Martine Pastuglia

The Arabidopsis TONNEAU2 Gene Encodes a Putative Novel Protein Phosphatase 2A Regulatory Subunit Essential for the Control of the Cortical Cytoskeleton

In Arabidopsis ton2 mutants, abnormalities of the cortical microtubular cytoskeleton, such as disorganization of the interphase microtubule array and lack of the preprophase band before mitosis, markedly affect cell shape and arrangement as well as overall plant morphology. We present the molecular isolation of the TON2 gene, which is highly conserved in higher plants and has a vertebrate homolog of unknown function. It encodes a protein similar in its C-terminal part to B″ regulatory subunits of type 2A protein phosphatases (PP2As). We show that the TON2 protein interacts with an Arabidopsis type A subunit of PP2A in the yeast two-hybrid system and thus likely defines a novel subclass of PP2A subunits that are possibly involved in the control of cytoskeletal structures in plants.

γ-Tubulin Is Essential for Microtubule Organization and Development in Arabidopsis

The process of microtubule nucleation in plant cells is still a major question in plant cell biology. γ-Tubulin is known as one of the key molecular players for microtubule nucleation in animal and fungal cells. Here, we provide genetic evidence that in Arabidopsis thaliana, γ-tubulin is required for the formation of spindle, phragmoplast, and cortical microtubule arrays. We used a reverse genetics approach to investigate the role of the two Arabidopsis γ-tubulin genes in plant development and in the formation of microtubule arrays. Isolation of mutants in each gene and analysis of two combinations of γ-tubulin double mutants showed that the two genes have redundant functions. The first combination is lethal at the gametophytic stage. Disruption of both γ-tubulin genes causes aberrant spindle and phragmoplast structures and alters nuclear division in gametophytes. The second combination of γ-tubulin alleles affects late seedling development, ultimately leading to lethality 3 weeks after germination. This partially viable mutant combination enabled us to follow dynamically the effects of γ-tubulin depletion on microtubule arrays in dividing cells using a green fluorescent protein marker. These results establish the central role of γ-tubulin in the formation and organization of microtubule arrays in Arabidopsis.

Arabidopsis TONNEAU1 Proteins Are Essential for Preprophase Band Formation and Interact with Centrin

Plant cells have specific microtubule structures involved in cell division and elongation. The tonneau1 (ton1) mutant of Arabidopsis thaliana displays drastic defects in morphogenesis, positioning of division planes, and cellular organization. These are primarily caused by dysfunction of the cortical cytoskeleton and absence of the preprophase band of microtubules. Characterization of the ton1 insertional mutant reveals complex chromosomal rearrangements leading to simultaneous disruption of two highly similar genes in tandem, TON1a and TON1b. TON1 proteins are conserved in land plants and share sequence motifs with human centrosomal proteins. The TON1 protein associates with soluble and microsomal fractions of Arabidopsis cells, and a green fluorescent protein–TON1 fusion labels cortical cytoskeletal structures, including the preprophase band and the interphase cortical array. A yeast two-hybrid screen identified Arabidopsis centrin as a potential TON1 partner. This interaction was confirmed both in vitro and in plant cells. The similarity of TON1 with centrosomal proteins and its interaction with centrin, another key component of microtubule organizing centers, suggests that functions involved in the organization of microtubule arrays by the centrosome were conserved across the evolutionary divergence between plants and animals.

A protein phosphatase 2A complex spatially controls plant cell division

In the absence of cell migration, the orientation of cell divisions is crucial for body plan determination in plants. The position of the division plane in plant cells is set up premitotically via a transient cytoskeletal array, the preprophase band, which precisely delineates the cortical plane of division. Here we describe a protein complex that targets protein phosphatase 2A activity to microtubules, regulating the transition from the interphase to the premitotic microtubule array. This complex, which comprises TONNEAU1 and a PP2A heterotrimeric holoenzyme with FASS as regulatory subunit, is recruited to the cytoskeleton via the TONNEAU1-recruiting motif family of proteins. Despite the acentrosomal nature of plant cells, all members of this complex share similarity with animal centrosomal proteins involved in ciliary and centriolar/centrosomal functions, revealing an evolutionary link between the cortical cytoskeleton of plant cells and microtubule organizers in other eukaryotes.

The Arabidopsis TRM1–TON1 Interaction Reveals a Recruitment Network Common to Plant Cortical Microtubule Arrays and Eukaryotic Centrosomes

TON1 is essential for cortical microtubule organization and preprophase band formation in plants. This work describes a superfamily of Arabidopsis thaliana proteins (TRMs) interacting with TON1 and show that TRM1 is able to target TON1 to microtubules. Partial sequence conservation of TON1 and TRMs with animal proteins points to a common protein network involved in centrosomal and cytoskeletal functions in eukaryotes. 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.

The function of TONNEAU1 in moss reveals ancient mechanisms of division plane specification and cell elongation in land plants

The preprophase band (PPB) is a transient ring of microtubules that forms before mitosis in land plants, and delineates the cytokinetic division plane established at telophase. It is one of the few derived traits specific to embryophytes, in which it is involved in the spatial control of cell division. Here we show that loss of function of Physcomitrella patens PpTON1 strongly affects development of the moss gametophore, phenocopying the developmental syndrome observed in Arabidopsis ton1 mutants: mutant leafy shoots display random orientation of cell division and severe defects in cell elongation, which are correlated with absence of PPB formation and disorganization of the cortical microtubule array in interphase cells. In hypomorphic Ppton1 alleles, PPB are still formed, whereas elongation defects are observed, showing the dual function of TON1 in organizing cortical arrays of microtubules during both interphase and premitosis. Ppton1 mutation has no impact on development of the protonema, which is consistent with the documented absence of PPB formation at this stage, apart from alteration of the gravitropic response, uncovering a new function of TON1 proteins in plants. Successful reciprocal cross-complementation between Physcomitrella and Arabidopsis shows conservation of TON1 function during land plant evolution. These results establish the essential role of the PPB in division plane specification in a basal land plant lineage, and provide new information on the function of TON1. They point to an ancient mechanism of cytoskeletal control of division plane positioning and cell elongation in land plants.

Molecular aspects of microtubule dynamics in plants.

Microtubules are highly dynamic structures that play a major role in a wide range of processes, including cell morphogenesis, cell division, intracellular transport and signaling. The recent identification in plants of proteins involved in microtubule organization has begun to reveal how cytoskeleton dynamics are controlled.

The preprophase band of microtubules controls the robustness of division orientation in plants

Refined understanding of the preprophase band Because plant cells do not move, plant tissues are constructed according to how they place the divisions of their constituent cells. Schaefer et al. found a mutation in the model plant Arabidopsis that abolishes a visible precursor of cell division, the preprophase band. Despite loss of the band—previously thought essential to define the division plane—the general orientations of cell division planes in the roots of these plants were normal. However, individual division orientations showed more variance than normal. Thus, the preprophase band serves to focus and refine the final orientation of the nascent cell division plane. Science, this issue p. 186 A structure thought to be required for division plane selection in plants merely refines division plane position by reducing spindle rotation. Controlling cell division plane orientation is essential for morphogenesis in multicellular organisms. In plant cells, the future cortical division plane is marked before mitotic entry by the preprophase band (PPB). Here, we characterized an Arabidopsis trm (TON1 Recruiting Motif) mutant that impairs PPB formation but does not affect interphase microtubules. Unexpectedly, PPB disruption neither abolished the capacity of root cells to define a cortical division zone nor induced aberrant cell division patterns but rather caused a loss of precision in cell division orientation. Our results advocate for a reassessment of PPB function and division plane determination in plants and show that a main output of this microtubule array is to limit spindle rotations in order to increase the robustness of cell division.

The Preprophase Band and Division Site Determination in Land Plants

In land plants, division plane is determined pre-mitotically, during the transition from G2 to M phase. A conspicuous spatial landmark of division plane determination is a narrow cortical band of microtubules, the preprophase band (PPB), a transient, premitotic array which precisely predicts the cortical region reached by the growing cell plate during cytokinesis. However, cells preparing for division exhibit a number of other cytological features contributing to division site establishment, such as nuclear migration, cytoplasmic modifications, and intra-cytoplasmic cytoskeleton reorganization. The spatial control of division plane is tightly linked to the temporal control of cell division by the cell cycle machinery. In this chapter, we review and discuss recently discovered cellular events and molecular partners potentially involved in division plane establishment and PPB function in land plants.

Forward and reverse genetics in Arabidopsis: isolation of cytoskeletal mutants

The availability of the complete genome sequence of the model plant Arabidopsis, together with powerful tools for functional genomics in this species, allows biological functions to be dissected with unprecedented precision. Devising specific genetic screens for the isolation of cytoskeletal mutants remains difficult, although several Arabidopsis and maize mutants affected in cytoskeletal structures have been isolated recently in screens for morphological defects. The Arabidopsis tonneau (ton1 and ton2=fass) mutants were originally isolated from visual screens for mutations affecting seedling organisation (Mayer et al., 1991; Traas et al., 1995). These mutations drastically change the shape of the seedling, resulting in dwarf and compressed plantlets (Fig. 1). Mutant cells are markedly altered in their size, shape and arrangement, in relation to defects in cell elongation and random orientation of division planes. Interestingly, mutant cells are unable to form the preprophase band, and the cortical microtubules are randomly arranged in interphase mutant cells, instead of forming regular arrays normally associated with cell elongation. Mitotic microtubule arrays seem unaffected by the mutation. The ton1 mutation disrupts two highly similar genes in tandem repeat (Nacry et al., 1998). The ton1 genes (ton1a and ton1b) encode small, acidic 30 kDa polypeptides with no extensive similarity with known proteins, although TON1 shares a LisH motif (Emes and Ponting, 2001) with other cytoskeleton proteins. TON1 proteins are associated with the cortical cytoskeleton. A number of putative TON1b interacting proteins have been identified using the two-hybrid system, including Centrin. Centrins are calcium-binding proteins associated with the centrosome in animals. Eleven other proteins interact with the TON1b protein in yeast. These proteins define a new, plant-specific family of large proteins containing five conserved motifs. Among these, several have a large basic domain resembling a microtubulebinding domain of mammal proteins. We are now using a microtubule-binding assay to determine whether these proteins can bind microtubules. The ton2 gene encodes a 55 kDa protein with high similarity to a human protein of unknown function (Camilleri et al, 2002). The C-terminal part of the TON2 protein is similar to B