Virginie Stoppin-Mellet

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.

Higher Plant Cells: Gamma-Tubulin and Microtubule Nucleation in the Absence of Centrosomes

The assembly of the higher plant cytoskeleton poses several fundamental questions. Since different microtubule arrays are successively assembled during the cell cycle in the absence of centrosomes, we can ask how these arrays are assembled and spatially organized. Two hypotheses are under debate. Either multiple nucleation sites are responsible for the assembly and organization of microtubule arrays or microtubule nucleation takes place at one site, the nuclear surface. In the latter case, microtubule nucleation and organization would be two distinct but coregulated processes. During recent years, novel approaches have provided entirely new insights to understand the assembly and dynamics of the plant cytoskeleton. In the present review, we summarize advances made in microscopy and in molecular biology which lead to novel hypotheses and open up new fields of investigation. From the results obtained, it is clear that the higher plant cell is a powerful model system to investigate cytoskeletal organization in acentrosomal eukaryotic cells. Microsc. Res. Tech. 49:487–495, 2000.

MAP65/Ase1 promote microtubule flexibility

Two microtubule cross-linkers of the major MAP65/PRC1/Ase1 family are found to modify the mechanical properties of dynamic microtubules (e.g., decrease the flexural rigidity of microtubules). This finding points to a role for these proteins in the formation of specific microtubule arrays in eukaryotic cells.

Arabidopsis Kinetochore Fiber-Associated MAP65-4 Cross-Links Microtubules and Promotes Microtubule Bundle Elongation

This study shows that Arabidopsis MAP65-4 associates with the forming spindle and kinetochore fibers during mitosis. In vitro, MAP65-4 induces microtubule (MT) bundling and 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, which results in the progressive lengthening of MT bundles. 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.

Characterization of microsome-associated tobacco BY-2 centrins

Centrin - higher plants - MTOCs - microtubules nucleation In most eukaryotic cells, the Ca(2+)-binding protein centrin is associated with structured microtubule-organizing centers (MTOCs) such as centrosomes. In these cells, centrin either forms centrosome-associated contractile fibers, or is involved in centrosome biogenesis. Our aim was to investigate the functions of centrin in higher plant cells which do not contain centrosome-like MTOCs. We have cloned two tobacco BY-2 centrin cDNAs and we show that higher plant centrins define a phylogenetic group of proteins distinct from centrosome-associated centrins. In addition, tobacco centrins were found primarily associated with microsomes and did not colocalize with gamma-tubulin, a known MTOC marker. While the overall level of centrin did not vary during the cell cycle, centrin was prominently detected at the cell plate during telophase. Our results suggest that in tobacco, the major portion of centrin is not MTOC-associated and could be involved in the formation of the cell plate during cytokinesis.

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

Tau antagonizes tracking of end-binding proteins (EBs) at microtubule ends, a process requiring the C-terminal part of EBs and the microtubule-binding sites of tau. The inhibiting activity of tau on EB properties is regulated by tau phosphorylation. The interplay between EBs and tau proteins results in modulation of microtubule dynamics.

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.

Plant katanin, a microtubule severing protein

Microtubules are highly dynamic polymers built from the addition and loss of tubulin dimers at their ends. Microtubule dynamics are essential to achieve fundamental cellular processes such as cell division. In vivo, microtubule assembly is tightly regulated. This regulation mainly results from the activity of microtubule effectors that interact with microtubules or tubulin dimers. There are two main types of microtubule effectors, those that favour the polymerised state of microtubules (mostly MAPs), and those that promote microtubule depolymerisation. Katanin is the best described microtubule-severing protein. Animal katanin is a hetero-dimer protein, composed of a catalytic subunit of 60 kDa (P60) and a regulatory subunit of 80 kDa (P80). P60 is an AAA (ATPases associated with various cellular activities) protein which hydrolyses ATP in a microtubule-dependent manner and is sufficient to sever microtubules in vitro (McNally et al., 2000). In animal cells, katanin is thought to be involved in the release of microtubules from centrosomes and the regulation of the number of microtubule ends in the spindle (Buster et al., 2002). In higher plants, no homologue of the P80 katanin regulatory subunit has been described so far. Recently two Arabidopsis thaliana mutants mutated in a gene showing significant homologies with the animal P60 catalytic katanin subunit have been described (Bichet et al., 2001; Burk et al., 2001; for a description of katanin mutants, see the abstract of D. Bouchez on the BOTERO mutant). Surprisingly, the microtubule cytoskeleton of these mutants has only very subtle defects, and cells seem to divide normally. Molecular effects of the plant P60 on microtubule assembly were not known. To study microtubule destabilization in plant cells, we cloned the cDNA encoding for the P60 orthologue in A. thaliana (AtP60) and functionally characterized the properties of a recombinant His-tagged AtP60 (Stoppin-Mellet et al., 2002). Using both video-microscopy assays and spectrofluorimetry, we showed for the first time that HisAtP60 can sever microtubules in vitro in the presence of ATP. His-AtP60 directly interacts with microtubules in co-sedimentation assays. In the presence of microtubules, the ATPase activity of AtP60 was stimulated in a non-hyperbolic way. The basal ATPase activity of AtP60 (calculated at one molecule ATP/AtP60/sec) was stimulated up to six times at low AtP60/tubulin molar ratio (maximum at 0.04), and inhibited at higher ratios. AtP60 is the first plant protein shown to fragment microtubules. The characterization of the functional domains of AtP60 is now under way, both in vitro and in vivo.

Tobacco BY-2 cell-free extracts induce the recovery of microtubule nucleating activity of inactivated mammalian centrosomes.

The structure and the molecular composition of the microtubule-organizing centers in acentriolar higher plant cells remain unknown. We developed an in vitro complementation assay where tobacco BY-2 extracts can restore the microtubule-nucleating activity of urea-inactivated mammalian centrosomes. Our results provide first evidence that soluble microtubule-nucleating factors are present in the plant cytosolic fraction. The implication for microtubule nucleation in higher plants is discussed.

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

Arabidopsis Aurora kinases phosphorylate MAP65-1 at its unfolded tail domain and dynamic switching of its phosphorylation status throughout mitosis is required for proper 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.