The plant microtubule-associated protein AtMAP65-3/PLE is essential for cytokinetic phragmoplast function

Directional cell expansion in interphase and nuclear and cell division in M-phase are mediated by four microtubule arrays, three of which are unique to plants: the interphase array, the preprophase band, and the phragmoplast. The plant microtubule-associated protein MAP65 has been identified as a key structural component in these arrays. The Arabidopsis genome has nine MAP65 genes, and here we show that one, AtMAP65-3/PLE, locates only to the mitotic arrays and is essential for cytokinesis. The Arabidopsis pleiade (ple) alleles are single recessive mutations, and we show that these mutations are in the AtMAP65-3 gene. Moreover, these mutations cause C-terminal truncations that abolish microtubule binding. In the ple mutants the anaphase spindle is normal, and the cytokinetic phragmoplast can form but is distorted; not only is it wider, but the midline, the region where oppositely oriented microtubules overlap, is unusually expanded. Here we present data that demonstrate an essential role for AtMAP65-3/PLE in cytokinesis in plant cells.     

Plant gamma-tubulin interacts with alphabeta-tubulin dimers and forms membrane-associated complexes

gamma-Tubulin is assumed to participate in microtubule nucleation in acentrosomal plant cells, but the underlying molecular mechanisms are still unknown. Here, we show that gamma-tubulin is present in protein complexes of various sizes and different subcellular locations in Arabidopsis and fava bean. Immunoprecipitation experiments revealed an association of gamma-tubulin with alphabeta-tubulin dimers. gamma-Tubulin cosedimented with microtubules polymerized in vitro and localized along their whole length. Large gamma-tubulin complexes resistant to salt treatment were found to be associated with a high-speed microsomal fraction. Blue native electrophoresis of detergent-solubilized microsomes showed that the molecular mass of the complexes was >1 MD. Large gamma-tubulin complexes were active in microtubule nucleation, but nucleation activity was not observed for the smaller complexes. Punctate gamma-tubulin staining was associated with microtubule arrays, accumulated with short kinetochore microtubules interacting in polar regions with membranes, and localized in the vicinity of nuclei and in the area of cell plate formation. Our results indicate that the association of gamma-tubulin complexes with dynamic membranes might ensure the flexibility of noncentrosomal microtubule nucleation. Moreover, the presence of other molecular forms of gamma-tubulin suggests additional roles for this protein species in microtubule organization.     

The microtubule cycle during successive mitotic waves in the syncytial female gametophyte of ginkgo

Plant morphogenesis is driven by a surprising number of microtubule arrays. The four arrays of vegetative tissues are hoop-like cortical, preprophase band (PPB), spindle, and phragmoplast. When syncytia occur during the reproductive phase of the plant life cycle, neither hoop-like corticals nor PPBs are present, and functional phragmoplasts fail to form following the proliferative mitoses that give rise to the multinucleate cytoplasm. Instead, the interphase microtubules are radial microtubule systems (RMSs) that emanate from the nuclei. These RMSs organize the cytoplasm into nascent cells and ultimately trigger phragmoplast formation at their boundaries. During investigations of the syncytial stage that initiates development of the female gametophyte in gymnosperms, we studied the large (3–4 mm) female gametophyte of Ginkgo biloba. Here we describe the microtubule cycle correlated with successive mitotic waves and discuss the importance of this system in studying the acentrosomal nucleation and organization of cycling microtubule arrays. Electronic Publication     

A mutation in gamma-tubulin alters microtubule dynamics and organization and is synthetically lethal with the kinesin-like protein pkl1p

Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. gamma-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in gamma-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30 degrees C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant gamma-tubulin is like the wild-type protein. Prediction of gamma-tubulin structure indicates that non-alpha/beta-tubulin protein-protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the gamma-tubulin mutant and in multicopy for normal cell morphology at 30 degrees C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for gamma-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of gamma-tubulin that involves non-tubulin protein-protein interactions, presumably with a second motor, MAP, or MTOC component.     

Expression of Arabidopsis gamma-tubulin in fission yeast reveals conserved and novel functions of gamma-tubulin

gamma-Tubulin localizes to microtubule-organizing centers in animal and fungal cells where it is important for microtubule nucleation. Plant cells do not have morphologically defined microtubule organizing centers, however, and gamma-tubulin is distributed in small, discrete structures along microtubules. The great difference in distribution has prompted speculation that plant gamma-tubulins function differently from animal and fungal gamma-tubulins. We tested this possibility by expressing Arabidopsis gamma-tubulin in the fission yeast Schizosaccharomyces pombe. At high temperatures, the plant gamma-tubulin was able to bind to microtubule-organizing centers, nucleate microtubule assembly, and support the growth and replication of S. pombe cells lacking endogenous gamma-tubulin. However, the distribution of microtubules was abnormal as was cell morphology, and at low temperatures, cells were arrested in mitosis. These results reveal that Arabidopsis gamma-tubulin can carry out essential functions in S. pombe and is, thus, functionally conserved. The morphological abnormalities reveal that it cannot carry out some nonessential functions, however, and they underscore the importance of gamma-tubulin in morphogenesis of fission yeast cells and in maintaining normal interphase microtubule arrays.     

Gamma-tubulin in barley and tobacco: sequence relationship and RNA expression patterns in developing leaves during mitosis and post-mitotic growth

gamma-Tubulin is typically associated with microtubule organising centres, such as the centrosome, and appears to mediate microtubule nucleation. Centrosomes are usually not found in higher plants, but active genes homologous to gamma-tubulin have been identified in the plant kingdom, including the angiosperms Arabidopsis, maize and rice. We have isolated and characterised gamma-tubulin cDNA sequences of two further angiosperm species, barley and tobacco. Sequence comparison revealed a phylogenetic tree with distinct clusters corresponding to the systematic position of the species. Furthermore, domains, thought to be exposed in the folded protein and to be candidates for interaction with associated, nucleation-site related proteins, exhibited motifs highly specific of multicellular plants. Strong expression of the gamma-tubulin genes, as determined by Northern blotting, correlated with mitotic activity. Expression dropped distinctly when mitotic activity ceased. Thus, in post-mitotic tissues that showed intricate reshuffling of cortical microtubule arrays related to cell shaping only very low gamma-tubulin steady-state RNA levels were found, contrasting with the situation for alpha-tubulin. The findings indicate that gamma-tubulin expression in plants may be more tightly linked to mitosis, although there is some gamma-tubulin expression at the RNA level even after mitosis. It follows that the post-mitotic changes in microtubular arrays may be less dependent on concurrent gamma-tubulin RNA expression than mitotic cells.     

Gamma-tubulin in basal land plants: characterization, localization, and implication in the evolution of acentriolar microtubule organizing centers

Although seed plants have gamma-tubulin, a ubiquitous component of centrosomes associated with microtubule nucleation in algal and animal cells, they do not have discrete microtubule organizing centers (MTOCs) comparable to animal centrosomes, and the organization of microtubule arrays in plants has remained enigmatic. Spindle development in basal land plants has revealed a surprising variety of MTOCs that may represent milestones in the evolution of the typical diffuse acentrosomal plant spindle. We have isolated and characterized the gamma-tubulin gene from a liverwort, one of the extant basal land plants. Sequence similarity to the gamma-tubulin gene of higher plants suggests that the gamma-tubulin gene is highly conserved in land plants. The G9 antibody to fission yeast gamma-tubulin recognized a single band of 55 kD in immunoblots from bryophytes. Immunohistochemistry with the G9 antibody clearly documented the association of gamma-tubulin with various MTOC sites in basal land plants (e.g., discrete centrosomes with and without centrioles and the plastid surface in monoplastidic meiosis of bryophytes). Changes in the distribution of gamma-tubulin occur in a cell cycle-specific manner during monoplastidic meiosis in the liverwort Dumortiera hirsuta. gamma-Tubulin changes its localization from the plastid surface in prophase I to the spindle, from the spindle to phragmoplasts and the nuclear envelope in telophase I, and back to the plastid surfaces in prophase II. In vitro experiments show that gamma-tubulin is detectable on the surface of isolated plastids and nuclei of D. hirsuta, and microtubules can be repolymerized from the isolated plastids. gamma-Tubulin localization patterns on plastid and nuclear surfaces are not affected by the destruction of microtubules by oryzalin. We conclude that gamma-tubulin is a highly conserved protein associated with microtubule nucleation in basal land plants and that it has a cell cycle-dependent distribution essential for the orderly succession of microtubule arrays.     

Characterization of the Arabidopsis augmin complex uncovers its critical function in the assembly of the acentrosomal spindle and phragmoplast microtubule arrays

Plant cells assemble the bipolar spindle and phragmoplast microtubule (MT) arrays in the absence of the centrosome structure. Our recent findings in Arabidopsis thaliana indicated that AUGMIN subunit3 (AUG3), a homolog of animal dim γ-tubulin 3, plays a critical role in γ-tubulin-dependent MT nucleation and amplification during mitosis. Here, we report the isolation of the entire plant augmin complex that contains eight subunits. Among them, AUG1 to AUG6 share low sequence similarity with their animal counterparts, but AUG7 and AUG8 share homology only with proteins of plant origin. Genetic analyses indicate that the AUG1, AUG2, AUG4, and AUG5 genes are essential, as stable mutations in these genes could only be transmitted to heterozygous plants. The sterile aug7-1 homozygous mutant in which AUG7 expression is significantly reduced exhibited pleiotropic phenotypes of seriously retarded vegetative and reproductive growth. The aug7-1 mutation caused delocalization of γ-tubulin in the mitotic spindle and phragmoplast. Consequently, spindles were abnormally elongated, and their poles failed to converge, as MTs were splayed to discrete positions rendering deformed arrays. In addition, the mutant phragmoplasts often had disorganized MT bundles with uneven edges. We conclude that assembly of MT arrays during plant mitosis depends on the augmin complex, which includes two plant-specific subunits.     

Alanine-scanning Mutagenesis of Aspergillus γ-Tubulin Yields Diverse and Novel Phenotypes

We have created 41 clustered charged-to-alanine scanning mutations of the mipA, gamma-tubulin, gene of Aspergillus nidulans and have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of gamma-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that gamma-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of gamma-tubulin and these data give a good initial indication of the functionally important regions of the molecule.     

The drosophila protein asp is involved in microtubule organization during spindle formation and cytokinesis

Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been suggested to be involved in microtubule nucleation from the centrosome. Here, we show that Asp is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules. Localization to these structures is independent of a functional centrosome. Moreover, colchicine treatment disrupts Asp localization to the centrosome, indicating that Asp is not an integral centrosomal protein. In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and gamma-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, a gene required for centrosome function, we have determined the role of Asp in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. Our data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis.     

Gamma-tubulin is essential for acentrosomal microtubule nucleation and coordination of late mitotic events in Arabidopsis

Gamma-tubulin is required for microtubule (MT) nucleation at MT organizing centers such as centrosomes or spindle pole bodies, but little is known about its noncentrosomal functions. We conditionally downregulated gamma-tubulin by inducible expression of RNA interference (RNAi) constructs in Arabidopsis thaliana. Almost complete RNAi depletion of gamma-tubulin led to the absence of MTs and was lethal at the cotyledon stage. After induction of RNAi expression, gamma-tubulin was gradually depleted from both cytoplasmic and microsomal fractions. In RNAi plants with partial loss of gamma-tubulin, MT recovery after drug-induced depolymerization was impaired. Similarly, immunodepletion of gamma-tubulin from Arabidopsis extracts severely compromised in vitro polymerization of MTs. Reduction of gamma-tubulin protein levels led to randomization and bundling of cortical MTs. This finding indicates that MT-bound gamma-tubulin is part of a cortical template guiding the microtubular network and is essential for MT nucleation. Furthermore, we found that cells with decreased levels of gamma-tubulin could progress through mitosis, but cytokinesis was strongly affected. Stepwise diminution of gamma-tubulin allowed us to reveal roles for MT nucleation in plant development, such as organization of cell files, anisotropic and polar tip growth, and stomatal patterning. Some of these functions of gamma-tubulin might be independent of MT nucleation.     

MICROTUBULE ORGANIZATION 1 regulates structure and function of microtubule arrays during mitosis and cytokinesis in the Arabidopsis root

MICROTUBULE ORGANIZATION 1 (MOR1) is a plant member of the highly conserved MAP215/Dis1 family of microtubule-associated proteins. Prior studies with the temperature-sensitive mor1 mutants of Arabidopsis (Arabidopsis thaliana), which harbor single amino acid substitutions in an N-terminal HEAT repeat, proved that MOR1 regulates cortical microtubule organization and function. Here we demonstrate by use of live cell imaging and immunolabeling that the mor1-1 mutation generates specific defects in the microtubule arrays of dividing vegetative cells. Unlike the universal cortical microtubule disorganization in elongating mor1-1 cells, disruption of mitotic and cytokinetic microtubule arrays was not detected in all dividing cells. Nevertheless, quantitative analysis identified distinct defects in preprophase bands (PPBs), spindles, and phragmoplasts. In nearly one-half of dividing cells at the restrictive temperature of 30 degrees C, PPBs were not detected prior to spindle formation, and those that did form were often disrupted. mor1-1 spindles and phragmoplasts were short and abnormally organized and persisted for longer times than in wild-type cells. The reduced length of these arrays predicts that the component microtubule lengths are also reduced, suggesting that microtubule length is a critical determinant of spindle and phragmoplast structure, orientation, and function. Microtubule organizational defects led to aberrant chromosomal arrangements, misaligned or incomplete cell plates, and multinucleate cells. Antiserum raised against an N-terminal MOR1 sequence labeled the full length of microtubules in interphase arrays, PPBs, spindles, and phragmoplasts. Continued immunolabeling of the disorganized and short microtubules of mor1-1 at the restrictive temperature demonstrated that the mutant mor1-1(L174F) protein loses function without dissociating from microtubules, providing important insight into the mechanism by which MOR1 may regulate microtubule length.     

Spindle Dynamics and the Role of γ-Tubulin in Early Caenorhabditis elegans Embryos

gamma-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that gamma-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans gamma-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::gamma-tubulin or GFP::beta-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth and the differential behavior of centrosomes destined for germ line and somatic blastomeres. To study the role of gamma-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegans embryos of gamma-tubulin. gamma-Tubulin (RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous long astral microtubules. Our results suggest that gamma-tubulin is not absolutely required for microtubule nucleation in C. elegans but is required for the normal organization and function of kinetochore and interpolar microtubules.     

Gamma-tubulin at ten: progress and prospects

The existence of gamma-tubulin was first reported approximately ten years ago, and it is appropriate to review the progress that has been made in gamma-tubulin research and to discuss some of the unanswered questions about gamma-tubulin function. gamma-Tubulin is ubiquitous in eukaryotes and is generally quite conserved. Two highly divergent gamma-tubulins have been discovered, however, one in Saccharomyces cerevisiae and one in Caenorhabditis elegans. Several organisms have two gamma-tubulin genes. In Drosophila melanogaster, the two gamma-tubulins differ significantly in sequence and expression pattern. In other organisms the two gamma-tubulins are almost identical and expression patterns have not been determined. gamma-Tubulin is located at microtubule organizing centers in many organisms, and it is also frequently associated with the mitotic spindle. gamma-Tubulin is essential for the formation of functional mitotic spindles in all organisms that have been examined to date. In animal cells, complexes containing gamma-tubulin are located at microtubule organizing centers where they nucleate the assembly of microtubules. In spite of the considerable progress that has been made in gamma-tubulin research important questions remain to be answered. The exact mechanisms of microtubule nucleation by gamma-tubulin complexes remain to be resolved as do the mechanisms by which microtubule nucleation from gamma-tubulin complexes is regulated. Finally, there is evidence that gamma-tubulin has important functions in addition to microtubule nucleation, and these functions are just beginning to be investigated.     

gamma-Tubulin in Arabidopsis: gene sequence, immunoblot, and immunofluorescence studies.

gamma-Tubulin is a protein associated with microtubule (Mt)-organizing centers in a variety of eukaryotic cells. Unfortunately, little is known about such centers in plants. Genomic and partial cDNA clones encoding two gamma-tubulins of Arabidopsis were isolated and sequenced. Comparisons of genomic and cDNA sequences showed that both genes, TubG1 and TubG2, contain nine introns at conserved locations. The sequences of the two genes both predict proteins containing 474 amino acids, with molecular masses of 53,250 and 53,280 D, respectively. The predicted gamma 1- and gamma 2-tubulins exhibit 98% amino acid identity with each other and approximately 70% amino acid identity with the gamma-tubulins of animals and fungi. RNA gel blot results demonstrated that both genes are transcribed in suspension culture cells, seedlings, and roots and flowers of mature plants. Immunoblots of Arabidopsis proteins using an antibody specific to a conserved peptide of gamma-tubulin showed a major cross-reacting polypeptide with an M(r) of 58,000. The same antibody stained all Mt arrays in tissue and suspension culture cells of this species. Binding was inhibited by the homologous oligopeptide in the gamma-tubulins predicted by the two Arabidopsis gene sequences. Antibody staining avoided the plus ends of Mts at the kinetochores and cell plate, but unlike the case in animal cells, seemed to be localized over broad stretches of the kinetochore fibers and phragmoplast toward the minus ends. We concluded that at least two gamma-tubulin protein homologs are present in Arabidopsis and that at least one of them is localized along Mt arrays. Its distribution is correlated with and may help explain unique characteristics of Mt organization in plants.     

Gamma-tubulin complexes and microtubule organization

Microtubule nucleation requires gamma-tubulin, which exists in two main protein complexes: the gamma-tubulin small complex, and the gamma-tubulin ring complex. During mitosis, these complexes accumulate at the centrosome to support spindle formation. Gamma-tubulin complexes are also present at non-centrosomal microtubule nucleation sites, both in interphase and in mitosis. In interphase, non-centrosomal nucleation enables the formation of microtubule bundles or networks of branched microtubules. Gamma-tubulin complexes may be involved not only in microtubule nucleation, but also in regulating microtubule dynamics. Recent findings indicate that the dynamics of microtubule plus-ends are altered, depending on the expression of gamma-tubulin complex proteins.     

GSK-3beta regulates proper mitotic spindle formation in cooperation with a component of the gamma-tubulin ring complex, GCP5

Glycogen synthase kinase-3beta (GSK-3beta) is known to play a role in the regulation of the dynamics of microtubule networks in cells. Here we show the role of GSK-3beta in the proper formation of the mitotic spindles through an interaction with GCP5, a component of the gamma-tubulin ring complex (gammaTuRC). GCP5 bound directly to GSK-3beta in vitro, and their interaction was also observed in intact cells at endogenous levels. Depletion of GCP5 dramatically reduced the GCP2 and gamma-tubulin in the gammaTuRC fraction of sucrose density gradients and disrupted gamma-tubulin localization to the spindle poles in mitotic cells. GCP5 appears to be required for the formation or stability of gammaTuRC and the recruitment of gamma-tubulin to the spindle poles. A GSK-3 inhibitor not only led to the accumulation of gamma-tubulin and GCP5 at the spindle poles but also enhanced microtubule nucleation activity at the spindle poles. Depletion of GCP5 rescued this disrupted organization of spindle poles observed in cells treated with the GSK-3 inhibitor. Furthermore, the inhibition of GSK-3 enhanced the binding of gammaTuRC to the centrosome isolated from mitotic cells in vitro. Our findings suggest that GSK-3beta regulates the localization of gammaTuRC, including GCP5, to the spindle poles, thereby controlling the formation of proper mitotic spindles.     

The microtubule‐associated kinase‐like protein RUNKEL functions in somatic and syncytial cytokinesis

The microtubule (MT)‐associated putative kinase RUNKEL (RUK) is an important component of the phragmoplast machinery involved in cell plate formation in Arabidopsis somatic cytokinesis. Since loss‐of‐function ruk mutants display seedling lethality, it was previously not known whether RUK functions in mature sporophytes or during gametophyte development. In this study we utilized RUK proteins that lack the N‐terminal kinase domain to further examine biological processes related to RUK function. Truncated RUK proteins when expressed in wild‐type Arabidopsis plants cause cellularization defects not only in seedlings and adult tissues but also during male meiocyte development, resulting in abnormal pollen and reduced fertility. Ultrastructural analysis of male tetrads revealed irregular and incomplete or absent intersporal cell walls, caused by disorganized radial MT arrays. Moreover, in ruk mutants endosperm cellularization defects were also caused by disorganized radial MT arrays. Intriguingly, in seedlings expressing truncated RUK proteins, the kinesin HINKEL, which is required for the activation of a mitogen‐activated protein kinase signaling pathway regulating phragmoplast expansion, was mislocalized. Together, these observations support a common role for RUK in both phragmoplast‐based cytokinesis in somatic cells and syncytial cytokinesis in reproductive cells.     

Identification of a phragmoplast-associated kinesin-related protein in higher plants

The phragmoplast executes cytokinesis in higher plants. The major components of the phragmoplast are microtubules, which are arranged in two mirror-image arrays perpendicular to the division plane [1]. The plus ends of these microtubules are located near the site of the future cell plate. Golgi-derived vesicles are transported along microtubules towards the plus ends to deliver materials bound for the cell plate [2] [3]. During cell division, rapid microtubule reorganization in the phragmoplast requires the orchestrated activities of microtubule motor proteins such as kinesins. We isolated an Arabidopsis cDNA clone of a gene encoding an amino-terminal motor kinesin, AtPAKRP1, and have determined the partial sequence of its rice homolog. Immunofluorescence experiments with two sets of specific antibodies revealed consistent localization of AtPAKRP1 and its homolog in Arabidopsis and rice cells undergoing anaphase, telophase and cytokinesis. AtPAKRP1 started to accumulate along microtubules towards the spindle midzone during late anaphase. Once the phragmoplast microtubule array was established, AtPAKRP1 conspicuously localized to microtubules near the future cell plate. Our results provide evidence that AtPAKRP1 is a hitherto unknown motor that may take part in the establishment and/or maintenance of the phragmoplast microtubule array.     

Fission yeast mto2p regulates microtubule nucleation by the centrosomin-related protein mto1p

From an insertional mutagenesis screen, we isolated a novel gene, mto2+, involved in microtubule organization in fission yeast. mto2Delta strains are viable but exhibit defects in interphase microtubule nucleation and in formation of the postanaphase microtubule array at the end of mitosis. The mto2Delta defects represent a subset of the defects displayed by cells deleted for mto1+ (also known as mod20+ and mbo1+), a centrosomin-related protein required to recruit the gamma-tubulin complex to cytoplasmic microtubule-organizing centers (MTOCs). We show that mto2p colocalizes with mto1p at MTOCs throughout the cell cycle and that mto1p and mto2p coimmunoprecipitate from cytoplasmic extracts. In vitro studies suggest that mto2p binds directly to mto1p. In mto2Delta mutants, although some aspects of mto1p localization are perturbed, mto1p can still localize to spindle pole bodies and the cell division site and to "satellite" particles on interphase microtubules. In mto1Delta mutants, localization of mto2p to all of these MTOCs is strongly reduced or absent. We also find that in mto2Delta mutants, cytoplasmic forms of the gamma-tubulin complex are mislocalized, and the gamma-tubulin complex no longer coimmunoprecipitates with mto1p from cell extracts. These experiments establish mto2p as a major regulator of mto1p-mediated microtubule nucleation by the gamma-tubulin complex.