Molecular aspects of microtubule dynamics in plants

Molecular aspects of microtubule dynamics in plants

513 Molecular aspects of microtubule dynamics in plants Juliette Azimzadeh*, Jan Traas† and Martine Pastuglia*‡ Microtubules are highly dynamic struc...

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Molecular aspects of microtubule dynamics in plants Juliette Azimzadeh*, Jan Traas† and Martine Pastuglia*‡ Microtubules are highly dynamic structures that play a major role in a wide range of processes, including cell morphogenesis, cell division, intracellular transport and signaling. The recent identification in plants of proteins involved in microtubule organization has begun to reveal how cytoskeleton dynamics are controlled. Addresses *Station de Génétique et Amélioration des Plantes, INRA, Route de Saint Cyr, 78026 Versailles Cedex, France † Laboratoire de Biologie Cellulaire, INRA, Route de Saint Cyr, 78026 Versailles Cedex, France ‡ e-mail: [email protected] Current Opinion in Plant Biology 2001, 4:513–519 1369-5266/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations AtKSS1 Arabidopsis thaliana KATANIN-LIKE PROTEIN SMALL SUBUNIT1 CaM calmodulin fra2 fragile fiber 2 gem1 gemini pollen 1 GFP green fluorescent protein KCBP kinesin-like calmodulin-binding protein KIPK KCBP-interacting protein kinase KRP kinesin-related protein MAP MT-associated protein mor1 microtubule organization 1 MT microtubule MTOC MT organizing center NPK1 Nicotiana Protein Kinase 1 PP1 Type 1 protein phosphatase PPB preprophase band spr spiral TAN1 TANGLED1 TON2 TONNEAU2

critical concentration, MTs alternate between phases of growth and rapid shortening, a phenomenon termed dynamic instability. Although these properties are the basis of MT dynamics, in vivo, interacting proteins are required for the nucleation, stabilization and (re)arrangement of MTs. Thus, in animals and fungi, MT organizing centers (MTOCs) are known to be required for the promotion of MT assembly in vivo. Moreover, many MT-associated proteins (MAPs) interact with the enormous MT protein surface to regulate tubulin assembly, as well as MT spatial distribution and stability [5,6]. In plants, MAPs and potential elements of MTOCs have now been identified. As expected, some of these are homologs of proteins identified in animals and fungi [7••,8••]. However, plants have specific cytoskeletal structures such as the preprophase band (PPB) and the phragmoplast. Reflecting these unique features, plant MAPs with no counterpart in other eukaryotes have also been found [9••,10••]. Here we discuss the role of these newly identified proteins in the molecular control of MT dynamics.

Nucleation

Studies using immunofluorescence, microinjection and, more recently, green fluorescent protein (GFP) fusions have been used to show that, in plants, microtubules (MTs) are highly dynamic structures, which constantly assemble, disassemble and rearrange in response to internal and external stimuli [1–4]. Perhaps the most striking example of MT dynamics in plants is seen during cell cycle progression, during which four different MT arrays succeed one another in a very short time frame (Figure 1). How are these MT rearrangements achieved?

Plant cells lack a well-defined MTOC, and bona fide plant MT nucleators remain to be identified. Nevertheless, several candidate MT nucleators have been isolated. These include homologs of γ-tubulin, which is involved in MT nucleation [11] and dynamics [12] in other eukaryotes. In addition, homologs of Spc97p and Spc98p, two γ-tubulininteracting proteins, are encoded in the Arabidopsis genome [13]. The first evidence for the existence in plants of complexes related to the animal γ-tubulin ring complex that is involved in the nucleation and anchorage of MT minus ends to the MTOC comes from work on maize. A soluble 1500-kDa γ-tubulin-containing protein complex has been purified from interphase maize cells [14]. This complex has a protein profile comparable to the composition of the animal γ-tubulin ring complex and could be its structural equivalent. Immunofluorescence studies have localized plant γ-tubulin in all arrays, in particular in sites that are supposedly involved in MT nucleation, such as the nuclear surface or the poles of the spindle [15]. However, localization of γ-tubulin along entire MTs, and as spots in G2-phase nuclei or at kinetochores, could suggest other roles for γ-tubulin, for instance in MT stabilization [16•,17•,18].

Part of the answer can be found in the intrinsic properties of MTs that can be visualized in vitro. At a critical concentration, α- and β-tubulin heterodimers spontaneously self-assemble to form hollow cylinders. Reflecting the orientation of αβ-heterodimers, MTs have a polar structure with a fast-growing end, the plus end, and a slow-growing end, the minus end. As such, MTs are labile structures: when polymerizing tubulin solutions are diluted below the

Although biochemical and molecular work has revealed some potential components involved in MT nucleation, the actual site of MT formation in plants is still under debate. There is a general consensus that the nuclear surface is a major MT initiator [19], but the existence of other nucleation sites is still an open question. In particular, the site of reassembly of cortical interphase MTs after mitosis has received considerable attention. After cytokinesis,

Introduction

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Figure 1

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Confocal imaging of GFP::MBD (i.e. GFP fused to the microtubule-binding domain of MAP4 [3]) expression revealing dynamic changes in MT organization during the cell cycle in Arabidopsis root cells. The two central cells in each frame are in mitosis. The dynamics of MT arrays during mitosis are visible in the right-hand cell from preprophase to the end of cytokinesis. (a–c) At preprophase, a transient cortical band of MTs, the preprophase band (PPB), encircles the cell and precisely marks the future site of division (asterisk). (c,d) As the cell enters mitosis, MTs assemble at the nuclear surface (indicated by an arrow), contributing to (e) the formation of the mitotic spindle after nuclear envelope breakdown ([f] shows the metaphase spindle and [g] the anaphase spindle). (h) At late anaphase, the phragmoplast (constituted by two sets of opposing MTs) appears between the spindle poles and (i,j) expands as a ring growing centrifugally toward the edges of the cell. (k) After cytokinesis, perinuclear MTs are assembled at the new nuclear envelope. (l) The new cell membrane is indicated with an arrowhead. Time points at which the scans were performed are indicated in minutes. Each image is a single optical section. Scale bar = 10 µM.

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MTs appear first in the perinuclear region, then radiate from the nucleus to the cortex, and are subsequently visualized in the cortex (e.g. [20,21]). This could imply that MTs are translocated from the nucleus to the cortex, but a scenario in which a new cortical interphase array is reassembled at the cortex cannot be discarded. The recent characterization of AtKSS1 (Arabidopsis thaliana KATANIN-LIKE PROTEIN SMALL SUBUNIT1) could shed new light on the establishment of cortical MT arrays during interphase. AtKSS1 is an Arabidopsis homolog of the 60-kDa subunit of the MT-severing katanin complex and localizes after cytokinesis at the perinuclear region [22•]. Burk et al. [8••] isolated a mutant allele of AtKSS1 called fragile fiber 2 (fra2) (which is allelic to botero1 [23•,24]). Interestingly, fra2 cells that are recently exited from cell division exhibit a delay in the disappearance of

perinuclear MTs, which form aster-like arrays running toward the cell cortex, concomitantly with a delay in the organization of transverse cortical MTs [8••]. This phenotype could be due to the retarded release of perinuclear MTs, leading to a delayed translocation to the cortex. In this scenario, FRA2/AtKSS1 would help to release the MTs from the nuclear surface. Such an activity has been shown in animals in which a heterodimeric katanin releases MTs from the centrosome [25]. However, other scenarios are also possible. The persistence of perinuclear MTs in the mutant could also be interpreted as a delay in their depolymerization. In that case, FRA2/AtKSS1 would induce depolymerization of perinuclear MTs by releasing their unstable minus ends. The tubulin dimers released by this process could then be reutilized by as yet unidentified nucleation sites at the cortex. Further studies

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Table 1 Plant MAPs and putative regulators of known sequence. Species

Gene product

Location

Reference(s)

KCBP/ZWICHEL

A. thaliana Nicotiana tabacum Solanum tuberosum

KRP

PPB, perinuclear region in prophase, spindle, phragmoplast

[58–61]

KatA

A. thaliana

KRP

PPB, spindle (concentrated near the midzone), phragmoplast

[62]

Motors

KatB

A. thaliana

KRP

ND

[63]

KatC

A. thaliana

KRP

ND

[63]

KatD

A. thaliana

KRP

ND

[64]

TKRP125

N. tabacum Daucus carota

KRP

Interphase array (S and G2 phases), PPB, spindle, phragmoplast

[34]

DcKRP120-2

D. carota

KRP

Interphase array and PPB (weakly), spindle, phragmoplast (concentrated near the midzone in some cells)

[35•]

AtPAKRP1

A. thaliana Oryza sativa

KRP

Midzone of the spindle (from anaphase), midzone of the phragmoplast

[36]

TBK5

N. tabacum

KRP

Interphase array

[65]

NtMAP65

N. tabacum

Plant specific

Interphase array, PPB, spindle, phragmoplast

[10••]

TAN1

Zea mays

Plant specific

Cytoplasm, PPB, spindle, phragmoplast

[9••]

MOR1

A. thaliana

Homologous to ch-TOGp

ND

[7••]

AtKSS1/AtKTN1

A. thaliana

Katanin

Perinuclear region in interphase, around the spindle poles

[8••,22•]

A. thaliana

PP2A regulatory subunit?

ND

(a)

Non-motors

Regulators? TON2

Kat A, kinesin-related A; At KTN1, A. thaliana KATANIN 1; TBK5, Tobacco BY-2 cells KINESIN-LIKE PROTEIN 5. ND, not determined. (a) C Camilleri et al., unpublished data.

of FRA2/AtKSS1 activity and fra2/bot1 mutants should distinguish between these two possibilities.

Organizing the microtubules The integration of the nucleated MTs into the different cytoskeletal arrays involves a number of MAPs. Two types are distinguished, motor and non-motor MAPs, which will be discussed separately (Table 1). Motors

Motor proteins use ATP hydrolysis to move unidirectionally along the surface of MTs (either to the plus or the minus end). They can function in the transport of ‘cargo’ along MTs, as well as in MT organization. Two classes of MT motor proteins exist in animal systems: a class including the kinesins and kinesin-related proteins (KRPs), and the dyneins. Of all the proteins present in dynein complexes, only one dynein light chain is found in angiosperm genomes. This evidence could indicate that higher plants have lost these protein complexes during evolution [26]. Conversely, plant KRPs have been identified, and some of their characteristics will be discussed here (for reviews, see [27,28]). Plant KRPs have been implicated in spindle and/or phragmoplast organization. Their roles often remain

unclear, and we will concentrate on two well-characterized examples, kinesin-like calmodulin-binding protein (KCBP) and TKRP125. In plants, the spindle forms through the rearrangement of existing MTs assembled at the premitotic nuclear envelope [29], a process that resembles spindle formation in meiotic acentriolar Drosophila cells. In these cells, the coordinated activities of plus- and minus-enddirected motors allows the grouping of MT minus ends at the poles by sliding apart two half-sets of antiparallel MTs [30]. Several plant KRPs could ensure the same function. KCBP is a plant-specific minus-end-directed KRP [31] that is able to bundle MTs in vitro [32•]. Constitutive activation of KCBP by microinjection of antibodies (see below) prematurely induces spindle formation, suggesting a role for KCBP in the rearrangement of MTs at the prometaphase transition [33••]. KCBP activity might also be involved in focusing the acentriolar poles, where it accumulates during anaphase [33••]. Another motor associated with spindle formation is TKRP125, a plus-end-directed motor that is present in all MT arrays from S-phase to the end of M-phase [34]. Because TKRP125 preferentially accumulates at the equatorial plane of the anaphase spindle, it is proposed to participate in spindle elongation. In addition, injection of TKRP125 antibodies inhibits phragmoplast MT translocation, and leads to the enlargement of the phragmoplast midzone where plus-end-growing

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MTs overlap [34]. Thus, TKRP125 is likely involved in maintenance of phragmoplast organization by sliding antiparallel MTs. On the basis of their localization, two other KRPs, DcKRP120-2 and AtPAKRP1, could also participate in phragmoplast organization [35•,36]. In other eukaryotes, some motors with gliding activity can also influence MT stability, depending on their spatial and temporal placement [37]. This could be the case for the KCBP motor, mentioned above, which might affect MT dynamics in the trichomes of Arabidopsis. This motor is impaired in zwichel (zwi) mutants, which exhibit decreased trichome branching [38], a defect that can be corrected by a short treatment with the MT-stabilizing drug taxol [39••]. It has been proposed that KCBP/ZWI could induce the transient and local stabilization of MTs, thus allowing the reorientation of MTs before trichome branch initiation. Interestingly, KCBP/ZWI, which also participates in spindle organization as indicated earlier, is a unique protein in Arabidopsis. Nevertheless, the zwi mutation does not affect mitosis, showing that other motors can compensate for the absence of the calcium-binding protein.

of the activity of Xenopus XMAP215 and human ch-TOGp, two members of this family, it appears that MOR1 could be involved in the promotion of tubulin assembly at MT plus ends [45,46]. It has been proposed that the mor1 mutation causes a conformational change in the MOR1 protein that specifically affects interphase MTs, as mitotic arrays appear normal [7••]. Recently, however, Twell and colleagues identified an allele of MOR1 that has an effect on gametophytic development (D Twell, personal communication; SK Park, D Twell, Abstract 49, 12th International Arabidopsis Meeting, 23–27 June 2001, Madison, WI). In plants that have this allele, called gemini pollen 1 (gem1), the cell plate forms ectopically at the end of the first division of the microspore. Cytokinesis appears to be spatially uncoupled from nuclear division as a result of mispositioning of the spindle or perturbation of phragmoplast guidance [47,48]. GEM1/MOR1 thus appears to be required for the proper functioning of the mitotic arrays in pollen. Although the gem1 mutation can be transmitted via the gametes, no plants that were homozygous for the mutation were found (D Twell, personal communication), indicating that GEM1/MOR1 is essential for embryo formation and that gem1 could also affect somatic cell division.

Non-motor MAPs

The control of MT dynamics and organization also depends on the stabilizing and/or bundling activities of non-motor MAPs. Several non-motor MAPs have been identified in plants on the basis of their homology to known animal proteins or of their ability to bind MTs [28,40•,41,42]. The most-studied MAPs are the MAP65 family of proteins, originally isolated from purified tobacco phragmoplasts [43]. Recently three tobacco genes encoding members of this family were cloned. These genes, named NtMAP65-1a–c, define a plant-specific gene family with no animal counterpart [10••]. Bacterially expressed NtMAP65-1a promotes polymerization of brain tubulin in vitro [10••] and bundles MTs at higher concentrations [28]. NtMAP65-1 proteins associate with a subset of interphase MTs and with the preprophase band, and accumulate at overlapping regions of antiparallel MT arrays (i.e. the midzone of the spindle and the phragmoplast), where they could be involved in stabilization of MTs and maintenance of antiparallel organization [10••]. The MAP65 protein family isolated from carrot interphase cells form 25–30 nm crossbridges between adjacent brain MTs, consistent with a role as MT spacers in the parallel arrays of cortical MTs [44]. Important new insights into the role of MAPs in the organization of plant MTs have started to arise from genetic approaches. The temperature-sensitive mutation microtubule organization 1 (mor1) in Arabidopsis leads to isotropic cell expansion and left-handed organ twisting. Altered cell elongation in mor1 mutants correlates with a disorganization of cortical interphase MTs, which shorten and lose their parallel alignment at the restrictive temperature. The MOR1 gene encodes a 217-kDa protein homologous to a family of MAPs from animals and fungi [7••]. On the basis

Another MAP that is required for MT organization is TANGLED1 (TAN1) in maize. The leaf cells of tan1 mutants form PPBs and phragmoplasts that are structurally normal but frequently misoriented [49]. TAN1s encodes a highly basic 43-kDa polypeptide that directly binds MTs in vitro [9••]. At least one other TAN1-related protein exists in maize. Immunolocalization showed that TAN1 and TAN1-related protein associate with the misoriented MT arrays (i.e. PPB, spindle and phragmoplast) in the mutant but do not localize to interphase MTs [9••]. The exact role of these proteins in the positioning of the division site is still a mystery, but they could mediate interactions between mitotic arrays and the cell cortex via interactions with other cytoskeletal elements, in particular the actin system [9••,49]. Calcium, phosphorylation and MT organization

In animals, reorganization of the cytoskeleton during the cell cycle or in response to environmental stimuli requires important changes in MAP activity. Two levels of regulation have been identified that include control by calcium and changes in the phosphorylation state. During mitosis in plants, cyclin-dependent kinases (CDKs) are supposedly involved in coordinated rearrangements of MTs. In particular, CDKs could be involved in the formation and degradation of the PPB and in the formation of a bipolar spindle (reviewed in [50]; see also Update). It is not known whether plant MAPs are substrates for CDKs, but phosphorylation sites for CDKs are found in the sequences of the motor proteins TKRP125 and DcKRP120-2 [34,35•]. Other non-mitotic kinases possibly control MAP activities. For example, the KCBP tail region interacts with a kinase in the two-hybrid system. KCBP-interacting protein kinase

Molecular aspects of microtubule dynamics in plants Azimzadeh, Traas and Pastuglia

(KIPK) could regulate KCBP or KCBP-associated proteins. Alternatively, KCBP could target KIPK to its proper cellular location [51•]. Another example is the NPK1 (Nicotiana Protein Kinase 1) mitogen-activated protein kinase kinase kinase that appears to control cell-plate expansion. NPK1 accumulates in the midline of the phragmoplast, and the expression of a kinase-negative mutant of NPK1 leads to the inhibition of lateral growth of the phragmoplast [52•]. Moreover, NPK1 is activated by its interaction with a newly identified KRP that could ensure the proper localization of the kinase to the phragmoplast [52•]. Type1 and type2A protein phosphatases (PP1 and PP2A) are also involved in regulating the dynamics and organization of interphase and mitotic MT arrays, as revealed by inhibitor studies [53,54,55•]. For example, inhibition of PP2A activities in alfalfa cells leads to the formation of abnormal PPBs and phragmoplasts, and to the precocious accumulation of MTs around the prophase nucleus [55•]. The first molecular evidence that PP2A complexes acting on the MT cytoskeleton might exist in plants arises from the cloning of the Arabidopsis TONNEAU2 (TON2) FASS gene, which encodes a putative B regulatory subunit of PP2A (C Camilleri, personal communication). TON2/FASS gene function is essential for the organization of cortical arrays, as mutants in which this gene is disrupted display disorganized interphase MT arrays in mitotic cells and lack PPBs [56]. Another mutant, ton1, is likely to be impaired in the same pathway as its phenotype is identical to a strong ton2 allele. Characterization of the TON1 gene product should permit further dissection of the molecular control of cortical MT organization. Ca2+

Other regulators involved in MT organization are and calmodulin (CaM), which directly control the activity of the motor KCBP. KCBP has a CaM-binding domain adjacent to its motor domain, and KCBP association with MTs is inhibited by Ca2+/CaM ([31], reviewed in [27]). Variations in Ca2+ concentration are proposed to regulate KCBP during mitosis. Activation of KCBP by blocking the CaM-binding site with a specific antibody (see also above) induces early nuclear envelope breakdown, metaphase arrest and abnormal phragmoplast formation, depending on the time of injection. In dividing cells, the increase in Ca2+ concentration could cause transient inactivation of KCBP at certain stages, allowing the completion of mitosis [33••].

Conclusions The integrated use of biochemical, cellular, molecular and genetic approaches has greatly improved our understanding of MT dynamics. Nevertheless, many questions remain, and more work is needed to analyze the function of known proteins and newly identified ones. In this context, the exploitation of genomic data will be of importance. Whereas work based on homologies with animal and yeast proteins concentrates on conserved aspects, the genetic approach might also lead to the identification of

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plant-specific mechanisms, possibly involving novel components. Typical examples of the genetic approach include work on the ton1 and spiral (spr) mutants [57••] in Arabidopsis. The SPR1 and SPR2 genes define two novel plant-specific gene families that are involved in the cortical organization of MTs and in cell expansion (I Furatani et al., Abstract 180, 12th International Arabidopsis Meeting, 23–27 June 2001, Madison, WI). Moreover, the identification of two suppressors of the spr1 phenotype, lefty1 and lefty2, will be of great interest (S Thitamadee et al., Abstract 296, 11th International Conference on Arabidopsis Research, 24–28 June 2000, Madison, WI). In this article, we leave aside functional aspects. The cytoskeleton has been associated with a wide range of processes, but often its precise role is poorly characterized. The next challenge, therefore, will be to understand how the plant cytoskeleton participates in processes such as intracellular transport, cell morphogenesis, cell division or signaling.

Update Recently, a CDK-A::GFP fusion protein has been used to precisely determine the localization of this CDK [66]. This study suggests that CDK-A is transiently recruited to the late PPB just before the disassembly of the PPB. After prometaphase, CDK-A associates physically with mitotic structures in a microtubule-dependent manner. Detailed pharmacological studies further suggest that CDK-A might regulate MT organization throughout mitosis.

Acknowledgements We wish to thank O Grandjean for his help with the confocal imaging system, G Wasteneys, T Hashimoto, H Tsukaya, D Twell and R McClinton for communicating their unpublished results, and H McKahnn for helpful comments on the manuscript. JA is recipient of a fellowship from the French Ministry for Research. MP and JT are members of the Institut National de la Recherche Agronomique.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

Zhang D, Wadsworth P, Hepler PK: Microtubule dynamics in living, dividing plant cells: confocal imaging of microinjected fluorescent brain tubulin. Proc Natl Acad Sci USA 1990, 87:8820-8824.

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Hush JM, Wadsworth P, Callaham DA, Hepler PK: Quantification of microtubule dynamics in living plant-cells using fluorescence redistribution after photobleaching. J Cell Sci 1994, 107:775-784.

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Marc J, Granger CL, Brincat J, Fisher DD, Kao T, McCubbin AG, Cyr RJ: A GFP–MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 1998, 10:1927-1939.

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Ueda K, Matsuyama T: Rearrangement of cortical microtubules from transverse to oblique or longitudinal in living cells of transgenic Arabidopsis thaliana. Protoplasma 2000, 213:28-38.

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Gundersen GG, Cook TA: Microtubules and signal transduction. Curr Opin Cell Biol 1999, 11:81-94.

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Cassimeris L: Accessory protein regulation of microtubule dynamics throughout the cell cycle. Curr Opin Cell Biol 1999, 11:134-141.

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7. ••

Whittington AT, Vugrek O, Wei KJ, Hasenbein NG, Sugimoto K, Rashbrooke MC, Wasteneys GO: MOR1 is essential for organizing cortical microtubules in plants. Nature 2001, 411:610-613. Using immunofluorescence microscopy, the authors screened an Arabidopsis mutant library for MT aberrant patterns and isolated two temperature-sensitive mutant alleles of the MOR1 gene. Shift to the restrictive temperature rapidly induces a reversible disruption of cortical MT arrays in mor1 mutants, which leads to isotropic cell expansion and left-handed twisting of organs. The MOR1 gene encodes a 217-kDa protein with similarity to animal MAPs. 8. ••

Burk DH, Liu B, Zhong R, Morrison WH, Ye ZH: A katanin-like protein regulates normal cell wall biosynthesis and cell elongation. Plant Cell 2001, 13:807-827. The authors characterize the fragile fiber 2 mutant, which was isolated in a screen for mutants with reduced mechanical strength of the influorescence stems. The fra2 mutant shows a general defect in anisotropic cell expansion. The FRA2 gene encodes a protein that is highly similar to the p60 subunit of katanin. Cells of fra2 mutants display abnormalities in the establishment of the interphase array of MTs after mitosis: disappearance of perinuclear MTs is delayed and MTs form an aster-like array in the perinuclear region. Cortical MTs in elongating cells are randomly arranged, but later a more transverse orientation of MT is reported. 9. ••

Smith LG, Gerttula SM, Han S, Levy J: TANGLED1: a microtubule binding protein required for the spatial control of cytokinesis in maize. J Cell Biol 2001, 152:231-236. The authors report the positional cloning of the TANGLED1 gene from maize, previously shown to be required for proper positioning of the preprophase band and for spatial guidance of expanding phragmoplasts [49]. The TAN1 gene encodes a highly basic protein that was shown to bind brain MTs in a blot overlay assay. TAN1 associates to mitotic MT arrays. 10. Smertenko A, Saleh N, Igarashi H, Mori H, Hauser-Hahn I, Jiang CJ, •• Sonobe S, Lloyd CW, Hussey PJ: A new class of microtubule-associated proteins in plants. Nat Cell Biol 2000, 2:750-753. The authors used an antiserum raised against the biochemically isolated tobacco MAP65 protein family to screen a tobacco library. They isolated cDNAs encoding three similar plant-specific microtubule-associated proteins: NtMAP65-1a, NtMAP65-1b and NtMAP65-1c. NtMAP65-1a binds MTs in vitro, and promotes the assembly of MTs but not MT bundling. AntiMAP65-1 stains only a subset of interphase cortical MTs, decorates the PPB, and strongly stains the midzone of the spindle and phragmoplast, where antiparallel MTs overlap. The authors discuss a possible role for the NtMAP65 proteins in the stabilization of overlapping MTs. 11. Pereira G, Schiebel E: Centrosome-microtubule nucleation. J Cell Sci 1997, 110:295-300. 12. Paluh JL, Nogales E, Oakley BR, McDonald K, Pidoux AL, Cande WZ: A mutation in gamma-tubulin alters microtubule dynamics and organization and is synthetically lethal with the kinesin-like protein Pkl1p. Mol Biol Cell 2000, 11:1225-1239. 13. The Arabidopsis Genome Initiative: Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 2000, 408:796-815. 14. Stoppin-Mellet V, Peter C, Lambert AM: Distribution of γ-tubulin in higher plant cells: cytosolic γ-tubulin is part of high molecular weight complexes. Plant Biol 2000, 2:290-296. 15. Liu B, Marc J, Joshi HC, Palevitz BA: A γ-tubulin-related protein associated with the microtubule arrays of higher plants in a cell cycle-dependent manner. J Cell Sci 1993, 104:1217-1228. 16. Panteris E, Apostolakos P, Graf R, Galatis B: Gamma-tubulin • colocalizes with microtubule arrays and tubulin paracrystals in dividing vegetative cells of higher plants. Protoplasma 2000, 210:179-187. The authors carefully observed the distribution of γ-tubulin throughout cell division in different plant species and showed that γ-tubulin is often found along the whole length of microtubules. They suggested that, in higher plants, γ-tubulin could have a role in MT stabilization in addition to or instead of a role in MT nucleation. 17. •

Binarova P, Cenklova V, Hause B, Kubatova E, Lysak M, Dolezel J, Bogre L, Draber P: Nuclear γ-tubulin during acentriolar plant mitosis. Plant Cell 2000, 12:433-442. γ-Tubulin was previously shown to associate with kinetochore/centromeric regions [18]. In this study, anti-γ-tubulin was shown to label discrete spots in G2-phase nuclei and prekinetochore regions in premitotic nuclei. Immunoblot analysis of flow-cytometric-sorted G1 and G2 nuclei confirmed an increased γ-tubulin content in G2-phase. The authors discuss the potential roles of nuclear γ-tubulin, that is, MT nucleation during spindle formation and/or MT stabilization at the kinetochore.

18. Binarova P, Hause B, Dolezel J, Draber P: Association of γ-tubulin with kinetochore/centromeric region of plant chromosomes. Plant J 1998, 14:751-757. 19. Stoppin V, Vantard M, Schmit AC, Lambert AM: Isolated plant nuclei nucleate microtubule assembly: the nuclear surface in higher plants has centrosome-like activity. Plant Cell 1994, 6:1099-1106. 20. Granger C, Cyr R: Microtubule reorganization in tobacco BY-2 cells stably expressing GFP–MBD. Planta 2000, 210:502-509. 21. Hasezawa S, Ueda K, Kumagai F: Time-sequence observations of microtubule dynamics throughout mitosis in living cell suspensions of stable transgenic Arabidopsis: direct evidence for the origin of cortical microtubules at M/G1 interface. Plant Cell Physiol 2000, 41:244-250. 22. McClinton RS, Chandler JS, Callis J: Isolation, characterization, and • intracellular localization of a katanin-like p60 subunit from Arabidopsis thaliana. Protoplasma 2001, 216:181-190. The authors report the identification of an Arabidopsis cDNA encoding a p60 katanin subunit named AtKSS1, whose sequence is identical to the deduced FRA2 protein [8••]. An antibody against AtKSS1 stained the perinuclear region in interphase cells. During mitosis, MT arrays were not stained and the signal was concentrated in cytoplasmic areas that surround the spindle poles. 23. Bichet A, Desnos T, Turner S, Grandjean O, Höfte H: BOTERO1 is • required for normal orientation of cortical microtubules and anisotropic cell expansion in Arabidopsis. Plant J 2001, 25:137-148. In Arabidopsis botero1 mutants, cortical MTs in root tip cells remain in random orientation after mitosis. The late organization of transverse array observed in the fra2 mutant, which is allelic to bot1 [8••], is not reported in this study. This may be explained either by the difference between the two alleles or because the root-tip area observed in this study does not cover the region observed in fra2 cells. Interestingly, the authors showed that the establishment of transverse cortical arrays in wild-type cells follows the cessation of radial expansion. 24. Zhong R, Burk DH, Ye ZH: Fibers. A model for studying cell differentiation, cell elongation, and cell wall biosynthesis. Plant Physiol 2001, 126:477-479. 25. Quarmby L: Cellular samurai: katanin and the severing of microtubules. J Cell Sci 2000, 113:2821-2827. 26. Lawrence CJ, Morris NR, Meagher RB, Dawe RK: Dyneins have run their course in plant lineage. Traffic 2001, 2:362-363. 27.

Reddy AS: Molecular motors and their functions in plants. Int Rev Cytol 2001, 204:97-178.

28. Lloyd C, Hussey P: Microtubule-associated proteins in plants — why we need a map. Nat Rev Mol Cell Biol 2001, 2:40-47. 29. Baskin TI, Cande WZ: The structure and function of the mitotic spindle in flowering plants. Annu Rev Plant Physiol Plant Mol Biol 1990, 41:277-315. 30. Vernos I, Karsenti E: Motors involved in spindle assembly and chromosome segregation. Curr Opin Cell Biol 1996, 8:4-9. 31. Song H, Golovkin M, Reddy AS, Endow SA: In vitro motility of AtKCBP, a calmodulin-binding kinesin protein of Arabidopsis. Proc Natl Acad Sci USA 1997, 94:322-327. 32. Kao YL, Deavours BE, Phelps KK, Walker RA, Reddy AS: Bundling of • microtubules by motor and tail domains of a kinesin-like calmodulin-binding protein from Arabidopsis: regulation by Ca(2+)/calmodulin. Biochem Biophys Res Commun 2000, 267:201-207. The authors show that bacterially expressed motor and tail regions of KCBP are both able to bundle MT. Using a protein that lacks the coil-coiled region of KCBP, they also showed that dimerization of the motor domain is not required for bundling activity. Moreover, bundles induced by the motor domain are dissociated by Ca2+/CaM. 33. Vos JW, Safadi F, Reddy AS, Hepler PK: The kinesin-like calmodulin •• binding protein is differentially involved in cell division. Plant Cell 2000, 12:979-990. In this study, an antibody that constitutively activates KCBP by binding to its CaM-binding domain was microinjected into Tradescantia stamen hair cells. Injection of antibodies during prophase induced nuclear envelope breakdown and, subsequently, cells were blocked in metaphase. When injected during anaphase, the antibodies caused aberrant phragmoplast formation. These results suggest that KCBP activity is downregulated during certain stages of the cell cycle by elevation of the Ca2+ level. During prophase and anaphase, KCBP could participate in spindle organization.

Molecular aspects of microtubule dynamics in plants Azimzadeh, Traas and Pastuglia

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Hunter AW, Wordeman L: How motor proteins influence microtubule polymerization dynamics. J Cell Sci 2000, 113:4379-4389.

38. Oppenheimer DG, Pollock MA, Vacik J, Szymanski DB, Ericson B, Feldmann K, Marks MD: Essential role of a kinesin-like protein in Arabidopsis trichome morphogenesis. Proc Natl Acad Sci USA 1997, 94:6261-6266. 39. Mathur J, Chua NH: Microtubule stabilization leads to growth •• reorientation in Arabidopsis trichomes. Plant Cell 2000, 12:465-477. Using MAP4::GFP, the authors showed that trichome branch initiation is accompanied by the reorientation of MTs at the branch junction. Branching is induced by the transient stabilization of MTs by taxol in the branchless mutants stichel and zwichel. STICHEL and ZWICHEL could provide the MT stability needed for branch initiation. α and 40. Moore RC, Cyr RJ: Association between elongation factor-1α • microtubules in vivo is domain dependent and conditional. Cell Motil Cytoskeleton 2000, 45:279-292. Elongation factor-1α (EF-1α) is a MAP that stabilizes MTs, promotes the assembly of MTs and bundles MTs. In this study, GFP fusion to truncated forms of EF-1α was used to identify two MT-binding domains. Both full length EF-1α and its amino-terminal domain bind MT only upon incubation in weak lipophilic organic acid, whereas binding of the carboxy-terminal domain is unconditional. These findings suggest that the EF-1α amino-terminal domain negatively regulates the binding of EF-1α to MT in vivo. 41. Freudenreich A, Nick P: Microtubular organization in tobacco cells: heat-shock protein 90 can bind to tubulin in vitro. Bot Acta 1998, 111:273-279. 42. Hugdahl JD, Bokros CL, Morejohn LC: End-to-end annealing of plant microtubules by the p86 subunit of eukaryotic initiation factor-(iso)4F. Plant Cell 1995, 7:2129-2138. 43. Jiang CJ, Sonobe S: Identification and preliminary characterization of a 65-kDa higher-plant microtubule-associated protein. J Cell Sci 1993, 105:891-901. 44. Chan J, Jensen CG, Jensen LCW, Bush M, Lloyd CW: The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules. Proc Natl Acad Sci USA 1999, 96:14931-14936. 45. Charrasse S, Schroeder M, Gauthier-Rouviere C, Ango F, Cassimeris L, Gard DL, Larroque C: The TOGp protein is a new human microtubule-associated protein homologous to the Xenopus XMAP215. J Cell Sci 1998, 111:1371-1383. 46. Vasquez RJ, Gard DL, Cassimeris L: XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover. J Cell Biol 1994, 127:985-993. 47.

Park SK, Howden R, Twell D: The Arabidopsis thaliana gametophytic mutation gemini pollen1 disrupts microspore polarity, division asymmetry and pollen cell fate. Development 1998, 125:3789-3799.

48. Park SK, Twell D: Novel patterns of ectopic cell plate growth and lipid body distribution in the Arabidopsis gemini pollen1 mutant. Plant Physiol 2001, 126:899-909. 49. Cleary AL, Smith LG: The Tangled1 gene is required for spatial control of cytoskeletal arrays associated with cell division during maize leaf development. Plant Cell 1998, 10:1875-1888. 50. Vantard M, Cowling R, Delichère C: Cell cycle regulation of the microtubular cytoskeleton. Plant Mol Biol 2000, 43:691-703.

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51. Day IS, Miller C, Golovkin M, Reddy ASN: Interaction of a kinesin• like calmodulin-binding protein with a protein kinase. J Biol Chem 2000, 275:13737-13745. Using the two-hybrid system, the authors identified a protein kinase, named KIPK, that interacts with the tail region of KCBP. This interaction is confirmed by co-precipitation assays. KIPK is also shown to undergo autophosphorylation. 52. Nishihama R, Ishikawa M, Araki S, Soyano T, Asada T, Machida Y: The • NPK1 mitogen-activated protein kinase kinase kinase is a regulator of cell-plate formation in plant cytokinesis. Genes Dev 2001, 15:352-363. The authors show that NPK1 kinase activity increases in late M phase. At anaphase, NPK1 accumulates in the spindle midzone, then at telophase, in the midzone of the phragmoplast. Inducible expression of a mutated form of NPK1 that cannot bind ATP in BY-2 cells generates defects in lateral expansion of the phragmoplast, and leads to a significant proportion of multinucleate cells with incomplete cell plates. The authors propose that NPK1 regulates the outward re-distribution of phragmoplast MTs. 53. Hasezawa S, Nagata T: Okadaic acid as a probe to analyse the cell cycle progression in plant cells. Bot Acta 1992, 105:63-69. 54. Baskin T, Wilson J: Inhibitors of protein kinases and phosphatases alter root morphology and disorganize cortical microtubules. Plant Physiol 1997, 113:493-502. 55. Ayaydin F, Vissi E, Meszaros T, Miskolczi P, Kovacs I, Feher A, • Dombradi V, Erdodi F, Gergely P, Dudits D: Inhibition of serine/threonine-specific protein phosphatases causes premature activation of cdc2MsF kinase at G2/M transition and early mitotic microtubule organisation in alfalfa. Plant J 2000, 23:85-96. Functions of serine/threonine phosphatases during the cell cycle were investigated in alfalfa suspension cells using the specific inhibitor endothall. The results obtained using 1 µM endothall, a concentration that inhibits most PP2A activity but does not affect PP1 activity, suggest that PP2A activities contribute to the control of MT rearrangements during mitosis. 56. Traas J, Bellini C, Nacry P, Kronenberger J, Bouchez D, Caboche M: Normal differentiation patterns in plants lacking microtubular preprophase bands. Nature 1995, 375:676-677. 57. ••

Furutani I, Watanabe Y, Prieto R, Masukawa M, Suzuki K, Naoi K, Thitamadee S, Shikanai T, Hashimoto T: The SPIRAL genes are required for directional control of cell elongation in Arabidopsis thaliana. Development 2000, 127:4443-4453. Arabidopsis sprl1 and spr2 mutants exhibit right-handed twisting of organs. This twisting seems to be connected to a defect in cell growth anisotropy that affects cortical cells more strongly than epidermal cells. In elongating epidermal cells of spr1, cortical MTs form left-handed helices instead of transverse arrays. The spr1 phenotype is reversed by treatments with low concentrations of both MT-stabilizing and MT-depolymerizing drugs. SPR1 and SPR2 are proposed to control the orientation of cortical arrays, possibly via the regulation of MT dynamics. 58. Bowser J, Reddy AS: Localization of a kinesin-like calmodulinbinding protein in dividing cells of Arabidopsis and tobacco. Plant J 1997, 12:1429-1437. 59. Wang W, Takezawa D, Narasimhulu SB, Reddy ASN, Poovaiah BW: A novel kinesin-like protein with a calmodulin-binding domain. Plant Mol Biol 1996, 31:87-100. 60. Reddy ASN, Narasimhulu F, Safadi F, Golovkin M: A plant kinesin heavy chain-like protein is a calmodulin-binding protein. Plant J 1996, 10:9-21. 61. Smirnova EA, Reddy AS, Bowser J, Bajer AS: Minus end-directed kinesin-like motor protein, Kcbp, localizes to anaphase spindle poles in Haemanthus endosperm. Cell Motil Cytoskeleton 1998, 41:271-280. 62. Liu B, Cyr RJ, Palevitz BA: A kinesin-like protein, KatAp, in the cells of Arabidopsis and other plants. Plant Cell 1996, 8:119-132. 63. Mitsui H, Nakatani K, Yamaguchi-Shinozaki K, Shinozaki K, Nishikawa K, Takahashi H: Sequencing and characterization of the kinesin-related genes katB and katC of Arabidopsis thaliana. Plant Mol Biol 1994, 25:865-876. 64. Tamura K, Nakatani K, Mitsui H, Ohashi Y, Takahashi H: Characterization of KatD, a kinesin-like protein gene specifically expressed in floral tissues of Arabidopsis thaliana. Gene 1999, 230:23-32. 65. Matsui K, Collings D, Asada T: Identification of a novel plant-specific kinesin-like protein that is highly expressed in interphase tobacco BY-2 cells. Protoplasma 2001, 215:105-115. 66. Weingartner M, Binarova P, Drykova D, Schweighofer A, David JP, Herberle-Bors E, Doonan J, Bögre L: Dynamic recruitment of Cdc2 to specific microtubule structures during mitosis. Plant Cell 2001, 13:1929-1943.