centers and reorganization of the interphase cfioskeleton and mitotic spindle in higher plant Haemanthus, Cell MotE. Cytoskeleton 27, 219-233 Mizuno, K. (1993) Microtubule-nucleation sites on nuclei of higher plant cells, Protoplasma 173, 77-85 Stoppin, V., Lambert, A-M. and Vantard, M. (1995) Plant microtubuleassociated proteins (MAPs) affect microtubule nucleation and growth at plant nuclei and mammalian centrosomes, Eur. J. Cell Biol. 69, 11-23 Gunning, B.E.S. and Hardham, A.R. (1982) Microtubules, Annu. Rev. Plant Physiol. 33, 651-698 Wasteneys, G.O., Gunning, B.E.S. and Hepler, P.K. (1993) Microinjection of fluorescent brain tubulin reveals dynamic properties of cortical microtubules in living plant cells, Cell MotE. Cytoskeleton 24, 205-213 Moritz, M. et al. (1995) Microtubule nucleation by ~-tubulin-containing rings in the centrosome, Nature 378, 638-640 Zheng, Y. et al. (1995) Nucleation of microtubule assembly by a ~-tubulin-containing ring complex, Nature 378, 578-583 June 1997,Vol 2, No. 6
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reviews 40 Detraves, C. et al. (1997) Protein complexes containing gamma-tubulin are present in mammalian brain microtubule protein preparation, Cell MotE. Cytoskeleton 36, 179-189 41 Erickson, H.P. and Stoffler, D. (1996) Protofilaments and rings, two conformations of the tubulin family conserved from bacterial FtsZ to ~/~ and ~ tubulin, J. Cell Biol. 135, 5-8 42 Heald, R. et al. (1996) Self-arganization of microtabules into bipolar spindles around artificial chromosomes in Xenopus egg extracts, Nature 382, 420-425 43 Hyman, A.A. and Karsenti, E. (1996) Morphogenetic properties of microtubules and mitotic spindle assembly, Cell 84, 401-410 44 Palevitz, B.A. (1991) Potential significance ofmicrotubule rearrangement, translocation and reutilization in plant cells, in The Cytosketetal Basis of Plant Growth and Form (Lloyd, C.W., ed.), pp. 45-55, Academic Press
45 Hepler, P.K. and Hush, J.M. (1996) Behavior of microtubules in living
plant cells, Plant Physiol. 112, 455-461 46 Durso, N.A., Leslie, J.D. and Cyr, R.J. (1996) In situ immunocytochemical evidence that a homolog of protein translation elongation factor EF-la is associated with microtubules in carrot cells, Protoptasma 190, 141-150 47 Marc, J. et al. (1996) Isolation ofa 90-kD microtubule-associated protein from tobacco membranes, Plant Cell 8, 2127-2138 48 Marc, J. and Gunning, B.E.S. (1988) Monoclonal antibodies to a fern spermatozoid detect novel components of the mitotic and cytokinetic apparatus in higher plant cells, Protoplasma 142, 15-24
Jan Ma~C:i~at the SChoOlof BiOlOgiCalSCiencesl Sydney Sydhey 2006
Regulatory phosphorylation of C 4 PEP carboxylase Joo° v.,o,oo,,oyoo,c,o..o. PEP carboxylase is of paramount importance in plant metabolism, and is one of only a few enzymes known to undergo regulatory phosphorylation in the living plant. In illuminated leaves of C4 species, a complex light-signal transduction cascade occurs, which possibly involves cross-talk between the two neighboring photosynthetic cell types. This process upregulates the activity of a Ca2*-independent, substrate-specific protein-Serfrhr kinase, and thereby increases the phosphorylation state of the photosynthesis-related, C4 PEP carboxylase isoform. This reversible covalent modification is superimposed on the opposing regulation of PEP carboxylase by carboxylic acids and phosphorylated metabolites, and is critical for the coordination and functioning of C4 photosynthesis. The general occurrence of PEP carboxylase-kinase and the conserved N-terminal phosphorylation domain of the various plant PEP carboxylase isoforms support the hypothesis that similar regulatory mechanisms are functional in the diverse physiological contexts involving this enzyme. he enzyme PEP (phosphoenolpyruvate) carboxylase (EC 4.1.1.31) is widely distributed in plant tissues, green algae and microorganisms, but is absent in animals'. It was first characterized in spinach leaves in 1953 (Ref. 2)~ and for a long time was considered a subsidiary plant carboxylase alongside Rubisco. However, the discovery of C4 photosynthesis in the mid-1960s and the involvement of a specific isoform of PEP carboxylase in this pathway [which led to the general photosynthesis groupings of C3, C4 and CAM (Crassulacean acid metabolism)] considerably boosted interest in this cytosolic enzymes. The ubiquitous occurrence of PEP carboxylase in plant cells and tissues and its multifaceted functions have since been established, and it is now recognized as playing a major role in plant metabolism. More recently, the extensive development and use of molecular and biochemical techniques has generated an impressive wealth of data, and significantly advanced our understanding of the nature of the regulatory processes for this enzyme.
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Properties of C 4 PEP carboxylase PEP carboxylase catalyzes the exergonic ~-carboxylation of PEP by HCQ (AG = - 7 kcal tool -~) in the presence of a divalent cation, which is generally Mg2+. The reaction proceeds through a stepwise mechanism that involves the reversible, rate-limiting formation of carboxyphosphate and 2:30
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the enolate of pyruvate (Fig. 1). An important mechanistic aspect not recognized in earlier studies is the decomposition of carboxyphosphate into inorganic phosphate and free CO2 within the active site, the latter then reacting with the enolate species to form the product oxaloacetate (OAA) (Ref. 4). PEP carboxylase is a homotetramer with each subunit having an approximate size of 110 kDa (Ref. 1). Because of its very low Kmfor the substrate bicarbonate (in the micromolar range), PEP carboxylase serves a general role in the carbon economy of plant cells by recapturing respiratory CO2. Several more specialized functions involve1'4-6: • Replenishing tricarboxylic acid cycle intermediates (an anaplerotic function). • A pH star. • Guard cell movements. • Nitrogen assimilation in C3 leaves. • Nitrogen fixation in legume root nodules. • Seed formation and maturation. • Fruit ripening. • C4 and CAM photosynthesis. Several isoforms of PEP carboxylase have been character. ized that presumably fulfil these specific tasks 5. Experiments with recombinant C4 PEP carboxylase from Sorghum have confirmed that it is subject to opposing feedback inhibition by the end-product L-malate (Ki = 0.17 raM) and atlosteric activation by glucose-6-phosphate
© 1997 Elsevier Science Ltd
PII $1360-1385(97)01046-7
Microtubule-organizing centres in plants , o o The organization of mierotubules into ordered arrays is essential for cell division and differentiation. Centrosomes have long been recognized as major components of this process in metazoans. Most plant cells, however, do not have such morphologically distinct organelles, although they do have functionally equivalent microtubuleorganizing centres. Recent discoveries have shed new light on these mysterious organelles. A key element is ~/-tubulin, which is ubiquitous in eukaryotes, including plants. Multiprotein complexes with ~-tubulin - '~/-somes' - probaby serve as templates for microtubule initiation. Microtubules can also self-organize into ordered arrays in the presence of motor proteins, providing evidence that the organization of microtubule arrays involves both nucleation and polymer translocation. These exciting findings pave the way for elucidating the identity of the diffuse microtubuleorganizing centres in plants and the principles of microtubule organization without centrosomes. icrotubules, which are dynamic polymers of a- and ~-tubulin heterodimers, operate in key developmental events, including cell division, growth and differentiation 1. Like dancers in a cytoplasmic ballet, they rearrange into new arrays according to each phase of the plant cell cycle. During G2, the interphase microtubule array is replaced by a preprophase band, which is soon replaced by two nuclear polar caps with microtubules oriented at right angles to the band. During prophase, the polar caps appear to give rise to the mitotic spindle. After anaphase, the spindle is replaced by the phragmoplast and, finally, a new cortical array is reinstated during the telophase-interphase transition. In cells that depart the division cycle (Go phase) and enter-the final act - differentiation - microtubules form cortical arrays with distinct geometry, including transversely oriented arrays in elongating plant tissues, banded patterns in epidermal cells and tracheary elements 1, or radial arrays in stomatal guard cells 2. Even in differentiating cells, microtubules still undergo dynamic interconversions 3. In mosses, ferns and zooidogamous gymnosperms, microtubules form spectacular, geometrically precise arrays during the differentiation of the motile sperm 4. How do microtubules generate such a multitude of patterns? Several basic principles have been established. First, microtubules have intrinsic polarity: a slow-growing (or slow-shrinking) minus end, and a fast-growing (or fastshrinking) plus end. Elongation is accomplished by the addition of heterodimers, with the ~-tubulin subunit containing an exchangeable GTP-binding site, at the growing end. Shrinkage, or loss of heterodimers, involves GTP hydrolysis and the loss of a GTP cap. Second, microtubules are highly dynamic. Stabilization of microtubules and organization into arrays is mediated by interactions with various partner molecules, termed microtubule-associated proteins 5. These may be structural, modulating microtubule dynamics and facilitating mechanical cross-linking with other organelles and cytoskeletal elements. Alternatively, they may be ATPpowered motor proteins ~, either travelling along the microtubules in a plus end or minus end direction and delivering specific cargo to specific locations, or driving intermicrotubular sliding. Finally, microtubule arrangements are facilitated by microtubule-organizing centres 7 (MTOCs),
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© 1997 Elsevier Science Ltd
entities that nucleate microtubules and influence their number, structure, polarity and spatial positioning s.
Morphological diversity of the microtubule-organizing centres The centrosome has long been regarded as the major microtubule organizer in metazoan cells s'9. Electron micrographs show that microtubules radiate from it in a star ('aster') formation. Following drug- or cold-induced depolymerization, new microtubules again originate from the centrosome. There is evidence that centrosomes nucleate the minus end of microtubules and orient the fast-growing plus ends distally. Morphologically and structurally diverse metazoan centrosomes share a common feature - a pair of centrioles surrounded by amorphous pericentriolar material. It is from this pericentriolar material that microtubules are nucleated 8. The centrosome, which remains in close association with the nucleus, replicates itself faithfully before each cell division, suggesting that it is an autonomous organelle. Plant cells do not have centrosomes, but they make flawless mitotic spindles and generate ordered interphase arrays. Do they contain organelles functionally homologous to the centrosome? Distinct microtubule-organizing centres are seen in algae and lower land plants in association with the nucleus. Two basal bodies function as templates for the assembly of fiage]lar axonemes in Chlamydomonas; remarkably, the axonemes are resorbed before mitosis, and the basal bodies convert into a pair of centrioles located at the poles of the mitotic spindle 1°. Another microtubuleorganizing centre, a multilayered structure, is involved in spermatogenesis in Characean algae and bryophytes. It generates a ribbon of microtubules, which is inserted at right angles to the lamellae of the Characean multilayered structure, but at a 45 ° angle in the bryophytes 1°, and it also carries two basal bodies. A distinct, spherical microtubuleorganizing centre - the blepharoplast - emerges during spermatogenous divisions in zooidogamous vascular plants (ferns, cycads and Ginkgo). It gives rise to a multilayered structure with a large ribbon of microtubules and cohorts of basal bodies4 (Fig. 1). Gigantic blepharoplasts, up to about 500 ~m in diameter and giving rise to many thousands of flagella, are typical for spermatogenesis in cycads. However, despite the suggestive location of blepharoplasts at the PII S1360-1385(97)01042-X
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reviews spindle poles of spermatogenous divisions, because they arise de novo they may be mere passengers rather than true spindle organizers. Clusters of small microtubule-organizing centres are involved in monoplastidic divisions in bryophytes and some primitive ferns 11.The nucleation and organization of spindle microtubules is associated entirely with the single plastid. Microtubules clearly emanate from close to the plastid envelope and, in plastids recovering from drug-induced depolymerization, microtubules again reassemble from numerous foci on the envelope. These microtubule-organizing centres are therefore particulate and dispersed, unlike the compact basal bodies, blepharoplasts and multilayered structures, although they still associate with a membranebound organelle. Conifers and flowering plants, along with acentriolar animal cells, such as early mouse embryos or Xenopus eggs8 (and including vegetative cells from the Charophytes upward to angiosperms), lack centrosomes or similarly distinct microtubule-organizing centres. Again, this has no apparent effect on cell division, raising a conceptual dilemma regarding the exact role of the centriolar centrosome. This dilemma was rationalized by Mazia 12in his illuminating concept of a 'flexible centrosome', where the nucleation of microtubules is accomplished by the amorphous pericentriolar material itself, regardless of the degree of its condensation or dispersion, or the presence of centrioles. Indeed, microtubule nucleation in plants is generally dispersed within the cell, possibly in relation to the evolution of rigid cell walls containing aligned microfibrils and the underlying, ordered cortical cytoskeleton1. Fittingly, the lack of a centriolar centrosome is in line not only with the absence of a centrally focused radial array of interphase microtubules, but also with a loosely distributed Golgi apparatus, which is in contrast to the microtubule motor-driven perinuclear location of Golgi in animal cells ~a. It is not surprising that the blepharoplasts, multilayered structures and basal bodies, which are all involved in the construction of a motile apparatus, have been lost, because a pollen tube that delivers the sperm to the egg has evolved in higher plants.
Fig. 1. Immunofluorescent images of developmental stages of spermatogenesis in the fern Pteridium, obtained with antibodies raised against [~-tubulin. (a) Spherical blepharoplasts (arrowheads) emerge at the spindle poles of a spermatogenous mitosis (left, prometaphase; right, metaphase). (b) A microtubule ribbon (R) extends from a multilayered structure (MLS) and carries basal bodies (arrow). (c) A microtubular ribbon with basal bodies (arrow) in the process of assembling flagellar axonemes (F). Bar represents 10 tzm. Reproduced, with permission, from Ref. 4.
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~,-Tubulin, the microtubule nucleator Many proteins have been implicated in microtubule nucleation. For metazoans, proteins that associate with centrosomes include microtubule-associated proteins, microtubule motors, other cytoskeletal components, pericentrin, elongation factor-l~, and nuclear matrix proteins s'9'14. Various regulatory proteins may also briefly associate with centrosomes, including epitopes that react with monoclonal antibody MPM-2, which recognizes phosphorylated proteins associated with centrosomes and other microtubuleorganizing centres ~5, and the cell division cycle regulators p34cdc2,p13sue1and cyclins 8. For plants, proteins that colocalize with perceived microtubule-organizing centres include: phosphoproteins that react with the antibody MPM-2 (Ref. 15); epitopes that react with autoimmune serum 5051, which recognizes pericentriolar material in animal cells (but see Ref. 16); and a 180-kDa protein detected with monoclonal antibody 6C6 that has been raised against isolated calf centrosomes 17. Centrin, a calcium-binding phosphoprotein, also colocalizes with certain plant microtubuleorganizing centres ~s'~9. However, centrin is absent from taxol-induced microtubule asters in metazoans 2°, and has
reviews been localized to the cytokinetic cell plate, but not the phragmoplast microtubules 21. Generally, the exact place, if any, for these proteins in the microtubule-organizing centre jigsaw puzzle remains to be established. Recent consensus has emerged that ~-tubulin, a unique member of the tubulin superfamily originally discovered as a suppressor of a ~-tubulin mutation in Aspergillus 2~'23, is essential for microtubule nucleation. It is now almost certain that ~-tubulin is the long sought, universal microtubule nucleator in morphologically diverse microtubuleorganizing centres: • Disruptions of the ~-tubulin gene in Aspergillus and yeast eliminate mitotic spindles and are lethal. • Xenopus sperm centrioles alone cannot nucleate astral microtubules, but are activated by recruiting ~-tubulin from egg extracts; conversely, immunodepletion of ~-tubulin inhibits the process2°. • ~-Tubulin localizes to 'classical' microtubule-organizing centres, such as centrosomes, centrioles, spindle poles and spindle-pole bodies23. • ~-Tubulin genes have been identified in various organisms, from yeast to mammals, indicating that it is widespread throughout the eukaryotes. • Nucleotide sequence analysis shows that ~-tubulin genes are highly conserved; indeed, a human ~-tubulin can rescue a yeast ~-tubulin mutant. • Experiments with labelled ~-tubulin confirm that it localizes to microtubule minus ends 23, which also fits neatly with the expected microtubule-organizing centre model. The exciting breakthrough in determining ~-tubulin's role in microtubule nucleation soon spread into the plant field. A ~-tubulin homologue was identified in various plant protein extracts using a peptide antibody raised against a conserved sequence of the ~-tubulin gene 24, although the immunoreactive polypeptides were unexpectedly-large - 58 kDa instead of the 48-50 kDa found in other eukaryotes. Immunofluorescence microscopy revealed an extensive, punctate staining pattern in association with microtubule arrays throughout the cell cycle24, with stronger labelling towards the expected microtubule minus ends ls'24'25. Some ~-tubulin signal may be located along microtubule bundles, presumably reflecting staggered microtubule minus ends; a secondary localization along individual microtubules may be transport or storage forms of ~-tubulin 25rather than incorporation into the ~ heterodimer lattice. An antibody, Rb27, raised to a ~tubulin peptide, specifically cross-reacts with ~-tubulin in algal basal bodies and mammalian centrioles 27. Sequencing of the first plant ~-tubulin gene from the fern Anemia revealed that it is approximately 20 amino acids longer than its fungal and metazoan counterparts 25. Subsequently, the genes TubG1 (encoding ~l-tubulin) and TubG2 (encoding 72tubulin) were isolated from Arabidopsis, each encoding a protein of approximately 53 kDa (see Ref. 25). A ~-tubulin gene has also been isolated from maize 23. Therefore, as in other eukaryotes, ~-tubulin is widespread throughout the plant kingdom 2s, Plant ~]-tubulin genes are highly conserved, sharing about 90% identity in amino acid sequence, and with only 60-70% identity to fungal, algal and metazoan ~-tubulins.
Dispersed plant microtubule-organizing centres: subcellular locations during the cell cycle The recognition of ~-tubulin as a universal microtubule nucleator has brought a fresh approach to the study of plant microtubule-organizing centres. Despite the absence of
Fig. 2. An immunofluorescent image of a wheat root-tip cell at prophase, stained with an antibody raised against ~tubulin. This is a polar view of a wide cell, surrounded by a preprophase band of microtubules and showing a polar cap with irregular margins around the central gap. Bar represents 10 t~m.Reproduced, with permission, from Ref 48.
visible morphological markers such as centrioles, it is now possible to locate microtubule-organizing centres within cells using antibodies raised against ~-tubulin. A consensus picture of microtubule-organizing centres in the various arrays has emerged, taking into account: ~-tubulin immunoreactions; loci of converging microtubules with vesicles and dense material, seen by electron microscopy; and results based on functional markers, such as points of microtubule reassembly after depolymerization. The mitotic apparatus During prophase, the nuclear envelope first develops uniform ~-tubulin staining, which progressively concentrates towards the opposite poles and eventually forms polar caps. Interestingly, each cap often has a central gap with an irregular margin (Fig. 2), which coincides with intense ~-tubulin staining 24. This gap may be a stage in a microtubule shift towards the pole, because the cap can also extend beyond the nucleus into a sharply focused pole29. Following breakdown of the nuclear envelope, the polar caps give rise to a broad ('anastral') prophase spindle, with most prominent staining accumulating along the kinetochore trunks and only a faint signal along the polar fibres tra= versing the equator u. As the kinetochore trunks shorten during anaphase, ~-tubulin staining also retreats towards the poles (Fig. 3a). Thus, microtubule-organizing centres are likely to redistribute along with the microtubules. The division site: the preprophase band and the phragmoplast The preprophase band has a pioneering role by marking out the cell division site. ~-Tubulin colocalizes with the band 24, and labelled tubulin incorporates into it 3°, suggesting that new microtubules are nucleated and assembled onsite. Nonetheless, the import of intact microtubules capped with ~-tubulin, from the cortex or from the nucleus, may contribute to the process. The cytokinetic phragmoplast facilitates the construction of the new cell wall. It consists of two sets of interdigitating June 1997, Vol. 2, No. 6
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reviews Furthermore, a phragmoplast can also form between non-sister nuclei 33, reinforcing the idea of its de novo origin. Nucleation of microtubules in interphase cells: perinuclear or cortical? The origin of cortical microtubules in interphase cells has been debated for some time. In the perinuclear model, microtubules initiate on the nuclear surface and then migrate to the cortex, where they are ordered into appropriate geometries by a separate mechanism 17'84'~5. Clearly, the nuclear envelope is a powerful microtubule nucleator and shows strong ~/-tubulin staining - particularly during preFig. 3. Onion root-tip cells stained with peptide antibodies raised against a conserved ~-tubulin sequence - EDFATQGGDRKDVFFY(J. Marc and T. Dibbayawan, prophase and prophase ~4'25. Isolated unpublished). (a) A cell at late anaphase, showing prominent accumulation of maize or tobacco nuclei have been immunoreactive signal at the spindle poles. (b) A cell at cytokinesis, showing two shown to initiate microtubules in vitro, rows of a punctate signal along the phragmoplast. Bar represents 10 ~m. although microinjected, fluorescently labelled tubulin failed to incorporate at the interphase nuclear envelope 1. antiparallel microtubules, their plus ends facing the cell Therefore, it remains to be established how many nuclei in plate, along with accessory motor proteins and other intact cells initiate microtubules during the G1 or Go cytoskeletal elements ~1. The origin of the phragmoplast is phases, when perinuclear ~-tubulin staining is weak and a unclear. Its initial, rod-shaped stage may arise from rem- new ~- and ~-tubulin pool is synthesized. nants of the spindle, although the subsequent ring-shaped In the cortical model, interphase microtubules are nuclestage is probably generated de novo by active microtubule- ated and ordered at the cell cortex 1. Foci of converging organizing centres. The phragmoplast ring grows centrifu- microtubules have been found along cell edges3~; microgally at about 0.2 ~m min 1, with a rapid microtubule tubules reassembling during recovery after depolymerizturnover (half-time of about 1 min; see Ref. 31). Again, the ation initiate from dispersed cortical sitesl; and microlocalization of ~/-tubulin (Fig. 3b) is redistributed along with injected, fluorescently labelled tubulin incorporates in the the microtubules of the expanding phragmoplast ring 24. cortex~7. Moreover, punctate ~/-tubulin staining is clearly also located in the cell cortex, including spots at the ends of microtubule bundles 2'~.Again, further analysis is needed to evaluate the generality of the cortical model for various tissues. A special case of cortical microtubule organization is seen in Alliurn stomatal guard cells2. Following division of the guard mother cell, new microtubules originate exclusively from a unique cortical domain along the new, longitudinal wall, and then elongate while spreading out into a radial array. In cells recovering from microtubule depolymerization, microtubules again regenerate from this domain, even if the nucleus has been displaced by centrifugation 2 (Fig. 4). The domain also reacts with antibodies against ~-tubulin 25, confirming that it represents an aggregation of microtubule-nucleating sites. Because it has a dual function - nucleation and spatial organization - the domain represents a true microtubule-organizing centre. Fig. 4. A pair of developingguard cells fromAllium cotyledons stained with an antibody raised against ~-tubulin [(b): differential interference contrast view of (a)]. The cotyledon was A ~,-tubulin ring complex: a template for microtubule incubated for 1 h with 200 ~M colchicineto depolymerizethe assembly microtubules completely,and then briefly centrifuged to sediHow does ~-tubutin nucleate microtubules? A ~/-tubulin ment the nuclei (n) to the bottom, along with small organelles ring model involving the nucleation of 13 protofilaments (vertical bar). Although only diffuse cytoplasmic staining was was proposed several years ago22, based on existing genetic, seen at first, after 10 min of ultraviolet irradiation (which biochemical and immunochemical evidence. Supporting evideactivates colchicine) a prominent layer of tubulin (arrowdence quickly followed. It was shown that Drosophila ~/heads) emerged on either side of the separating wall. The laytubulin binds to a tubulin affinity column as part of a proers are positioned centrally, even though the nuclei were distein complex8 and, similarly, Xenopus ~-tubulin binds to placed by the centrifugation. Bar represents 10 ~m. microtubules and to centrosomes as a large, 25S complex, or Reproduced, with permission, from Ref. 2. '~-some '2°. Recently, two papers have provided ultrastructural and biochemical evidence that ~/-tubulin does indeed 226
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reviews form a multiprotein ring complex (~/-TuRC). Moritz and colleagues "s used immune-electron microscopic tomography of Drosophila centrosomes to identify hundreds of microtubule-nucleating sites within the pericentriolar material. These were mostly ring-shaped, 25-30 nm in diameter and contained multiple copies of ~/-tubulin. Using Xenopus eggs, Zheng and colleagues ~ purified a 2000-kDa complex that consists of seven different proteins, including ~- and ~-tubulin and 10-13 molecules of ~-tubulin. The proposed ~/-TuRC model is a helical template that nucleates a three-start helix with a B-surface microtubule lattice and caps the microtubule minus end. Given the highly conserved nature of the tubulin superfamily, it is probable that other eukaryotes will also have followed this pattern 4°. A simple working model of ~-some assembly is shown in Box 1; assembly of microtubule-organizing centres and microtubule arrays is shown in Box 2. This outstanding breakthrough has elevated ~-tubulin to the status of the anticipated minus-end nucleator of microtubule assembly. Nonetheless, as a testimony to the rapid development of this area, the ~-TuRC model has already been challenged 4~. The counter-argument is that the microtubule minus end terminates with ~- instead of B-tubulin, and that ring structures are also formed by a- and B-tubulins, and by a bacterial relative of eukaryotic tubulins, FtsZ (Ref. 41). In the proposed alternative model, ~-tubulin forms a straight protofilament onto which a[~-tubulin heterodimers assemble laterally, forming a sheet. Because the lattice naturally curls, the edges of the laterally expanding sheet eventually touch and seal into a tubule 41. One could counter, however, that a straight, longitudinal ~-tubulin protofilament could not easily explain the specific number of ~ protofilaments in the tubule. Self-organization of microtubules into ordered arrays Ironically, while ~/-somes have clearly substantiated the function of microtubule-organizing centres in microtubule nucleation, another recent discovery has shaken the concept of the centrosome as a master microtubule organizer. In an experiment employing a mixture of plasmid DNA-coated beads, cytoplasmic extract of meiotic frog eggs and fluorescently labelled tubulin or polarity-marked microtubules, Heald and colleagues4~'4~showed that microtubules can selforganize into a bipolar spindle. Initially, microtubules assembled at random orientations on the surface of the beads. The microtubules then gradually coalesced, presumably by crosslinking with microtubule-associated proteins, into bundles of mixed parallel and antiparallel polarity. In the presence of the minus end-directed motor, cytoplasmic dynein, the mixed microtubules segregated by sliding with their minus ends in two opposite directions; these eventually formed a bipolar spindle, each half spindle containing microtubules of uniform polarity. This was achieved without centrosomes. This result is particularly gratifying for plant cell biologists. It is certainly consistent with the dispersed plant microtubule-organizing centres, but is also consistent with several types of microtubule behaviour that are arguably best explained by self-organization. First, various rearrangements ofmicrotubules are known to precede mitesis ~, including: transformation of three or more polar caps into two poles in some endosperm cells; extension of annular polar caps away from the nuclear envelope, drawing out into a tight focus; and invasion of microtubules into the nucleoplasm following breakdown of the nuclear envelope.
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Second, microtubuIes in Haemanthus endosperm and other plant cells can self-organize into 'fir-tree' configurations, with their presumed minus ends forming 'converging centres' that point towards the spindle poles33. Third, there are examples of ATP-driven sliding of antiparallel microtubules in the spindle and in isolated phragmoplasts, presumably involving the action of plus end- and minus enddirected motors 6. In addition to mitosis, 'fir trees' also appear during interphase in Haemanthus 3~ and, similarly, V- or Y-shaped structures or entire branching clusters appear in the cortex of Nitella 37, suggesting that selforganization operates throughout the cell cycle. Various reorientations of interphase cortical microtubules are also commonTM. It is therefore likely that microtubule selforganization does occur in plants, although relatively little is known about the partner proteins involved. June 1997, Voi. 2, No. 6
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reviews connections to anchoring points within the cell. In addition, the activity of microtubule-organizing centres may be modulated, possibly by reacting with proteins that act as a scaffold, such as (D Assembly of e - o r g a n i z ~ centres pericentrin, which is essential for the assembly of centrosomes 2°. Components of microtubule-organizing centres are also regulated by phosphorylations. Recruitment of ~/-tubulin to centrosomes requires ATP, presumably for the phosphorylation of centrosomal proteins 2°. Phosphoprotein epiMembrane Firm support Matrix topes that react with the MPM-2 A microtubule-organizing centre is envisaged as a cluster of several to many antibody have been localized to centrohundreds of v-somes. The ~/-somes(circled) within the cluster are presumably crosssomes, and also to the nuclear envelope, linked inone of three ways: by flexible or contractile fibrous proteins; by a complex spindle and phragmoplast in plants 15, matrix; or by being attached to membranes. A rigid attachment of ~-somes to a in a pattern that resembles the distrifirm support would facilitate precise ordering of microtubutes along the multibution of ~/-tubulin. The highly conlayered structure of bryophytes and ferns. Because of their large mass, isolated served regulators of the cell cycle, microtubule-organizing centres may sediment by a mild centrifugation (e.g. cyclin-dependent kinase p34 ode2and its 10000g for 10 min). tn immunofluorescence microscopy using antibodies raised regulatory subunit cyclin B, associate against ~/-tubulin, microtubule-organizmg centres would appear as dots or spots~.2~; immuno-electron microscopy would show large aggregates of gold both with centrosomes 9 and with particles. mitotic arrays in plants 45. Interestingly, microinjection of Chlamydomonas (2) 0 ation of mierotubules into arrays p34CdC2/cyclin B-like kinase into Tradescantia staminal hair cells induced a rapid degradation of the preprophase band 3~, although it is unknown whether the treatment Microtubule Membrane affected the nucleation of microtubules or their stability. MicrotubNe arrays generated by microtubule-organizing centres reflect the geomet~ of these centres. Individual microtubules may be released from microtubuteThe net product of nucleation is con°rganizing centresS, however, presumably with their minus ends still capped by founded by microtubule dynamics. The ~/-somes (circled~. Such mierat-ubutes may interact with structural microtubule~/-tubulin molecule may nucleate either assoeiated proteins (NAPs, shaded boxes), facilitating mntuat cross-linking or dynamically unstable or intrinsically a~achments to membranes'.5 Interactions with motor proteins 6 (cireles surrounded stable microtubules according to its by shaded boxes) facilitate intermicrotubule sliding or sliding over a solid supporL alternative conformational states, driving the self-organization ofmicrotubules into new arrays. which are regulated by the phosphorylation of a centrosomal protein 26. Microtubule dynamics in metazoans is also modulated by a variety of cytoplasmic and centrosomal Regulation of microtubule organization Microtubule organization involves their nucleation, microtubule-associated proteins 9, some of which involve cell dynamics, association with structural and motor proteins, cycle-dependent phosphorylation5. Microtubule-associated and interactions with other cytoskeletal elements, proteins have been described in plants, and currently organelles and membranes. How are all these processes include a 76 kDa protein from carrot, a 65 kDa protein from choreographed? This area is largely not understood, but tobacco, 50 and 100 kDa proteins from maize, elongation factor-l~ (Refs 1 and 46) and a 90 kDa protein recently isosome basic principles are emerging. Nucleation, the key step, is facilitated by the location and lated from tobacco membranes 47. The mechanism of action activity of the disperse microtubule-organizing centres and of these proteins remains to be established. possibly also free ~/-somes2°. Their essential component, soluble ~/-tubulin, is presumably the net product of its syn- Future directions Recent work has brought remarkable discoveries. We thesis, post-translational modifications and chaperoninassisted folding26'2s. In cultured animal cells, about half of now know that ~-tubulin is a key element of microtubulethe cellular ~-tubulin is distributed in the cytoplasm in a organizing centres, that it functions as a complex of several soluble forms'2°. In plants, the succession of distinct micro- proteins in stoichiometric proportions, that the complex is tubule arrays and corresponding rearrangements of ~/-tubu- probably ring-shaped and that it nucleates microtubule lin staining during the cell cycle mean that ~/-somes or assembly and caps the minus end. Microtubules can initiate microtubule-organizing centres in various stages of their from microtubule-organizing centres that are dispersed in construction (see Boxes 1 and 2) are relocated within the the cell, and assembled microtubules can self-organize into cell. It can be speculated that free ~/-somes may migrate a bipolar spindle, without a centriolar centrosome. These within the cytoplasm, ~/-somes still attached to assembled exciting findings open doors for further exploration. The microtubules may travel together during self-organization, basic challenge will be to identify plant ~/-somes and deterand whole microtubule-organizing centres may be pulled by mine their molecular composition. Given the disperse 228
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reviews nature of plant microtubule-organizing centres, it will be important to elucidate how ~-somes become organized into larger assemblies - microtubule-organizing centres - and how these are targeted to specific locations in the cell. We also need to know how the activity of microtubuleorganizing centres is regulated, and how dynamic microtubules become stabilized. Also, because microtubules can self-organize into arrays by interacting with partner proteins, we need to know who the partners are and what the necessary conditions are for initiating the interaction. The recent molecular advances put this research onto solid ground and set the stage for making further progress and new discoveries.
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Dedication Dedicated to the memory of Daniel Mazia.
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Acknowledgements
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Only a small proportion of the vast literature on microtubule-organizing centres is cited here because of space limitations; my apologies to all authors whose work could not be directly cited. I thank Dr John Harper for providing valuable suggestions on the manuscript, Dr Teresa Dibbayawan for preparing Fig. 3 and Dr Richard Cyr for valuable comments and suggestions on the manuscript, and for expert preparation of electronic versions of the figures. I also thank three anonymous reviewers for their suggestions. Financial support was provided by the Australian Research Council.
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Jan Ma~C:i~at the SChoOlof BiOlOgiCalSCiencesl Sydney Sydhey 2006
Regulatory phosphorylation of C 4 PEP carboxylase Joo° v.,o,oo,,oyoo,c,o..o. PEP carboxylase is of paramount importance in plant metabolism, and is one of only a few enzymes known to undergo regulatory phosphorylation in the living plant. In illuminated leaves of C4 species, a complex light-signal transduction cascade occurs, which possibly involves cross-talk between the two neighboring photosynthetic cell types. This process upregulates the activity of a Ca2*-independent, substrate-specific protein-Serfrhr kinase, and thereby increases the phosphorylation state of the photosynthesis-related, C4 PEP carboxylase isoform. This reversible covalent modification is superimposed on the opposing regulation of PEP carboxylase by carboxylic acids and phosphorylated metabolites, and is critical for the coordination and functioning of C4 photosynthesis. The general occurrence of PEP carboxylase-kinase and the conserved N-terminal phosphorylation domain of the various plant PEP carboxylase isoforms support the hypothesis that similar regulatory mechanisms are functional in the diverse physiological contexts involving this enzyme. he enzyme PEP (phosphoenolpyruvate) carboxylase (EC 4.1.1.31) is widely distributed in plant tissues, green algae and microorganisms, but is absent in animals'. It was first characterized in spinach leaves in 1953 (Ref. 2)~ and for a long time was considered a subsidiary plant carboxylase alongside Rubisco. However, the discovery of C4 photosynthesis in the mid-1960s and the involvement of a specific isoform of PEP carboxylase in this pathway [which led to the general photosynthesis groupings of C3, C4 and CAM (Crassulacean acid metabolism)] considerably boosted interest in this cytosolic enzymes. The ubiquitous occurrence of PEP carboxylase in plant cells and tissues and its multifaceted functions have since been established, and it is now recognized as playing a major role in plant metabolism. More recently, the extensive development and use of molecular and biochemical techniques has generated an impressive wealth of data, and significantly advanced our understanding of the nature of the regulatory processes for this enzyme.
T
Properties of C 4 PEP carboxylase PEP carboxylase catalyzes the exergonic ~-carboxylation of PEP by HCQ (AG = - 7 kcal tool -~) in the presence of a divalent cation, which is generally Mg2+. The reaction proceeds through a stepwise mechanism that involves the reversible, rate-limiting formation of carboxyphosphate and 2:30
June 1997,Vol. 2, No. 6
the enolate of pyruvate (Fig. 1). An important mechanistic aspect not recognized in earlier studies is the decomposition of carboxyphosphate into inorganic phosphate and free CO2 within the active site, the latter then reacting with the enolate species to form the product oxaloacetate (OAA) (Ref. 4). PEP carboxylase is a homotetramer with each subunit having an approximate size of 110 kDa (Ref. 1). Because of its very low Kmfor the substrate bicarbonate (in the micromolar range), PEP carboxylase serves a general role in the carbon economy of plant cells by recapturing respiratory CO2. Several more specialized functions involve1'4-6: • Replenishing tricarboxylic acid cycle intermediates (an anaplerotic function). • A pH star. • Guard cell movements. • Nitrogen assimilation in C3 leaves. • Nitrogen fixation in legume root nodules. • Seed formation and maturation. • Fruit ripening. • C4 and CAM photosynthesis. Several isoforms of PEP carboxylase have been character. ized that presumably fulfil these specific tasks 5. Experiments with recombinant C4 PEP carboxylase from Sorghum have confirmed that it is subject to opposing feedback inhibition by the end-product L-malate (Ki = 0.17 raM) and atlosteric activation by glucose-6-phosphate
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