Specific Targeting of Plant and Apicomplexa Parasite Tubulin through Differential Screening Using In Silico and Assay-Based Approaches.

Dinitroanilines are chemical compounds with high selectivity for plant cell α-tubulin in which they promote microtubule depolymerization. They target α-tubulin regions that have diverged over evolution and show no effect on non-photosynthetic eukaryotes. Hence, they have been used as herbicides over decades. Interestingly, dinitroanilines proved active on microtubules of eukaryotes deriving from photosynthetic ancestors such as Toxoplasma gondii and Plasmodium falciparum, which are responsible for toxoplasmosis and malaria, respectively. By combining differential in silico screening of virtual chemical libraries on Arabidopsis thaliana and mammal tubulin structural models together with cell-based screening of chemical libraries, we have identified dinitroaniline related and non-related compounds. They inhibit plant, but not mammalian tubulin assembly in vitro, and accordingly arrest A. thaliana development. In addition, these compounds exhibit a moderate cytotoxic activity towards T. gondii and P. falciparum. These results highlight the potential of novel herbicidal scaffolds in the design of urgently needed anti-parasitic drugs.

Tau can switch microtubule network organizations: from random networks to dynamic and stable bundles.

In neurons, microtubule networks alternate between single filaments and bundled arrays under the influence of effectors controlling their dynamics and organization. Tau is a microtubule bundler that stabilizes microtubules by stimulating growth and inhibiting shrinkage. The mechanisms by which tau organizes microtubule networks remain poorly understood. Here, we studied the self-organization of microtubules growing in the presence of tau isoforms and mutants. The results show that tau's ability to induce stable microtubule bundles requires two hexapeptides located in its microtubule-binding domain and is modulated by its projection domain. Site-specific pseudophosphorylation of tau promotes distinct microtubule organizations: stable single microtubules, stable bundles, or dynamic bundles. Disease-related tau mutations increase the formation of highly dynamic bundles. Finally, cryo-electron microscopy experiments indicate that tau and its variants similarly change the microtubule lattice structure by increasing both the protofilament number and lattice defects. Overall, our results uncover novel phosphodependent mechanisms governing tau's ability to trigger microtubule organization and reveal that disease-related modifications of tau promote specific microtubule organizations that may have a deleterious impact during neurodegeneration.

Phosphorylation of MAP65-1 by Arabidopsis Aurora Kinases Is Required for Efficient Cell Cycle Progression.

Aurora kinases are key effectors of mitosis. Plant Auroras are functionally divided into two clades. The alpha Auroras (Aurora1 and Aurora2) associate with the spindle and the cell plate and are implicated in controlling formative divisions throughout plant development. The beta Aurora (Aurora3) localizes to centromeres and likely functions in chromosome separation. In contrast to the wealth of data available on the role of Aurora in other kingdoms, knowledge on their function in plants is merely emerging. This is exemplified by the fact that only histone H3 and the plant homolog of TPX2 have been identified as Aurora substrates in plants. Here we provide biochemical, genetic, and cell biological evidence that the microtubule-bundling protein MAP65-1-a member of the MAP65/Ase1/PRC1 protein family, implicated in central spindle formation and cytokinesis in animals, yeasts, and plants-is a genuine substrate of alpha Aurora kinases. MAP65-1 interacts with Aurora1 in vivo and is phosphorylated on two residues at its unfolded tail domain. Its overexpression and down-regulation antagonistically affect the alpha Aurora double mutant phenotypes. Phospho-mutant analysis shows that Aurora contributes to the microtubule bundling capacity of MAP65-1 in concert with other mitotic kinases.

Resolving bundled microtubules using anti-tubulin nanobodies.

Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50-70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.

Microtubule-sliding activity of a kinesin-8 promotes spindle assembly and spindle-length control.

Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by crosslinking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report an antiparallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule-destabilizing activity. In conjunction with Cin8, a kinesin-5 family member, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a slide-disassemble model where the sliding and destabilizing activity of Kip3 balance during pre-anaphase. This facilitates normal spindle assembly. However, the destabilizing activity of Kip3 dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly.

MAP65 coordinate microtubule growth during bundle formation.

Microtubules (MTs) are highly dynamical structures that play a crucial role in cell physiology. In cooperation with microtubule-associated proteins (MAPs), MTs form bundles endowing cells with specific mechanisms to control their shape or generate forces. Whether the dynamics of MTs is affected by the lateral connections that MAPs make between MTs during bundle formation is still under debate. Using in vitro reconstitution of MT bundling, we analyzed the dynamics of MT bundles generated by two plant MAP65 (MAP65-1/4), MAP65-1 being the plant ortholog of vertebrate PRC1 and yeast Ase1. MAP65-1/4 limit the amplitude of MT bundle depolymerization and increase the elongation phases. The subsequent sustained elongation of bundles is governed by the coordination of MT growth, so that MT ends come in close vicinity. We develop a model based on the assumption that both MAP65-1/4 block MT depolymerization. Model simulations reveal that rescue frequencies are higher between parallel than between anti-parallel MTs. In consequence the polarity of bundled MTs by MAP65 controls the amplitude of bundle's growth. Our results illustrate how MAP-induced MT-bundling, which is finely tuned by MT polarity, robustly coordinates MT elongation within bundles.

Quantification of MAP and molecular motor activities on geometrically controlled microtubule networks.

The spatial organization of the microtubule (MT) network directs cell polarity and mitosis. It is finely regulated by hundreds of different types of microtubule-associated proteins and molecular motors whose specific functions are difficult to investigate directly in cells. Here, we have investigated their functions using geometrically controlled MT networks in vitro in cell-free assay. This was achieved by developing a new method to spatially define MT nucleation using MT microseeds adsorbed on a micropatterned glass substrate. This method could be used to control MT growth and the induction of complex MT networks. We selected the interaction of two radial arrays of dynamic and polarized MTs to analyze the formation of the central antiparallel MT bundle. We investigated the effects of the MT cross-linker anaphase spindle elongation 1 (Ase1) and the kinesin motor Klp2, which are known to regulate MT organization in the spindle midzone. We thus identified the respective roles of each protein and revealed their synergy on the establishment of stable antiparallel MT bundles by quantifying MT interactions over hundreds of comparable MT networks.

The Arabidopsis TRM1-TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes.

Land plant cells assemble microtubule arrays without a conspicuous microtubule organizing center like a centrosome. In Arabidopsis thaliana, the TONNEAU1 (TON1) proteins, which share similarity with FOP, a human centrosomal protein, are essential for microtubule organization at the cortex. We have identified a novel superfamily of 34 proteins conserved in land plants, the TON1 Recruiting Motif (TRM) proteins, which share six short conserved motifs, including a TON1-interacting motif present in all TRMs. An archetypal member of this family, TRM1, is a microtubule-associated protein that localizes to cortical microtubules and binds microtubules in vitro. Not all TRM proteins can bind microtubules, suggesting a diversity of functions for this family. In addition, we show that TRM1 interacts in vivo with TON1 and is able to target TON1 to cortical microtubules via its C-terminal TON1 interaction motif. Interestingly, three motifs of TRMs are found in CAP350, a human centrosomal protein interacting with FOP, and the C-terminal M2 motif of CAP350 is responsible for FOP recruitment at the centrosome. Moreover, we found that TON1 can interact with the human CAP350 M2 motif in yeast. Taken together, our results suggest conservation of eukaryotic centrosomal components in plant cells.

Differential interactions of the formins INF2, mDia1, and mDia2 with microtubules.

A number of cellular processes use both microtubules and actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be elucidated in detail. Formins are actin-binding proteins that have multiple effects on actin dynamics, and one formin, mDia2, has been shown to bind and stabilize microtubules through its formin homology 2 (FH2) domain. Here we show that three formins, INF2, mDia1, and mDia2, display important differences in their interactions with microtubules and actin. Constructs containing FH1, FH2, and C-terminal domains of all three formins bind microtubules with high affinity (K(d) < 100 nM). However, only mDia2 binds microtubules at 1:1 stoichiometry, with INF2 and mDia1 showing saturating binding at approximately 1:3 (formin dimer:tubulin dimer). INF2-FH1FH2C is a potent microtubule-bundling protein, an effect that results in a large reduction in catastrophe rate. In contrast, neither mDia1 nor mDia2 is a potent microtubule bundler. The C-termini of mDia2 and INF2 have different functions in microtubule interaction, with mDia2's C-terminus required for high-affinity binding and INF2's C-terminus required for bundling. mDia2's C-terminus directly binds microtubules with submicromolar affinity. These formins also differ in their abilities to bind actin and microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia2, whereas they moderately inhibit mDia1 and have no effect on INF2. Conversely, actin monomers inhibit microtubule binding/bundling by INF2 but do not affect mDia1 or mDia2. These differences in interactions with microtubules and actin suggest differential function in cellular processes requiring both cytoskeletal elements.

Arabidopsis kinetochore fiber-associated MAP65-4 cross-links microtubules and promotes microtubule bundle elongation.

The acentrosomal plant mitotic spindle is uniquely structured in that it lacks opposing centrosomes at its poles and is equipped with a connective preprophase band that regulates the spatial framework for spindle orientation and mobility. These features are supported by specialized microtubule-associated proteins and motors. Here, we show that Arabidopsis thaliana MAP65-4, a non-motor microtubule associated protein (MAP) that belongs to the evolutionarily conserved MAP65 family, specifically associates with the forming mitotic spindle during prophase and with the kinetochore fibers from prometaphase to the end of anaphase. In vitro, MAP65-4 induces microtubule (MT) bundling through the formation of cross-bridges between adjacent MTs both in polar and antipolar orientations. The association of MAP65-4 with an MT bundle is concomitant with its elongation. Furthermore, MAP65-4 modulates the MT dynamic instability parameters of individual MTs within a bundle, mainly by decreasing the frequency of catastrophes and increasing the frequency of rescue events, and thereby supports the progressive lengthening of MT bundles over time. These properties are in line with its role of initiating kinetochore fibers during prospindle formation.

Two microtubule-associated proteins of Arabidopsis MAP65s promote antiparallel microtubule bundling.

The Arabidopsis MAP65s are a protein family with similarity to the microtubule-associated proteins PRC1/Ase1p that accumulate in the spindle midzone during late anaphase in mammals and yeast, respectively. Here we investigate the molecular and functional properties of AtMAP65-5 and improve our understanding of AtMAP65-1 properties. We demonstrate that, in vitro, both proteins promote the formation of a planar network of antiparallel microtubules. In vivo, we show that AtMAP65-5 selectively binds the preprophase band and the prophase spindle microtubule during prophase, whereas AtMAP65-1-GFP selectively binds the preprophase band but does not accumulate at the prophase spindle microtubules that coexists within the same cell. At later stages of mitosis, AtMAP65-1 and AtMAP65-5 differentially label the late spindle and phragmoplast. We present evidence for a mode of action for both proteins that involves the binding of monomeric units to microtubules that "zipper up" antiparallel arranged microtubules through the homodimerization of the N-terminal halves when adjacent microtubules encounter.

Analysis of cortical arrays from Tradescantia virginiana at high resolution reveals discrete microtubule subpopulations and demonstrates that confocal images of arrays can be misleading.

Cortical microtubule arrays are highly organized networks involved in directing cellulose microfibril deposition within the cell wall. Their organization results from complex interactions between individual microtubules and microtubule-associated proteins. The precise details of these interactions are often not evident using optical microscopy. Using high-resolution scanning electron microscopy, we analyzed extensive regions of cortical arrays and identified two spatially discrete microtubule subpopulations that exhibited different stabilities. Microtubules that lay adjacent to the plasma membrane were often bundled and more stable than the randomly aligned, discordant microtubules that lay deeper in the cytoplasm. Immunolabeling revealed katanin at microtubule ends, on curves, or at sites along microtubules in line with neighboring microtubule ends. End binding 1 protein also localized along microtubules, at microtubule ends or junctions between microtubules, and on the plasma membrane in direct line with microtubule ends. We show fine bands in vivo that traverse and may encircle microtubules. Comparing confocal and electron microscope images of fluorescently tagged arrays, we demonstrate that optical images are misleading, highlighting the fundamental importance of studying cortical microtubule arrays at high resolution.

Arabidopsis katanin binds microtubules using a multimeric microtubule-binding domain.

Katanin is a heterodimeric protein that mediates ATP-dependent destabilization of microtubules in animal cells. In plants, the catalytic subunit of Arabidopsis thaliana katanin (AtKSS, Arabidopsis thaliana Katanin Small Subunit) has been identified and its microtubule-severing activity has been demonstrated in vitro. In vivo, plant katanin plays a role in the organization of cortical microtubules, but the way it achieves this function is unknown. To go further in our understanding of the mechanisms by which katanin severs microtubules, we analyzed the functional domains of Arabidopsis katanin. We characterized the microtubule-binding domain of katanin both in vitro and in vivo. It corresponds to a poorly conserved sequence between plant and animal katanins that is located in the N-terminus of the protein. This domain interacts with cortical microtubules in vivo and has a low affinity for microtubules in vitro. We also observed that katanin microtubule-binding domain oligomerizes into trimers. These results show that, besides being involved in the interaction of katanin with microtubules, the microtubule-binding domain may also participate in the oligomerization of katanin. At the structural level, we observed that AtKSS forms ring-shaped oligomers.

Katanin's severing activity favors bundling of cortical microtubules in plants.

Higher plant cells exhibit interphase microtubule arrays specific to plants, which are essential for their developmental program. These cortical microtubules (CMT) consist of a population of highly dynamic microtubules that are usually organized into bundles in the cortex of the cells. The organization of CMT is intimately linked to the acquisition of specialized functions, and subsequentchanges in their distribution affect their properties. The mechanisms underlying the formation and the distribution of CMT are still unclear, and little is known about the proteins that are involved in this phenomenon. Here we investigated the putative role of katanin, the only known plant microtubule-severing protein, in the organization of CMT. We generated transgenic Arabidopsis lines that overexpress katanin under the control of an ethanol-inducible promoter. In response to an induced overexpression of katanin, CMT organized into numerous and thick bundles, which ultimately depolymerized. From the analyses of CMT patterns together with recent data on CMT dynamics, we propose that, in interphase cells, katanin's main activity is to free CMT, generating motile microtubules that incorporate into bundles.

Arabidopsis CUL3A and CUL3B genes are essential for normal embryogenesis.

Cullin (CUL)-dependent ubiquitin ligases form a class of structurally related multisubunit enzymes that control the rapid and selective degradation of important regulatory proteins involved in cell cycle progression and development, among others. The CUL3-BTB ligases belong to this class of enzymes and despite recent findings on their molecular composition, our knowledge on their functions and substrates remains still very limited. In contrast to budding and fission yeast, CUL3 is an essential gene in metazoans. The model plant Arabidopsis thaliana encodes two related CUL3 genes, called CUL3A and CUL3B. We recently reported that cul3a loss-of-function mutants are viable but exhibit a mild flowering and light sensitivity phenotype. We investigated the spatial and temporal expression of the two CUL3 genes in reproductive tissues and found that their expression patterns are largely overlapping suggesting possible functional redundancy. Thus, we investigated the consequences on plant development of combined Arabidopsis cul3a cul3b loss-of-function mutations. Homozygous cul3b mutant plants developed normally and were fully fertile. However, the disruption of both the CUL3A and CUL3B genes reduced gametophytic transmission and caused embryo lethality. The observed embryo abortion was found to be under maternal control. Arrest of embryogenesis occurred at multiple stages of embryo development, but predominantly at the heart stage. At the cytological level, CUL3 loss-of-function mutations affected both embryo pattern formation and endosperm development.

Identification and characterization of plasma membrane proteins that bind to microtubules in pollen tubes and generative cells of tobacco.

The organization and function of microtubules in plant cells are important in many developmental stages. Connections between microtubules and the endomembrane system of plant cells have been discovered by microscopy, but the molecular characteristics of these relationships are mostly unknown except for a few cases. Using two antibodies raised against microtubule-associated proteins (MAPs) from maize, we have identified two polypeptides that share properties of the MAP family in the pollen tube of Nicotiana tabacum. The two polypeptides (with an apparent Mr of 161 and 90 kDa) bind efficiently to animal and plant microtubules and are found in association with the cellular membranes of the pollen tube, from which they can be solubilized with a zwitterionic detergent. One of these proteins has been purified and shown to promote the assembly of tubulin and, to a lesser extent, the bundling of microtubules. Subcellular fractionation indicated that the two proteins are associated with the plasma membrane compartment. The two proteins are found to co-localize in situ with cortical microtubules in the vegetative cytoplasm of tobacco pollen tubes; co-localization is also evident in the generative cell. According to these data, both the 161 and 90 kDa polypeptides are likely to mediate the interactions between the plasma membrane and microtubules in pollen tubes. In addition, functional data indicate that these MAP-like proteins take part in the process of microtubule assembly and reorganization occurring during cell growth. The evidence that both proteins associate with different cellular compartments also suggests a broad-spectrum role in mediating the dynamic relationships between microtubules and plant cell membranes.

Interactions of tobacco microtubule-associated protein MAP65-1b with microtubules.

Tobacco microtubule associated protein (MAP65) (NtMAP65s) constitute a family of microtubule-associated proteins with apparent molecular weight around 65 kDa that collectively induce microtubule bundling and promote microtubule assembly in vitro. They are associated with most of the tobacco microtubule arrays in situ. Recently, three NtMAP65s belonging to the NtMAP65-1 subfamily have been cloned. Here we investigated in vitro the biochemical properties of one member of this family, the tobacco NtMAP65-1b. We demonstrated that recombinant NtMAP65-1b is a microtubule-binding and a microtubule-bundling protein. NtMAP65-1b has no effect on microtubule polymerization rate and binds microtubules with an estimated equilibrium constant of dissociation (K(d)) of 0.57 micro m. Binding of NtMAP65-1b to microtubules occurs through the carboxy-terminus of tubulin, as NtMAP65-1b was no longer able to bind subtilisin-digested tubulin. In vitro, NtMAP65-1b stabilizes microtubules against depolymerization induced by cold, but not against katanin-induced destabilization. The biological implications of these results are discussed.

Actin polymerization processes in plant cells.

Growing evidence shows that the actin cytoskeleton is a key effector of signal transduction, which controls and maintains the shape of plant cells, as well as playing roles in plant morphogenesis. Recently, several signaling pathways, including those triggered by hormones, Ca(2+), and cAMP, have been reported to be connected to the reorganization of the actin cytoskeleton. The molecular mechanisms involved in such signaling cascades are, however, largely unknown. The Arabidopsis genome sequence is a valuable tool for identifying some of the highly conserved molecules that are involved in such signaling cascades. Recent work has begun to unravel these complex pathways using a panoply of techniques, including genetic analysis, live-cell imaging of intracellular actin dynamics, in vivo localization of factors that are involved in the control of actin dynamics, and the biochemical characterization of how these factors function.

Functional evidence for in vitro microtubule severing by the plant katanin homologue.

Temporal and spatial assembly of microtubules in plant cells depends mainly on the activity of microtubule-interacting proteins, which either stabilize, destabilize or translocate microtubules. Recent data have revealed that the thale cress (Arabidopsis thaliana) contains a protein related to the p60 catalytic subunit of animal katanin, a microtubule-severing protein. However, effects of the plant p60 on microtubule assembly are not known. We report the first functional evidence that the recombinant A. thaliana p60 katanin subunit, Atp60, binds to microtubules and severs them in an ATP-dependent manner in vitro. ATPase activity of Atp60 is stimulated by low tubulin/katanin ratios, and is inhibited at higher ratios. Considering its properties in vitro, several functions of Atp60 in vivo are discussed.