Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis.

Plant vascular cells, or tracheary elements (TEs), rely on circumferential secondary cell wall thickenings to maintain sap flow. The patterns in which TE thickenings are organized vary according to the underlying microtubule bundles that guide wall deposition. To identify microtubule interacting proteins present at defined stages of TE differentiation, we exploited the synchronous differentiation of TEs in Arabidopsis thaliana suspension cultures. Quantitative proteomic analysis of microtubule pull-downs, using ratiometric (14)N/(15)N labeling, revealed 605 proteins exhibiting differential accumulation during TE differentiation. Microtubule interacting proteins associated with membrane trafficking, protein synthesis, DNA/RNA binding, and signal transduction peaked during secondary cell wall formation, while proteins associated with stress peaked when approaching TE cell death. In particular, CELLULOSE SYNTHASE-INTERACTING PROTEIN1, already associated with primary wall synthesis, was enriched during secondary cell wall formation. RNAi knockdown of genes encoding several of the identified proteins showed that secondary wall formation depends on the coordinated presence of microtubule interacting proteins with nonoverlapping functions: cell wall thickness, cell wall homogeneity, and the pattern and cortical location of the wall are dependent on different proteins. Altogether, proteins linking microtubules to a range of metabolic compartments vary specifically during TE differentiation and regulate different aspects of wall patterning.

Arabidopsis KCBP interacts with AIR9 but stays in the cortical division zone throughout mitosis via its MyTH4-FERM domain.

The preprophase band of microtubules performs the crucial function of marking the plane of cell division. Although the preprophase band depolymerises at the onset of mitosis, the division plane is 'memorized' by a cortical division zone to which the phragmoplast is attracted during cytokinesis. Proteins have been discovered that are part of the molecular memory but little is known about how they contribute to phragmoplast guidance. Previously, we found that the microtubule-associated protein AIR9 is found in the cortical division zone at preprophase and returns during cell plate insertion but is absent from the cortex during the intervening mitosis. To identify new components of the preprophase memory, we searched for proteins that interact with AIR9. We detected the kinesin-like calmodulin-binding protein, KCBP, which can be visualized at the predicted cortical site throughout division. A truncation study of KCBP indicates that its MyTH4-FERM domain is required for linking the motor domain to the cortex. These results suggest a mechanism by which minus-end-directed KCBP helps guide the centrifugally expanding phragmoplast to the cortical division site.

Actin-Dependent and -Independent Functions of Cortical Microtubules in the Differentiation of Arabidopsis Leaf Trichomes.

Arabidopsis thaliana tortifolía2 carries a point mutation in α-tubulin 4 and shows aberrant cortical microtubule dynamics. The microtubule defect of tortifolia2 leads to overbranching and right-handed helical growth in the single-celled leaf trichomes. Here, we use tortifolia2 to further our understanding of microtubules in plant cell differentiation. Trichomes at the branching stage show an apical ring of cortical microtubules, and our analyses support that this ring is involved in marking the prospective branch site. tortifolia2 showed ectopic microtubule bundles at this stage, consistent with a function for microtubules in selecting new branch sites. Overbranching of tortifolia2 required the C-terminal binding protein/brefeldin A-ADP ribosylated substrate protein ANGUSTIFOLIA1, and our results indicate that the angustifolia1 mutant is hypersensitive to alterations in microtubule dynamics. To analyze whether actin and microtubules cooperate in the trichome cell expansion process, we generated double mutants of tortifolia2 with distorted1, a mutant that is defective in the actin-related ARP2/3 complex. The double mutant trichomes showed a complete loss of growth anisotropy, suggesting a genetic interaction of actin and microtubules. Green fluorescent protein labeling of F-actin or microtubules in tortifolia2 distorted1 double mutants indicated that F-actin enhances microtubule dynamics and enables reorientation. Together, our results suggest actin-dependent and -independent functions of cortical microtubules in trichome differentiation.

Mechanisms for shaping, orienting, positioning and patterning plant secondary cell walls.

Xylem vessels are cells that develop a specifically ornamented secondary cell wall to ensure their vascular function, conferring both structural strength and impermeability. Further plasticity is given to these vascular cells by a range of different patterns described by their secondary cell walls that-as for the growth of all plant organs-are developmentally regulated. Microtubules and their associated proteins, named MAPs, are essential to define the shape, the orientation, the position and the overall pattern of these secondary cell walls. Key actors in this process are the land-plant specific MAP70 proteins which not only allow the secondary cell wall to be positioned at the cell cortex but also determine the overall pattern described by xylem vessel secondary cell walls. 

Microtubule dynamics in plant cells.

This chapter describes some of the choices and unavoidable compromises to be made when studying microtubule dynamics in plant cells. The choice of species still depends very much on the ability to produce transgenic plants and most work has been done in the relatively small cells of Arabidopsis plants or in tobacco BY-2 suspension cells. Fluorescence-tagged microtubule proteins have been used to label entire microtubules, or their plus ends, but there are still few minus-end markers for these acentrosomal cells. Pragmatic decisions have to be made about probes, balancing the efficacy of microtubule labeling against a tendency to overstabilize and bundle the microtubules and even induce helical plant growth. A key limitation in visualizing plant microtubules is the ability to keep plants alive for long periods under the microscope and we describe a biochamber that allows for plant cell growth and development while allowing gas exchange and reducing evaporation. Another major difficulty is the limited fluorescence lifetime and we describe imaging strategies to reduce photobleaching in long-term imaging. We also discuss methods of measuring microtubule dynamics, with emphasis on the behavior of plant-specific microtubule arrays.

The microtubule-associated protein AtMAP70-5 regulates secondary wall patterning in Arabidopsis wood cells.

Xylem tracheary elements (TEs) form hollow, sap-conducting tubes kept open by thickened ribs of secondary cell wall that provide the major structural element in wood. These ribs are enriched with cellulose and lignin, molecules that utilize more atmospheric CO(2) than any other biopolymer on Earth. The thickenings form characteristic patterns (e.g., spiral and pitted) that depend upon the bundling of underlying microtubules [1, 2]. To identify microtubule-associated proteins (MAPs) involved in patterning microtubules, we optimized an in vitro system for triggering single Arabidopsis cells to differentiate synchronously into TEs. From more than 200 microtubule-implicated proteins, AtMAP70-5 was the only MAP upregulated upon, and specific to, TE differentiation. It lines the borders of each microtubule bundle and forms C-shaped "spacers" between adjacent bundles. Manipulating levels of AtMAP70-5 and its binding partner AtMAP70-1 by overexpression or RNA interference (RNAi) silencing shifted the balance between the characteristic patterns. RNAi silencing produced stunted plants with disorganized vascular bundles. In culture, RNAi knockdown caused ribs of secondary cell wall, surrounded by microtubules, to invaginate and fall into the cytoplasm. These results suggest that AtMAP70-5 and AtMAP70-1 are essential for defining where secondary cell wall polymers are applied at the cell cortex in wood-forming cells.

Helical growth of the Arabidopsis mutant tortifolia2 does not depend on cell division patterns but involves handed twisting of isolated cells.

Several factors regulate plant organ growth polarity. tortifolia2 (tor2), a right-handed helical growth mutant, has a conservative replacement of Arg-2 with Lys in the alpha-tubulin 4 protein. Based on a published high-resolution (2.89 A) tubulin structure, we predict that Arg-2 of alpha-tubulin forms hydrogen bonds with the GTPase domain of beta-tubulin, and structural modeling suggests that these contacts are interrupted in tor2. Consistent with this, we found that microtubule dynamicity is reduced in the tor2 background. We investigated the developmental origin of the helical growth phenotype using tor2. One hypothesis predicts that cell division patterns cause helical organ growth in Arabidopsis thaliana mutants. However, cell division patterns of tor2 root tips appear normal. Experimental uncoupling of cell division and expansion suggests that helical organ growth is based on cell elongation defects only. Another hypothesis is that twisting is due to inequalities in expansion of epidermal and cortical tissues. However, freely growing leaf trichomes of tor2 mutants show right-handed twisting and cortical microtubules form left-handed helices as early as the unbranched stage of trichome development. Trichome twisting is inverted in double mutants with tor3, a left-handed mutant. Single tor2 suspension cells also exhibit handed twisting. Thus, twisting of tor2 mutant organs appears to be a higher-order expression of the helical expansion of individual cells.

Selective recruitment of proteins to 5' cap complexes during the growth cycle in Arabidopsis.

Translation of most mRNAs is performed in a cap-dependent manner, requiring a protein complex, the cap complex, to regulate the accessibility of the message to the 40S ribosome. The cap complex initiates protein translation by binding to the 5' cap of an mRNA and recruiting ribosomes to begin translation. Compared to animals and yeast, there are significant plant-specific differences in the regulation of cap-dependent mRNA translation, but these are poorly understood. Here, we purified proteins that bind to the 5' cap during the Arabidopsis growth cycle. The protein profile of the cap-binding complexes varies during the various stages of the growth cycle in suspension culture cells. Using Western blotting, the cap complexes of quiescent cells were found to be composed of only three major proteins: eIF4isoE, which is primarily a cytoplasmic protein, and eIF4E and CBP80, which accumulate in the nucleus. However, when cells proliferate, at least 10 major proteins bind directly or indirectly to the 5' cap. Proteomic, Western blotting and immunoprecipitation data establish that the spectrum of RNA helicases in the cap complexes also changes during the growth cycle. Cap complexes from proliferating cultures mainly contain eIF4A, which associates with at least four cap complexes, but eIF4A is replaced by additional helicases in quiescent cells. These findings suggest that the dynamic and selective recruitment of various proteins to mRNA 5' cap complexes could play an important role in the regulation of gene expression.

ENDOSPERM DEFECTIVE1 Is a Novel Microtubule-Associated Protein Essential for Seed Development in Arabidopsis.

Early endosperm development involves a series of rapid nuclear divisions in the absence of cytokinesis; thus, many endosperm mutants reveal genes whose functions are essential for mitosis. This work finds that the endosperm of Arabidopsis thaliana endosperm-defective1 (ede1) mutants never cellularizes, contains a reduced number of enlarged polyploid nuclei, and features an aberrant microtubule cytoskeleton, where the specialized radial microtubule systems and cytokinetic phragmoplasts are absent. Early embryo development is substantially normal, although occasional cytokinesis defects are observed. The EDE1 gene was cloned using a map-based approach and represents the pioneer member of a conserved plant-specific family of genes of previously unknown function. EDE1 is expressed in the endosperm and embryo of developing seeds, and its expression is tightly regulated during cell cycle progression. EDE1 protein accumulates in nuclear caps in premitotic cells, colocalizes along microtubules of the spindle and phragmoplast, and binds microtubules in vitro. We conclude that EDE1 is a novel plant-specific microtubule-associated protein essential for microtubule function during the mitotic and cytokinetic stages that generate the Arabidopsis endosperm and embryo.

Arabidopsis mutants and the network of microtubule-associated functions.

In early eukaryotes, the microtubule system was engaged in mitosis, intracellular transport, and flagellum-based motility. In the plant lineage, the evolution of a multicellular body involved the conservation of some core functions, the loss of others, and the elaboration of new microtubule functions associated with the multicellular plant habit. This diversification is reflected by the presence of both conserved (animal/fungi-like) and novel (plant-like) sequences encoding microtubule-related functions in the Arabidopsis genome. The collection of microtubule mutants has grown rapidly over recent years. These mutants present a wide range of phenotypes, consistent with the hypothesis of a functional diversification of the microtubule system. In this review, we focus on mutant analysis and, in particular, discuss double mutant analysis as a valuable tool for pinpointing pathways of gene function. A future challenge will be to define the complete network of genetic and physical interactions of microtubule function in plants. In addition to reviewing recent progress in the functional analysis of the 'MAPome', we present an online database of Arabidopsis mutants impaired in microtubule functions.

AtMAP70-5, a divergent member of the MAP70 family of microtubule-associated proteins, is required for anisotropic cell growth in Arabidopsis.

AtMAP70-5 is the most divergent of a recently described multigene family of plant-specific microtubule-associated proteins (MAPs). It is significantly smaller than other members and has several isoform-specific sequence features. To confirm that this protein still functions as a MAP we show that it directly binds microtubules in vitro and decorates microtubules in vivo. When added to tubulin polymerization assays, AtMAP70-5 increases the length distribution profile of microtubules indicating that it stabilizes microtubule dynamics. The overexpressed fusion protein perturbs cell polarity in cell suspensions by inducing extra poles for growth. Similarly, in Arabidopsis plants the overexpression of AtMAP70-5 causes epidermal cells to swell; it also stunts growth and induces right-handed organ twisting. RNAi-mediated downregulation of AtMAP70-5 results in reduced inflorescence stem length and diameter and individual cells are inhibited in their capacity for expansion. These observations suggest that the control over AtMAP70-5 expression levels is important in order to maintain axial polarity and to ensure regular extension of plant organs.

Homologues of Arabidopsis Microtubule-Associated AIR9 in Trypanosomatid Parasites: Hints on Evolution and Function.

AIR9 is an essential microtubule-associated protein from Arabidopsis. Sequence similarity searches indicate homologues of AIR9 in land plants and in excavate protists, including trypanosomatid parasites and Trichomonas. The AIR9-like protein from Trypanosoma brucei was recently detected in the proteome of the trypanosome flagellum, raising the possibility that trypanosomatid AIR9-like proteins also associate with microtubules. Because microtubule functions are essential to the viability of trypanosomatid parasites AIR9-like proteins may be exploited as drug targets without homology in humans. We further discuss the unexpected phylogeny of AIR9-like proteins from plants and protozoans.

Microtubule-associated AIR9 recognizes the cortical division site at preprophase and cell-plate insertion.

In plants, the preprophase band (PPB) of microtubules marks the cortical site where the cross-wall will fuse with the parental wall during cytokinesis . This band disappears before metaphase, and it is not known how the division plane is "memorized". One idea is that the PPB leaves behind molecules involved in the maturation of the cell plate . Here, we report on the proteomic isolation of a novel 187 kDa microtubule-associated protein, AIR9, conserved in land plants and trypanosomatid parasites. AIR9 decorates cortical microtubules and the PPB but is downregulated during mitosis. AIR9 reappears at the former PPB site precisely when the cortex is contacted by the outwardly growing cytokinetic apparatus. AIR9 then moves inward on the new cross-wall and thus forms a torus. Truncation studies show that formation of the torus requires a repeated domain separate from AIR9's microtubule binding site. Cell plates induced to insert outside the predicted division site do not elicit an AIR9 torus, suggesting that AIR9 recognizes a component of the former PPB. Such misplaced walls remain immature, based on their prolonged staining for the cell-plate polymer callose. We propose that AIR9 may be part of the mechanism ensuring the maturation of those cell plates successfully contacting the "programmed" cortical division site.

The role of MAP65-1 in microtubule bundling during Zinnia tracheary element formation.

The MAP65 family of microtubule-associated proteins performs various functions at different stages of the cell cycle and differentiation. In this study, we have investigated the synchronous transdifferentiation of Zinnia mesophyll cells into tracheary elements in vitro. This allowed us to examine the role of the microtubule-associated protein MAP65 during the characteristic bunching of cortical microtubules that underlie the developing ribs of secondarily thickened cell wall. Immunofluorescence confirmed the microtubule bundles to be decorated with anti-MAP65 antibodies. Three Zinnia MAP65 genes were examined; the expression of ZeMAP65-1 was found to match that of the differentiation marker TED2 and both were found to be upregulated upon addition of inductive hormones. We cloned the full-length sequence of ZeMAP65-1 and found it to be most similar to other MAP65 isoforms known to bundle microtubules in other plant species. However, not all MAP65 proteins crosslink cortical microtubules and so, to confirm its potential bundling capacity, ZeMAP65-1 was transiently overexpressed in Arabidopsis suspension cells. This resulted in the super-bundling of microtubules in patterns resembling those in differentiating xylem cells. These findings establish that the MAP65-1 group of proteins is responsible for the bundling of cortical microtubules during secondary cell wall formation of xylogenesis as well as during the expansion of primary cell walls.

Modulated targeting of GFP-AtMAP65-1 to central spindle microtubules during division.

AtMAP65-1 bundles cortical microtubules and we examined how this property is regulated during division in time-lapse studies of Arabidopsis suspension cells expressing GFP-AtMAP65-1. Spindle fluorescence is diffuse during metaphase, restored to the central spindle at anaphase and then compacted at the midline during late anaphase/early telophase. However, mutagenesis of the microtubule-associated protein (MAP) consensus Cdk site to a non-phosphorylatable form allows premature decoration of microtubules traversing the central region of the metaphase spindle without affecting the timing of the subsequent compaction. This suggests that mutagenesis does not affect compaction but does affect a phosphorylation/dephosphorylation switch that normally targets AtMAP65-1 to the central spindle at the metaphase/anaphase transition. GFP-AtMAP65-1 continues to label the midline of the early phragmoplast, suggesting a structural continuity with the central spindle - both structures being composed of anti-parallel microtubules. However, once the cytokinetic apparatus expands into a ring the MAP becomes depleted at the midline. Despite this, cytokinesis is not arrested and membrane and callose are deposited at the cell plate. It is concluded that AtMAP65-1 plays a role in the central spindle at anaphase to early cytokinesis but is not essential at the midline of the phragmoplast at later stages.

Identification of a novel family of 70 kDa microtubule-associated proteins in Arabidopsis cells.

Most plant microtubule-associated proteins (MAPs) have homologues across the phylogenetic spectrum. To find potential plant-specific MAPs that will have evaded bioinformatic searches we devised a low stringency method for isolating proteins from an Arabidopsis cell suspension on endogenous taxol-microtubules. By tryptic peptide mass fingerprinting we identified 55 proteins that were enriched on taxol-microtubules. Amongst a range of known MAPs, such as kinesins, MAP65 isoforms and MOR1, we detected 'unknown' 70 kDa proteins that belong to a family of five closely related Arabidopsis proteins having no known homologues amongst non-plant organisms. To verify that AtMAP70-1 associates with microtubules in vivo, it was expressed as a GFP fusion. This confirmed that the protein decorates all four microtubule arrays in both transiently infected Arabidopsis and stably transformed tobacco BY-2 suspension cells. Microtubule-directed drugs perturbed the localization of AtMAP70-1 but cytochalasin D did not. AtMAP70-1 contains four predicted coiled-coil domains and truncation studies identified a central domain that targets the fusion protein to microtubules in vivo. This study therefore introduces a novel family of plant-specific proteins that interact with microtubules.

Helical growth of the Arabidopsis mutant tortifolia1 reveals a plant-specific microtubule-associated protein.

Plants can grow straight or in the twisted fashion exhibited by the helical growth of some climbing plants. Analysis of helical-growth mutants from Arabidopsis has indicated that microtubules are involved in the expression of the helical phenotype. Arabidopsis mutants growing with a right-handed twist have been reported to have cortical microtubules that wind around the cell in left-handed helices and vice versa. Microtubular involvement is further suspected from the finding that some helical mutants are caused by single amino acid substitutions in alpha-tubulin and because of the sensitivity of the growth pattern to anti-microtubule drugs. Insight into the roles of microtubules in organ elongation is anticipated from analyses of genes defined by helical mutations. We investigated the helical growth of the Arabidopsis mutant tortifolia1/spiral2 (tor1/spr2), which twists in a right-handed manner, and found that this correlates with a complex reorientation of cortical microtubules. TOR1 was identified by a map-based approach; analysis of the TOR1 protein showed that it is a member of a novel family of plant-specific proteins containing N-terminal HEAT repeats. Recombinant TOR1 colocalizes with cortical microtubules in planta and binds directly to microtubules in vitro. This shows that TOR1 is a novel, plant-specific microtubule-associated protein (MAP) that regulates the orientation of cortical microtubules and the direction of organ growth.

In vivo interaction between CDKA and eIF4A: a possible mechanism linking translation and cell proliferation.

In a proteomics-based screen for proteins interacting with cyclin-dependent protein kinase (CDK), we have identified a novel CDK complex containing the eukaryotic translation initiation factor, eIF4A. Reciprocal immunoprecipitations using antibodies against eIF4A indicate that the interaction is specific. The CDKA-eIF4A complex is abundant in actively proliferating and growing cells but is absent from cells that have ceased dividing. The CDKA-eIF4A complex contains kinase activity that is sensitive to the CDK-specific inhibitor roscovitine. This interaction points to a possible molecular mechanism linking cell proliferation with translational control.

EB1 reveals mobile microtubule nucleation sites in Arabidopsis.

In plants, it is unclear how dispersed cortical microtubules are nucleated, polarized and organized in the absence of centrosomes. In Arabidopsis thaliana cells, expression of a fusion between the microtubule-end-binding protein AtEB1a and green fluorescent protein (GFP) results in labelling of spindle poles, where minus ends gather. During interphase, AtEB1a-GFP labels the microtubule plus end as a comet, but also marks the minus end as a site from which microtubules can grow and shrink. These minus-end nucleation sites are mobile, explaining how the cortical array can redistribute during the cell cycle and supporting the idea of a flexible centrosome in plants.