Encounters between dynamic cortical microtubules promote ordering of the cortical array through angle-dependent modifications of microtubule behavior

Ordered cortical microtubule arrays are essential for normal plant morphogenesis, but how these arrays form is unclear. The dynamics of individual cortical microtubules are stochastic and cannot fully account for the observed order; however, using tobacco (Nicotiana tabacum) cells expressing either the MBD-DsRed (microtubule binding domain of the mammalian MAP4 fused to the Discosoma sp red fluorescent protein) or YFP-TUA6 (yellow fluorescent protein fused to the Arabidopsis alpha-tubulin 6 isoform) microtubule markers, we identified intermicrotubule interactions that modify their stochastic behaviors. The intermicrotubule interactions occur when the growing plus-ends of cortical microtubules encounter previously existing cortical microtubules. Importantly, the outcome of such encounters depends on the angle at which they occur: steep-angle collisions are characterized by approximately sevenfold shorter microtubule contact times compared with shallow-angle encounters, and steep-angle collisions are twice as likely to result in microtubule depolymerization. Hence, steep-angle collisions promote microtubule destabilization, whereas shallow-angle encounters promote both microtubule stabilization and coalignment. Monte Carlo modeling of the behavior of simulated microtubules, according to the observed behavior of transverse and longitudinally oriented cortical microtubules in cells, reveals that these simple rules for intermicrotubule interactions are necessary and sufficient to facilitate the self-organization of dynamic microtubules into a parallel configuration.     

Higher plant cortical microtubule array analyzed in vitro in the presence of the cell wall

Plant morphogenesis depends on an array of microtubules in the cell cortex, the cortical array. Although the cortical array is known to be essential for morphogenesis, it is not known how the array becomes organized or how it functions mechanistically. Here, we report the development of an in vitro model that provides good access to the cortical array while preserving the array's organization and, importantly, its association with the cell wall. Primary roots of maize (Zea mays) are sectioned, without fixation, in a drop of buffer and then incubated as desired before eventual fixation. Sectioning removes cytoplasm except for a residuum comprising cortical microtubules, vesicles, and fragments of plasma membrane underlying the microtubules. The majority of the cortical microtubules remain in the cut-open cells for more than 1 h, fully accessible to the incubation solution. The growth zone or more mature tissue can be sectioned, providing access to cortical arrays that are oriented either transversely or obliquely to the long axis of the root. Using this assay, we report, first, that cortical microtubule stability is regulated by protein phosphorylation; second, that cortical microtubule stability is a function of orientation, with divergent microtubules within the array depolymerizing within minutes of sectioning; and third, that the polarity of microtubules in the cortical array is not uniform. These results suggest that the organization of the cortical array involves random nucleation followed by selective stabilization of microtubules formed at the appropriate orientation, and that the signal specifying alignment must treat orientations of +/- 180 degrees as equivalent.     

Progressive Transverse Microtubule Array Organization in Hormone-Induced Arabidopsis Hypocotyl Cells

The acentriolar cortical microtubule arrays in dark-grown hypocotyl cells organize into a transverse coaligned pattern that is critical for axial plant growth. In light-grown Arabidopsis thaliana seedlings, the cortical array on the outer (periclinal) cell face creates a variety of array patterns with a significant bias (>3:1) for microtubules polymerizing edge-ward and into the side (anticlinal) faces of the cell. To study the mechanisms required for creating the transverse coalignment, we developed a dual-hormone protocol that synchronously induces ∼80% of the light-grown hypocotyl cells to form transverse arrays over a 2-h period. Repatterning occurred in two phases, beginning with an initial 30 to 40% decrease in polymerizing plus ends prior to visible changes in the array pattern. Transverse organization initiated at the cell’s midzone by 45 min after induction and progressed bidirectionally toward the apical and basal ends of the cell. Reorganization corrected the edge-ward bias in polymerization and proceeded without transiting through an obligate intermediate pattern. Quantitative comparisons of uninduced and induced microtubule arrays showed a limited deconstruction of the initial periclinal array followed by a progressive array reorganization to transverse coordinated between the anticlinal and periclinal cell faces.     

Plasma Membrane-Associated Actin in Bright Yellow 2 Tobacco Cells : Evidence for Interaction with Microtubules

Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K⁺ and Ca²⁺ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.     

Arabidopsis MICROTUBULE-ASSOCIATED PROTEIN18 functions in directional cell growth by destabilizing cortical microtubules

Microtubule-associated proteins (MAPs) play important roles in the regulation of microtubule function in cells. We describe Arabidopsis thaliana MAP18, which binds to microtubules and inhibits tubulin polymerization in vitro and colocalizes along cortical microtubules as patches of dot-like structures. MAP18 is expressed mostly in the expanding cells. Cells overexpressing MAP18 in Arabidopsis exhibit various growth phenotypes with loss of polarity. Cortical microtubule arrays were significantly altered in cells either overexpressing MAP18 or where it had been downregulated by RNA interference (RNAi). The cortical microtubules were more sensitive to treatment with microtubule-disrupting drugs when MAP18 was overexpressed, but more resistant when MAP18 was eliminated in cells expressing MAP18 RNAi. Our study demonstrated that MAP18 may play a role in regulating directional cell growth and cortical microtubule organization by destabilizing microtubules.     

Molecular encounters at microtubule ends in the plant cell cortex

The cortical arrays that accompany plant cell division and elongation are organized by a subtle interplay between intrinsic properties of microtubules, their self-organization capacity and a variety of cellular proteins that interact with them, modify their behaviour and drive organization of diverse, higher order arrays during the cell cycle, cell growth and differentiation. As a polar polymer, the microtubule has a minus and a plus end, which differ in structure and dynamic characteristics, and to which different sets of partners and activities associate. Recent advances in characterization of minus and plus end directed proteins provide insights into both plant microtubule properties and the way highly organized cortical arrays emerge from the orchestrated activity of individual microtubules.     

Altered microtubule dynamics by expression of modified alpha-tubulin protein causes right-handed helical growth in transgenic Arabidopsis plants

The proper organization of cortical microtubule arrays is essential for anisotropic growth in plants but how distinct array patterns are formed is not understood. Here, we report a relationship between microtubule dynamics and array organization using transgenic plants expressing modified tubulins. When green fluorescent protein (GFP) or a hemaglutinin epitope tag was fused to the N-terminus of tubulins and expressed in Arabidopsis plants, these tubulins were incorporated into microtubules along with endogenous tubulins. Plants expressing the modified beta-tubulins were phenotypically normal and possessed transversely oriented cortical arrays in the epidermal cells of the root elongation zone; however, the expression of modified alpha-tubulins caused right-handed helical growth, increased trichome branching, and a shallow left-handed (S-form) helical array organization. In cells expressing the modified alpha-tubulins, microtubule dynamicity was suppressed and polymerization was promoted, and GFP-EB1 (End Binding 1) labeled larger regions of the microtubule end more frequently, when compared with control cells. We propose that the N-terminal appendage introduced into alpha-tubulin inhibits GTP hydrolysis, thus producing polymerization-prone microtubules with an extended GTP cap. Consistent with this interpretation, plants expressing an alpha-tubulin mutated in the GTPase-activating domain exhibited similar microtubule properties, with regard to dynamics and the localization of GFP-EB1, and showed right-handed helical growth.     

Consistent handedness of microtubule helical arrays in maize and Arabidopsis primary roots

Summary The orientation of cortical microtubules in plant cells has been extensively studied, in part because of their influence on the expansion of most plant cell types. Cortical microtubules are often arranged in helical arrays, which are well known to occur with a specific pitch as a function of development or experimental treatment; however, it is not known if the handedness of helical arrays can also be specified. We have studied the handedness of helical arrays by using Vibratome sectioning of maize primary roots and confocal microscopy of Arabidopsis primary roots. In cortical cells of maize roots, the helical array was found to have the same handedness at a given position, not only for the cells of a single root, but also for the cells of more than one hundred roots examined. Quantification of angular distribution of apparent individual microtubules showed that defined regions of the root were composed of cells with highly uniform microtubule orientation. In the region between transverse and longitudinal microtubules (5–10.5 mm from the tip), the array formed a right-handed helix, and basal of cells with longitudinal microtubules (11.5–15 mm from the tip), the array formed a left-handed helix. Similarly, in epidermal cells of Arabidopsis roots right-handed helical arrays were found in the region between transverse and longitudinal microtubules. These results suggest that, in addition to the orientation of microtubules, the handedness of helical microtubule arrays is under cellular control.Abbreviations Cy3 indocarbocyanine - PBS phosphate-buffered saline - PIPES piperazine-N,N-bis-[2-ethanesulfonic acid]     

Microtubule organization in three-dimensional confined geometries: evaluating the role of elasticity through a combined in vitro and modeling approach

Microtubules or microtubule bundles in cells often grow longer than the size of the cell, which causes their shape and organization to adapt to constraints imposed by the cell geometry. We test the reciprocal role of elasticity and confinement in the organization of growing microtubules in a confining box-like geometry, in the absence of other (active) microtubule organizing processes. This is inspired, for example, by the cortical microtubule array of elongating plant cells, where microtubules are typically organized in an aligned array transverse to the cell elongation axis. The method we adopt is a combination of analytical calculations, in which the polymers are modeled as inextensible filaments with bending elasticity confined to a two-dimensional surface that defines the limits of a three-dimensional space, and in vitro experiments, in which microtubules are polymerized from nucleation seeds in microfabricated chambers. We show that these features are sufficient to organize the polymers in aligned, coiling configurations as for example observed in plant cells. Though elasticity can account for the regularity of these arrays, it cannot account for a transverse orientation of microtubules to the cell's long axis. We therefore conclude that an additional active, force-generating process is necessary to create a coiling configuration perpendicular to the long axis of the cell.     

BOTERO1 is required for normal orientation of cortical microtubules and anisotropic cell expansion in Arabidopsis

Mutants at the BOTERO1 locus are affected in anisotropic growth in all non-tip-growing cell types examined. Mutant cells are shorter and broader than those of the wild type. Mutant inflorescence stems show a dramatically reduced bending modulus and maximum stress at yield. Our observations of root epidermis cells show that the cell expansion defect in bot1 is correlated with a defect in the orientation of the cortical microtubules. We found that in cells within the apical portion of the root, which roughly corresponds to the meristem, microtubules were loosely organized and became much more highly aligned in transverse arrays with increasing distance from the tip. Such a transition was not observed in bot1. No defect in microtubule organization was observed in kor-1, another mutant with a radial cell expansion defect. We also found that in wild-type root epidermal cells, cessation of radial expansion precedes the increased alignment of cortical microtubules into transverse arrays. Bot1 roots still show a gravitropic response, which indicates that ordered cortical microtubules are not required for differential growth during gravitropism. Interestingly, the fact that in the mutant, these major changes in microtubule organization cause relatively subtle changes in cell morphology, suggest that other levels of control of growth anisotropy remain to be discovered. Together, these observations suggest that BOT1 is required for organizing cortical microtubules into transverse arrays in interphase cells, and that this organization is required for consolidating, rather than initiating, changes in the direction of cell expansion.     

Microtubule cortical array organization and plant cell morphogenesis

Plant cell cortical microtubule arrays attain a high degree of order without the benefit of an organizing center such as a centrosome. New assays for molecular behaviors in living cells and gene discovery are yielding insight into the mechanisms by which acentrosomal microtubule arrays are created and organized, and how microtubule organization functions to modify cell form by regulating cellulose deposition. Surprising and potentially important behaviors of cortical microtubules include nucleation from the walls of established microtubules, and treadmilling-driven motility leading to polymer interaction, reorientation, and microtubule bundling. These behaviors suggest activities that can act to increase or decrease the local level of order in the array. The SPIRAL1 (SPR1) and SPR2 microtubule-localized proteins and the radial swollen 6 (rsw-6) locus are examples of new molecules and genes that affect both microtubule array organization and cell growth pattern. Functional tagging of cellulose synthase has now allowed the dynamic relationship between cortical microtubules and the cell-wall-synthesizing machinery to be visualized, providing direct evidence that cortical microtubules can organize cellulose synthase complexes and guide their movement through the plasma membrane as they create the cell wall.     

A Three-Dimensional Computer Simulation Model Reveals the Mechanisms for Self-Organization of Plant Cortical Microtubules into Oblique Arrays

The noncentrosomal cortical microtubules (CMTs) of plant cells self-organize into a parallel three-dimensional (3D) array that is oriented transverse to the cell elongation axis in wild-type plants and is oblique in some of the mutants that show twisted growth. To study the mechanisms of CMT array organization, we developed a 3D computer simulation model based on experimentally observed properties of CMTs. Our computer model accurately mimics transverse array organization and other fundamental properties of CMTs observed in rapidly elongating wild-type cells as well as the defective CMT phenotypes observed in the Arabidopsis mor1-1 and fra2 mutants. We found that CMT interactions, boundary conditions, and the bundling cutoff angle impact the rate and extent of CMT organization, whereas branch-form CMT nucleation did not significantly impact the rate of CMT organization but was necessary to generate polarity during CMT organization. We also found that the dynamic instability parameters from twisted growth mutants were not sufficient to generate oblique CMT arrays. Instead, we found that parameters regulating branch-form CMT nucleation and boundary conditions at the end walls are important for forming oblique CMT arrays. Together, our computer model provides new mechanistic insights into how plant CMTs self-organize into specific 3D arrangements.     

Microtubule-dependent microtubule nucleation in plant cells

Regulation of microtubule nucleation sites is an essential step in microtubule organization. Cortical microtubule arrays in green plant cells at inter-phase are organized in a distinct manner—the array is formed in the absence of previously recognized organelles for microtubule nucleation, for example the centrosome and spindle pole body. Microtubules in the cortical array were recently found to be nucleated as branches on pre-existing microtubules via recruitment of cytosolic γ-tubulin. In this review we briefly summarize the mechanism of microtubule-dependent microtubule nucleation and discuss a possible role of this mechanism in other cellular processes and their evolution.     

The Type II Arabidopsis Formin14 Interacts with Microtubules and Microfilaments to Regulate Cell Division

Formins have long been known to regulate microfilaments but have also recently been shown to associate with microtubules. In this study, Arabidopsis thaliana FORMIN14 (AFH14), a type II formin, was found to regulate both microtubule and microfilament arrays. AFH14 expressed in BY-2 cells was shown to decorate preprophase bands, spindles, and phragmoplasts and to induce coalignment of microtubules with microfilaments. These effects perturbed the process of cell division. Localization of AFH14 to microtubule-based structures was confirmed in Arabidopsis suspension cells. Knockdown of AFH14 in mitotic cells altered interactions between microtubules and microfilaments, resulting in the formation of an abnormal mitotic apparatus. In Arabidopsis afh14 T-DNA insertion mutants, microtubule arrays displayed abnormalities during the meiosis-associated process of microspore formation, which corresponded to altered phenotypes during tetrad formation. In vitro biochemical experiments showed that AFH14 bound directly to either microtubules or microfilaments and that the FH2 domain was essential for cytoskeleton binding and bundling. However, in the presence of both microtubules and microfilaments, AFH14 promoted interactions between microtubules and microfilaments. These results demonstrate that AFH14 is a unique plant formin that functions as a linking protein between microtubules and microfilaments and thus plays important roles in the process of plant cell division.     

TONNEAU2/FASS regulates the geometry of microtubule nucleation and cortical array organization in interphase Arabidopsis cells

Organization of microtubules into ordered arrays involves spatial and temporal regulation of microtubule nucleation. Here, we show that acentrosomal microtubule nucleation in plant cells involves a previously unknown regulatory step that determines the geometry of microtubule nucleation. Dynamic imaging of interphase cortical microtubules revealed that the ratio of branching to in-bundle microtubule nucleation on cortical microtubules is regulated by the Arabidopsis thaliana B' subunit of protein phosphatase 2A, which is encoded by the TONNEAU2/FASS (TON2) gene. The probability of nucleation from γ-tubulin complexes localized at the cell cortex was not affected by a loss of TON2 function, suggesting a specific role of TON2 in regulating the nucleation geometry. Both loss of TON2 function and ectopic targeting of TON2 to the plasma membrane resulted in defects in cell shape, suggesting the importance of TON2-mediated regulation of the microtubule cytoskeleton in cell morphogenesis. Loss of TON2 function also resulted in an inability for cortical arrays to reorient in response to light stimulus, suggesting an essential role for TON2 and microtubule branching nucleation in reorganization of microtubule arrays. Our data establish TON2 as a regulator of interphase microtubule nucleation and provide experimental evidence for a novel regulatory step in the process of microtubule-dependent nucleation.     

Regional variation of microtubule flux reveals microtubule organization in the metaphase meiotic spindle

Continuous poleward movement of tubulin is a hallmark of metaphase spindle dynamics in higher eukaryotic cells and is essential for stable spindle architecture and reliable chromosome segregation. We use quantitative fluorescent speckle microscopy to map with high resolution the spatial organization of microtubule flux in Xenopus laevis egg extract meiotic spindles. We find that the flux velocity decreases near spindle poles by ~20%. The regional variation is independent of functional kinetochores and centrosomes and is suppressed by inhibition of dynein/dynactin, kinesin-5, or both. Statistical analysis reveals that tubulin flows in two distinct velocity modes. We propose an association of these modes with two architecturally distinct yet spatially overlapping and dynamically cross-linked arrays of microtubules: focused polar microtubule arrays of a uniform polarity and slower flux velocities are interconnected by a dense barrel-like microtubule array of antiparallel polarities and faster flux velocities.     

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.     

Functions of the Arabidopsis kinesin superfamily of microtubule-based motor proteins

Plants possess a large number of microtubule-based kinesin motor proteins. While the kinesin-2, 3, 9, and 11 families are absent from land plants, the kinesin-7 and 14 families are greatly expanded. In addition, some kinesins are specifically present only in land plants. The distinctive inventory of plant kinesins suggests that kinesins have evolved to perform specialized functions in plants. Plants assemble unique microtubule arrays during their cell cycle, including the interphase cortical microtubule array, preprophase band, anastral spindle and phragmoplast. In this review, we explore the functions of plant kinesins from a microtubule array viewpoint, focusing mainly on Arabidopsis kinesins. We emphasize the conserved and novel functions of plant kinesins in the organization and function of the different microtubule arrays.     

Formation and functions of asymmetric microtubule organization in polarized cells

Although microtubules are known to be essential for chromosome segregation during cell division, they also play important roles in the regulation and function of cell polarity. Cell polarization is fundamental to appropriate tissue patterning and the regulation of cellular diversity during animal development. In polarized cells, microtubules are often organized asymmetrically along the polarity axis. Recent studies show that such asymmetry in microtubule organization is important to connect a cell's polarization with its polarized functions. In some cases, asymmetrically organized microtubule arrays themselves induce cell polarity. Here we present an overview of the mechanisms and functions of asymmetric microtubule organization and discuss the possible role of microtubule asymmetry in the symmetry-breaking that leads to cell polarization.     

Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

 Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927–1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array. Received: 2 June 1999 / Accepted: 13 August 1999