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.

Stable GDP-tubulin islands rescue dynamic microtubules.

Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they provide structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. Here, using in vitro reconstitution assays, we show that GDP-tubulin, usually considered inactive, can itself assemble into microtubules, preferentially at the minus end, and promote persistent growth. GDP-tubulin-assembled microtubules are highly stable, displaying no detectable spontaneous shrinkage. Strikingly, islands of GDP-tubulin within dynamic microtubules stop shrinkage events and promote rescues. Microtubules thus possess an intrinsic capacity for stability, independent of accessory proteins. This finding provides novel mechanisms to explain microtubule dynamics.

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.

VASH1-SVBP and VASH2-SVBP generate different detyrosination profiles on microtubules.

The detyrosination/tyrosination cycle of α-tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzymes involved in microtubule detyrosination, whose mode of action is little known. Here, we show in reconstituted systems and cells that VASH1-SVBP and VASH2-SVBP drive the global and local detyrosination of microtubules, respectively. We solved the cryo-electron microscopy structure of VASH2-SVBP bound to microtubules, revealing a different microtubule-binding configuration of its central catalytic region compared to VASH1-SVBP. We show that the divergent mode of detyrosination between the two enzymes is correlated with the microtubule-binding properties of their disordered N- and C-terminal regions. Specifically, the N-terminal region is responsible for a significantly longer residence time of VASH2-SVBP on microtubules compared to VASH1-SVBP. We suggest that this VASH region is critical for microtubule detachment and diffusion of VASH-SVBP enzymes on lattices. Our results suggest a mechanism by which VASH1-SVBP and VASH2-SVBP could generate distinct microtubule subpopulations and confined areas of detyrosinated lattices to drive various microtubule-based cellular functions.

Studying Tau-Microtubule Interaction Using Single-Molecule TIRF Microscopy.

Microtubule architecture depends on a complex network of microtubule-associated proteins (MAPs) that act in concert to modulate microtubule assembly/disassembly and spatial arrangement. In vitro reconstitution of cytoskeleton dynamics coupled to single-molecule fluorescence assays has opened new perspectives to quantify the interaction of MAPs with microtubules. Here, we present a Total Internal Reflection Fluorescence (TIRF) microscopy-based assay enabling the characterization of Tau interaction with dynamic microtubules at the single-molecule level. We describe protein sample preparation in flow cells, single-molecule acquisitions by TIRF microscopy, and quantitative analysis of Tau oligomerization states and dwell time on microtubules.

Plant and mouse EB1 proteins have opposite intrinsic properties on the dynamic instability of microtubules.

OBJECTIVE: Most eukaryotic cells contain microtubule filaments, which play central roles in intra-cellular organization. However, microtubule networks have a wide variety of architectures from one cell type and organism to another. Nonetheless, the sequences of tubulins, of Microtubule Associated proteins (MAPs) and the structure of microtubules are usually well conserved throughout the evolution. MAPs being known to be responsible for regulating microtubule organization and dynamics, this raises the question of the conservation of their intrinsic properties. Indeed, knowing how the intrinsic properties of individual MAPs differ between organisms might enlighten our understanding of how distinct microtubule networks are built. End-Binding protein 1 (EB1), first described as a MAP in yeast, is conserved in plants and mammals. The intrinsic properties of the mammalian and the yeast EB1 proteins have been well described in the literature but, to our knowledge, the intrinsic properties of EB1 from plant and mammals have not been compared thus far. RESULTS: Here, using an in vitro assay, we discovered that plant and mammalian EB1 purified proteins have different intrinsic properties on microtubule dynamics. Indeed, the mammalian EB1 protein increases microtubules dynamic while the plant EB1 protein stabilizes them.

Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue.

The stress-induced c-Jun N-terminal kinase (JNK) controls microtubule dynamics by enhancing both microtubule growth and rescues. Here, we show that upon cell stress, JNK directly phosphorylates the microtubule rescue factor CLIP-170 in its microtubule-binding domain to increase its rescue-promoting activity. Phosphomimetic versions of CLIP-170 enhance its ability to promote rescue events in vitro and in cells. Furthermore, while phosphomimetic mutations do not alter CLIP-170's capability to form comets at growing microtubule ends, both phosphomimetic mutations and JNK activation increase the occurrence of CLIP-170 remnants on the microtubule lattice at the rear of comets. As the CLIP-170 remnants, which are potential sites of microtubule rescue, display a shorter lifetime when CLIP-170 is phosphorylated, we propose that instead of acting at the time of rescue occurrence, CLIP-170 would rather contribute in preparing the microtubule lattice for future rescues at these predetermined sites.

Adenomatous Polyposis Coli as a Scaffold for Microtubule End-Binding Proteins.

End-binding proteins (EBs), referred to as the core components of the microtubule plus-end tracking protein network, interact with the C-terminus of the adenomatous polyposis coli (APC) tumor suppressor. This interaction is disrupted in colon cancers expressing truncated APC. APC and EBs act in synergy to regulate microtubule dynamics during spindle formation, chromosome segregation and cell migration. Since EBs autonomously end-track microtubules and partially co-localize with APC at microtubule tips in cells, EBs have been proposed to direct APC to microtubule ends. However, the interdependency of EB and APC localization on microtubules remains elusive. Here, using in vitro reconstitution and single-molecule imaging, we have investigated the interplay between EBs and the C-terminal domain of APC (APC-C) on dynamic microtubules. Our results show that APC-C binds along the microtubule wall but does not accumulate at microtubule tips, even when EB proteins are present. APC-C was also found to enhance EB binding at the extremity of growing microtubules and on the microtubule lattice: APC-C promotes EB end-tracking properties by increasing the time EBs spend at microtubule growing ends, whereas a pool of EBs with a fast turnover accumulates along the microtubule surface. Overall, our results suggest that APC is a promoter of EB interaction with microtubules, providing molecular determinants to reassess the relationship between APC and EBs.

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.

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.

TIRF assays for real-time observation of microtubules and actin coassembly: Deciphering tau effects on microtubule/actin interplay.

Microtubule and actin cytoskeletons are key players in vital processes in cells. Although the importance of microtubule-actin interaction for cell development and function has been highlighted for years, the properties of these two cytoskeletons have been mostly studied separately. Thus we now need procedures to simultaneously assess actin and microtubule properties to decipher the basic mechanisms underlying microtubule-actin crosstalk. Here we describe an in vitro assay that allows the coassembly of both filaments and the real-time observation of their interaction by TIRF microscopy. We show how this assay can be used to demonstrate that tau, a neuronal microtubule-associated protein, is a bona fide actin-microtubule cross-linker. The procedure relies on the use of highly purified proteins and chemically passivated perfusion chambers. We present a step-by-step protocol to obtain actin and microtubule coassembly and discuss the major pitfalls. An ImageJ macro to quantify actin and microtubule interaction is also provided.

A TIRF microscopy assay to decode how tau regulates EB's tracking at microtubule ends.

Tau is a major microtubule-associated protein (MAP) mainly expressed in the brain. Tau binds the lattice of microtubules and favors their elongation and bundling. Recent studies have shown that tau is also a partner of end-binding proteins (EBs) in neurons. EBs belong to the protein family of the plus-end tracking proteins that preferentially associate with the growing plus-ends of microtubules and control microtubule end behavior and anchorage to intracellular organelles. Reconstituted cell-free systems using purified proteins are required to understand the precise mechanisms by which tau influences EB localization on microtubules and how the concerted activity of these two MAPs modulates microtubule dynamics. We developed an in vitro assay combining TIRF microscopy and site-directed mutagenesis to dissect the interaction of tau with EBs and to study how this interaction affects microtubule dynamics. Here, we describe the detailed procedures to purify proteins (tubulin, tau, and EBs), prepare the samples for TIRF microscopy, and analyze microtubule dynamics, and EB binding at microtubule ends in the presence of tau.

Tau antagonizes end-binding protein tracking at microtubule ends through a phosphorylation-dependent mechanism.

Proper regulation of microtubule dynamics is essential for cell functions and involves various microtubule-associated proteins (MAPs). Among them, end-binding proteins (EBs) accumulate at microtubule plus ends, whereas structural MAPs bind along the microtubule lattice. Recent data indicate that the structural MAP tau modulates EB subcellular localization in neurons. However, the molecular determinants of EB/tau interaction remain unknown, as is the effect of this interplay on microtubule dynamics. Here we investigate the mechanisms governing EB/tau interaction in cell-free systems and cellular models. We find that tau inhibits EB tracking at microtubule ends. Tau and EBs form a complex via the C-terminal region of EBs and the microtubule-binding sites of tau. These two domains are required for the inhibitory activity of tau on EB localization to microtubule ends. Moreover, the phosphomimetic mutation S262E within tau microtubule-binding sites impairs EB/tau interaction and prevents the inhibitory effect of tau on EB comets. We further show that microtubule dynamic parameters vary, depending on the combined activities of EBs and tau proteins. Overall our results demonstrate that tau directly antagonizes EB function through a phosphorylation-dependent mechanism. This study highlights a novel role for tau in EB regulation, which might be impaired in neurodegenerative disorders.

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.

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.

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.

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.

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.

The plant Spc98p homologue colocalizes with gamma-tubulin at microtubule nucleation sites and is required for microtubule nucleation.

The molecular basis of microtubule nucleation is still not known in higher plant cells. This process is better understood in yeast and animals cells. In the yeast spindle pole body and the centrosome in animal cells, gamma-tubulin small complexes and gamma-tubulin ring complexes, respectively, nucleate all microtubules. In addition to gamma-tubulin, Spc98p or its homologues plays an essential role. We report here the characterization of rice and Arabidopsis homologues of SPC98. Spc98p colocalizes with gamma-tubulin at the nuclear surface where microtubules are nucleated on isolated tobacco nuclei and in living cells. AtSpc98p-GFP also localizes at the cell cortex. Spc98p is not associated with gamma-tubulin along microtubules. These data suggest that multiple microtubule-nucleating sites are active in plant cells. Microtubule nucleation involving Spc98p-containing gamma-tubulin complexes could then be conserved among all eukaryotes, despite differences in structure and spatial distribution of microtubule organizing centers.