Cortical Microtubule Organization Research Articles

Cell Edges Accumulate Gamma Tubulin Complex Components and Nucleate Microtubules following Cytokinesis in Arabidopsis thaliana

Microtubules emanate from distinct organizing centers in fungal and animal cells. In plant cells, by contrast, microtubules initiate from dispersed sites in the cell cortex, where they then self-organize into parallel arrays. Previous ultrastructural evidence suggested that cell edges participate in microtubule nucleation but so far there has been no direct evidence for this. Here we use live imaging to show that components of the gamma tubulin nucleation complex (GCP2 and GCP3) localize at distinct sites along the outer periclinal edge of newly formed crosswalls, and that microtubules grow predominantly away from these edges. These data confirm a role for cell edges in microtubule nucleation, and suggest that an asymmetric distribution of microtubule nucleation factors contributes to cortical microtubule organization in plants, in a manner more similar to other kingdoms than previously thought.

Control of cortical microtubule organization and desmosome stability by centrosomal proteins

In many tissues microtubules reorganize into non-centrosomal arrays in differentiated cells. In the epidermis, proliferative basal cells have a radial array of microtubules organized around a centrosome, while differentiated cells have cortical microtubules. The desmosomal protein desmoplakin is required for the microtubules to organize around the cell cortex. Furthermore, the centrosomal and/or microtubule-associated proteins ninein, Lis1, Ndel1, and CLIP170 are recruited to the cell cortex, where they have been implicated in the cortical organization of microtubules. Recently, it has been shown that in Lis1-null epidermis, microtubules are disorganized in the differentiated layers of the epidermis. Furthermore, Lis1-null mice die perinatally due to dehydration. This is due, in part, to the unexpected desmosome phenotype observed in Lis1-null skin. Upon loss of Lis1, desmosomal proteins become less stable. Here, we propose that Lis1 may regulate desmosomal stability through its binding partners Nde1/Ndel1 and dynein.

A CLASP-modulated cell edge barrier mechanism drives cell-wide cortical microtubule organization in Arabidopsis.

It is well known that the parallel order of microtubules in the plant cell cortex defines the direction of cell expansion, yet it remains unclear how microtubule orientation is controlled, especially on a cell-wide basis. Here we show through 4D imaging and computational modelling that plant cell polyhedral geometry provides spatial input that determines array orientation and heterogeneity. Microtubules depolymerize when encountering sharp cell edges head-on, whereas those oriented parallel to those sharp edges remain. Edge-induced microtubule depolymerization, however, is overcome by the microtubule-associated protein CLASP, which accumulates at specific cell edges, enables microtubule growth around sharp edges and promotes formation of microtubule bundles that span adjacent cell faces. By computationally modelling dynamic 'microtubules on a cube' with edges differentially permissive to microtubule passage, we show that the CLASP-edge complex is a 'tuneable' microtubule organizer, with the inherent flexibility to generate the numerous cortical array patterns observed in nature.

Lis1 is essential for cortical microtubule organization and desmosome stability in the epidermis

Desmosomes are cell-cell adhesion structures that integrate cytoskeletal networks. In addition to binding intermediate filaments, the desmosomal protein desmoplakin (DP) regulates microtubule reorganization in the epidermis. In this paper, we identify a specific subset of centrosomal proteins that are recruited to the cell cortex by DP upon epidermal differentiation. These include Lis1 and Ndel1, which are centrosomal proteins that regulate microtubule organization and anchoring in other cell types. This recruitment was mediated by a region of DP specific to a single isoform, DPI. Furthermore, we demonstrate that the epidermal-specific loss of Lis1 results in dramatic defects in microtubule reorganization. Lis1 ablation also causes desmosomal defects, characterized by decreased levels of desmosomal components, decreased attachment of keratin filaments, and increased turnover of desmosomal proteins at the cell cortex. This contributes to loss of epidermal barrier activity, resulting in completely penetrant perinatal lethality. This work reveals essential desmosome-associated components that control cortical microtubule organization and unexpected roles for centrosomal proteins in epidermal function.

Microtubule configurations and nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv. Topas microspores

In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A ‘mild’ heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3–8 celled.

NIMA‐related kinases 6, 4, and 5 interact with each other to regulate microtubule organization during epidermal cell expansion in Arabidopsis thaliana

NimA-related kinase 6 (NEK6) has been implicated in microtubule regulation to suppress the ectopic outgrowth of epidermal cells; however, its molecular functions remain to be elucidated. Here, we analyze the function of NEK6 and other members of the NEK family with regard to epidermal cell expansion and cortical microtubule organization. The functional NEK6-green fluorescent protein fusion localizes to cortical microtubules, predominantly in particles that exhibit dynamic movement along microtubules. The kinase-dead mutant of NEK6 (ibo1-1) exhibits a disturbance of the cortical microtubule array at the site of ectopic protrusions in epidermal cells. Pharmacological studies with microtubule inhibitors and quantitative analysis of microtubule dynamics indicate excessive stabilization of cortical microtubules in ibo1/nek6 mutants. In addition, NEK6 directly binds to microtubules in vitro and phosphorylates β-tubulin. NEK6 interacts and co-localizes with NEK4 and NEK5 in a transient expression assay. The ibo1-3 mutation markedly reduces the interaction between NEK6 and NEK4 and increases the interaction between NEK6 and NEK5. NEK4 and NEK5 are required for the ibo1/nek6 ectopic outgrowth phenotype in epidermal cells. These results demonstrate that NEK6 homodimerizes and forms heterodimers with NEK4 and NEK5 to regulate cortical microtubule organization possibly through the phosphorylation of β-tubulins.

A role for katanin in plant cell division: Microtubule organization in dividing root cells of fra2 and lue1Arabidopsis thaliana mutants

Severing of microtubules by katanin has proven to be crucial for cortical microtubule organization in elongating and differentiating plant cells. On the contrary, katanin is currently not considered essential during cell division in plants as it is in animals. However, defects in cell patterning have been observed in katanin mutants, implying a role for it in dividing plant cells. Therefore, microtubule organization was studied in detail by immunofluorescence in dividing root cells of fra2 and lue1 katanin mutants of Arabidopsis thaliana. In both, early preprophase bands consisted of poorly aligned microtubules, prophase spindles were multipolar, and the microtubules of expanding phragmoplasts were elongated, bended toward and connected to the surface of daughter nuclei. Accordingly, severing by katanin seems to be necessary for the proper organization of these microtubule arrays. In both fra2 and lue1, metaphase/anaphase spindles and initiating phragmoplasts exhibited typical organization. However, they were obliquely oriented more frequently than in the wild type. It is proposed that this oblique orientation may be due to prophase spindle multipolarity and results in a failure of the cell plate to follow the predetermined division plane, during cytokinesis, producing oblique cell walls in the roots of both mutants. It is therefore concluded that, like in animal cells, katanin is important for plant cell division, influencing the organization of several microtubule arrays. Moreover, failure in microtubule severing indirectly affects the orientation of the division plane.

Differential regulation of cellulose orientation at the inner and outer face of epidermal cells in the Arabidopsis hypocotyl.

It is generally believed that cell elongation is regulated by cortical microtubules, which guide the movement of cellulose synthase complexes as they secrete cellulose microfibrils into the periplasmic space. Transversely oriented microtubules are predicted to direct the deposition of a parallel array of microfibrils, thus generating a mechanically anisotropic cell wall that will favor elongation and prevent radial swelling. Thus far, support for this model has been most convincingly demonstrated in filamentous algae. We found that in etiolated Arabidopsis thaliana hypocotyls, microtubules and cellulose synthase trajectories are transversely oriented on the outer surface of the epidermis for only a short period during growth and that anisotropic growth continues after this transverse organization is lost. Our data support previous findings that the outer epidermal wall is polylamellate in structure, with little or no anisotropy. By contrast, we observed perfectly transverse microtubules and microfibrils at the inner face of the epidermis during all stages of cell expansion. Experimental perturbation of cortical microtubule organization preferentially at the inner face led to increased radial swelling. Our study highlights the previously underestimated complexity of cortical microtubule organization in the shoot epidermis and underscores a role for the inner tissues in the regulation of growth anisotropy.

A Barley ROP GTPase ACTIVATING PROTEIN Associates with Microtubules and Regulates Entry of the Barley Powdery Mildew Fungus into Leaf Epidermal Cells

Little is known about the function of host factors involved in disease susceptibility. The barley (Hordeum vulgare) ROP (RHO of plants) G-protein RACB is required for full susceptibility of the leaf epidermis to invasion by the biotrophic fungus Blumeria graminis f. sp hordei. Stable transgenic knockdown of RACB reduced the ability of barley to accommodate haustoria of B. graminis in intact epidermal leaf cells and to form hairs on the root epidermis, suggesting that RACB is a common element of root hair outgrowth and ingrowth of haustoria in leaf epidermal cells. We further identified a barley MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN (MAGAP1) interacting with RACB in yeast and in planta. Fluorescent MAGAP1 decorated cortical microtubules and was recruited by activated RACB to the cell periphery. Under fungal attack, MAGAP1-labeled microtubules built a polarized network at sites of successful defense. By contrast, microtubules loosened where the fungus succeeded in penetration. Genetic evidence suggests a function of MAGAP1 in limiting susceptibility to penetration by B. graminis. Additionally, MAGAP1 influenced the polar organization of cortical microtubules. These results add to our understanding of how intact plant cells accommodate fungal infection structures and suggest that RACB and MAGAP1 might be antagonistic players in cytoskeleton organization for fungal entry.

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.

ArabidopsisHomologs of Nucleus- and Phragmoplast-Localized Kinase 2 and 3 and Mitogen-Activated Protein Kinase 4 Are Essential for Microtubule Organization

A double homozygous recessive mutant in the Arabidopsis thaliana homologs of nucleus- and phragmoplast-localized kinase 2 (ANP2) and 3 (ANP3) genes and a homozygous recessive mutant in the mitogen-activated protein kinase 4 (MPK4) gene of Arabidopsis exhibit deficiencies in the overall microtubule (MT) organization, which result in abnormal cell growth patterns, such as branching of root hairs and swelling of diffusely growing epidermal cells. Genetic, pharmacological, molecular, cytological, and biochemical analyses show that the major underlying mechanism for these phenotypes is excessive MT stabilization manifested in both mutants as heavy MT bundling, disorientation, and drug stability. The above defects in MAPK signaling result in the adverse regulation of members of the microtubule-associated protein (MAP65) protein family, including strongly diminished phosphorylation of MAP65-1. These data suggest that ANP2/ANP3, MPK4, and the microtubule-associated protein MAP65-1, a putative target of MPK4 signaling, are all essential for the proper organization of cortical microtubules in Arabidopsis epidermal cells.

The mechanics behind plant development

Morphogenesis in living organisms relies on the integration of both biochemical and mechanical signals. During the last decade, attention has been mainly focused on the role of biochemical signals in patterning and morphogenesis, leaving the contribution of mechanics largely unexplored. Fortunately, the development of new tools and approaches has made it possible to re-examine these processes. In plants, shape is defined by two local variables: growth rate and growth direction. At the level of the cell, these variables depend on both the cell wall and turgor pressure. Multidisciplinary approaches have been used to understand how these cellular processes are integrated in the growing tissues. These include quantitative live imaging to measure growth rate and direction in tissues with cellular resolution. In parallel, stress patterns have been artificially modified and their impact on strain and cell behavior been analysed. Importantly, computational models based on analogies with continuum mechanics systems have been useful in interpreting the results. In this review, we will discuss these issues focusing on the shoot apical meristem, a population of stem cells that is responsible for the initiation of the aerial organs of the plant.

Mechanisms of Self-Organization of Cortical Microtubules in Plants Revealed by Computational Simulations

Microtubules confined to the two-dimensional cortex of elongating plant cells must form a parallel yet dispersed array transverse to the elongation axis for proper cell wall expansion. Some of these microtubules exhibit free minus-ends, leading to migration at the cortex by hybrid treadmilling. Collisions between microtubules can result in plus-end entrainment ("zippering") or rapid depolymerization. Here, we present a computational model of cortical microtubule organization. We find that plus-end entrainment leads to self-organization of microtubules into parallel arrays, whereas catastrophe-inducing collisions do not. Catastrophe-inducing boundaries (e.g., upper and lower cross-walls) can tune the orientation of an ordered array to a direction transverse to elongation. We also find that changes in dynamic instability parameters, such as in mor1-1 mutants, can impede self-organization, in agreement with experimental data. Increased entrainment, as seen in clasp-1 mutants, conserves self-organization, but delays its onset and fails to demonstrate increased ordering. We find that branched nucleation at acute angles off existing microtubules results in distinctive sparse arrays and infer either that microtubule-independent or coparallel nucleation must dominate. Our simulations lead to several testable predictions, including the effects of reduced microtubule severing in katanin mutants.

A ROP GTPase Signaling Pathway Controls Cortical Microtubule Ordering and Cell Expansion in Arabidopsis

Cortical microtubules (MTs) participate in the spatial control of cell expansion and division that is required for plant growth and morphogenesis. Well-ordered transverse cortical MTs promote cell elongation and restrict radial cell expansion. The molecular mechanism controlling their ordering is poorly understood. We report the first known signaling pathway that promotes the organization of cortical MTs into parallel arrays oriented perpendicular to the axis of cell elongation in plants. Well-ordered MTs locally restrict cell expansion to promote indentation formation in the jigsaw-puzzle-shaped pavement cells of Arabidopsis leaves. Deleting ROP6, a Rho-family GTPase, randomized cortical MTs and released the localized restriction of cell expansion, whereas ROP6 overexpression enhanced MT ordering, turning the jigsaw-puzzle appearance of cells into a cylindrical shape. ROP6 directly binds and activates MT-associated RIC1 to achieve the MT ordering. The ROP6-RIC1 pathway also affects MT ordering of hypocotyl cells, showing a broad role for this pathway in the spatial regulation of cell expansion.

Microtubule reorganization as a response to implementation of NO signals in plant cells

The effects of an exogeneous NO donor, sodium nitroprusside, on the orientation and organization of cortical microtubules in Arabidopsis thaliana root cells expressing GFP-MAP4 were studied in vivo. It was found that sodium nitroprusside treatment (10–500 μM, 24 h) caused the acceleration of primary root growth and enhanced initiation of root hairs in the differentiation zone. The influence of sodium nitroprusside revealed in changes in the orientation and organization of cortical microtubules in different types of cells of A. thaliana root. The most sensitive to sodium nitroprusside exposure were microtubules in epidermal cells of the elongation zone, where native transverse orientation of cortical microtubules turned into random, oblique, or longitudinal relative to the primary root axis. We suggest that NO, as one of the intracellular secondary messengers, triggers cell differentiation by reorientation of cortical microtubules, possibly via tubulin nitrotyrosination.

Microtubule-associated protein AtMPB2C plays a role in organization of cortical microtubules, stomata patterning, and tobamovirus infectivity.

AtMPB2C is the Arabidopsis (Arabidopsis thaliana) homolog of MPB2C, a microtubule-associated host factor of tobacco mosaic virus movement protein that was been previously identified in Nicotiana tabacum. To analyze the endogenous function of AtMPB2C and its role in viral infections, transgenic Arabidopsis plant lines stably overexpressing green fluorescent protein (GFP)-AtMPB2C were established. The GFP-AtMPB2C fusion protein was detectable in various cell types and organs and localized at microtubules in a punctuate pattern or in filaments. To determine whether overexpression impacted on the cortical microtubular cytoskeleton, GFP-AtMPB2C-overexpressing plants were compared to known microtubular marker lines. In rapidly elongated cell types such as vein cells and root cells, GFP-AtMPB2C overexpression caused highly unordered assemblies of cortical microtubules, a disturbed, snake-like microtubular shape, and star-like crossing points of microtubules. Phenotypically, GFP-AtMPB2C transgenic plants showed retarded growth but were viable and fertile. Seedlings of GFP-AtMPB2C transgenic plants were characterized by clockwise twisted leaves, clustered stomata, and enhanced drought tolerance. GFP-AtMPB2C-overexpressing plants showed increased resistance against oilseed rape mosaic virus, a close relative of tobacco mosaic virus, but not against cucumber mosaic virus when compared to Arabidopsis wild-type plants. These results suggest that AtMPB2C is involved in the alignment of cortical microtubules, the patterning of stomata, and restricting tobamoviral infections.

MOR1, theArabidopsis thalianahomologue ofXenopusMAP215, promotes rapid growth and shrinkage, and suppresses the pausing of microtubules in vivo

MOR1, the Arabidopsis thaliana homologue of the Xenopus microtubule-associated protein MAP215, is required for spatial organization of the acentrosomal microtubule arrays of plant cells. To determine how loss of MOR1 function affects microtubule dynamics, we compared various parameters of microtubule dynamics in the temperature-sensitive mor1-1 mutant at its permissive and restrictive temperatures, 21 degrees C and 31 degrees C, respectively. Dynamic events were tracked in live cells expressing either GFP-tagged beta-tubulin or the plus end tracking EB1. Microtubule growth and shrinkage velocities were both dramatically reduced in mor1-1 at 31 degrees C and the incidence and duration of pause events increased. Interestingly, the association of EB1 with microtubule plus ends was reduced in mor1-1 whereas side wall binding increased, suggesting that MOR1 influences the association of EB1 with microtubules either by modulating microtubule plus end structure or by interacting with EB1. Although mor1-1 microtubules grew and shrank more slowly than wild-type microtubules at 21 degrees C, the incidence of pause was not altered, suggesting that pause events, which occur more frequently at 31 degrees C, have a major detrimental role in the spatial organization of cortical microtubules. Extensive increases in microtubule dynamics in wild-type cells when shifted from 21 degrees C to 31 degrees C underline the importance of careful temperature control in live cell imaging.

Arabidopsis Casein Kinase 1-Like 6 Contains a Microtubule-Binding Domain and Affects the Organization of Cortical Microtubules

Members of the casein kinase 1 (CK1) family are evolutionarily conserved eukaryotic protein kinases that are involved in various cellular, physiological, and developmental processes in yeast and metazoans, but the biological roles of CK1 members in plants are not well understood. Here, we report that an Arabidopsis (Arabidopsis thaliana) CK1 member named casein kinase 1-like 6 (CKL6) associates with cortical microtubules in vivo and phosphorylates tubulins in vitro. The unique C-terminal domain of CKL6 was shown to contain the signal that allows localization of CKL6 to the cortical microtubules. This domain on its own was sufficient to associate with microtubules in vivo and to bind tubulins in vitro. CKL6 was able to phosphorylate soluble tubulins as well as microtubule polymers, and its endogenous activity was found to associate with a tubulin-enriched subcellular fraction. Two major in vitro phosphorylation sites were mapped to serine-413 and serine-420 of tubulin beta. Ectopic expression of wild-type CKL6 or a kinase-inactive mutant form induced alterations in cortical microtubule organization and anisotropic cell expansion. Collectively, these results demonstrate that CKL6 is a protein kinase containing a novel tubulin-binding domain and plays a role in anisotropic cell growth and shape formation in Arabidopsis through the regulation of microtubule organization, possibly through the phosphorylation of tubulins.

Tubulin Cytoskeleton in Arabidopsis thaliana Root Cells Under Clinorotation

The effect of horizontal clinorotation (2 rpm) on the spatial organization of cortical microtubules (CMTs) in the living Arabidopsis root cells has been investigated. MTs were visualized by using a stably transformed line of transgenic Arabidopsis thaliana expressing GFP-MAP4 fusion protein. To monitor total changes in MT dynamics, the sensitivity to MT-inhibitor 5 μM/l oryzalin was used as an indicator. It was shown that CMTs in the distal elongation zone (DEZ) of clinorotated 5 day old seedlings exhibited a higher degree of disorder and also they were more sensitive to oryzalin. We suggest that rearrangement of CMTs in DEZ cells under clinorotation is caused by fast rate of MT dynamic turnover.

Effects of tyrosine kinase and phosphatase inhibitors on microtubules in Arabidopsis root cells

To investigate the role of tyrosine phosphorylation/dephosphorylation processes in plant cells the morphology of Arabidopsis thaliana primary roots and the organization of cortical microtubules (MTs) were studied after inhibition of protein tyrosine kinases (PTKs) and tyrosine phosphatases (PTPs). It was found that all tested types of PTKs inhibitors (herbimycin A, genistein and tyrphostin AG 18) altered root hair growth and development, probably as a result of their significant influences on MTs organization in root hairs. The treatment also led to MTs reorientation and disruption in epidermis and cortex cells of both elongation and differentiation zones of primary roots. Enhanced tyrosine phosphorylation after treatment with a PTPs inhibitor (sodium orthovanadate) resulted in intense induction of root hair development and growth and caused a significant shortening of the elongation zone. It also led to changes of MTs orientation from transverse to longitudinal in epidermis and cortex cells of the elongation and differentiation zones of the root. From the data obtained we can suppose that tyrosine phosphorylation can be involved in the dynamics and organization of MTs in different types of plant cells.