Super-resolved imaging and Bayesian analysis of single exocytosis events reveals molecular-scale patterning by cortical microtubule arrays.

Indirect immunofluorescence using a-tubulin antibodies applied to sections of maize roots prepared using low melting point wax was found to give excellent visualisation of both cortical and endoplasmic microtubule (MT) arrays. This allows, for the first time, these arrays to be investigated in cells of the different tissues at various stages of their growth and development. Many cells in the zone between the meristem and the cell elongation region develop a highly ordered transversal bundling of cortical MTs, which we suggest is related to vacuolation of the cytoplasm and which may also be indispensable for the ensuing rapid cell elongation. On the other hand, there are subtle differences between the cells of the individual tissues regarding the arrangement of their cortical MTs in this zone. The possible physiological significance of these tissue-specific MT arrays is discussed. Endoplasmic MTs were seen to encircle and to connect the nucleus with the cortical MT arrays in both dividing and elongating cells. Even the G1 phase nuclei of the slowly dividing cells of the quiescent centre were encircled by endoplasmic MTs. The continuity of the two MT systems may provide the cell with an important signalling system whereby mechanical and physiological information is relayed from the exterior of the cell to the nucleus.

External Mechanical Cues Reveal a Katanin-Independent Mechanism behind Auxin-Mediated Tissue Bending in Plants.

Microtubule-reporter plants expressing green fluorescent protein-α-TUBULIN fusion protein (GFP-TUA6) in male gametophytic cells of tobacco and Arabidopsis provide new tools for studying the native organization of microtubule (MT) arrays during reproductive development. These plants reveal unique features of gametophytic MT arrays including a basket-like cortical MT array in polarized microspores at interphase, an asymmetric spindle and curved phragmoplast MTs at microspore division and an assembly of bundled cortical MTs during germ cell morphogenesis. The application of these MT-reporter plants has been demonstrated by RNAi-mediated knockdown of the microtubule-associated protein TMBP200, the tobacco orthologue of the conserved MAP215/Dis1 family protein. The double transgenic lines display defects in nuclear positioning, division asymmetry and cytokinesis that are associated with striking defects in spindle and phragmoplast position and organization. This study reveals native and altered MT arrays in unprecedented detail and clarifies the essential functions of MAP215/Dis1 protein function in successive steps in male germline establishment. Such gametophytic MT-reporter lines should accelerate studies of the dynamic regulation of MT arrays by microtubule associated proteins and other effectors during male gametophyte development.

The plant cortical microtubule array is a unique acentrosomal array that is essential for plant morphogenesis. To understand how this array is organized, we exploited the microtubule (+)-end tracking activity of two Arabidopsis EB1 proteins in combination with FRAP (fluorescence recovery after photobleaching) experiments of GFP-tubulin to examine the relationship between cortical microtubule array organization and polarity. Significantly, our observations show that the majority of cortical microtubules in ordered arrays, within a particular cell, face the same direction in both Arabidopsis plants and cultured tobacco cells. We determined that this polar microtubule coalignment is at least partially due to a selective stabilization of microtubules, and not due to a change in microtubule polymerization rates. Finally, we show that polar microtubule coalignment occurs in conjunction with parallel grouping of cortical microtubules and that cortical array polarity is progressively enhanced during array organization. These observations reveal a novel aspect of plant cortical microtubule array organization and suggest that selective stabilization of dynamic cortical microtubules plays a predominant role in the self-organization of cortical arrays.

The infected cells of mature soybean root nodules have no vacuole; instead, the main body of the cell is packed with symbiosomes. Symbiosomes are the basic units of nitrogen fixation, comprising the nitrogen-fixing bacteroids and the surrounding peribacteroid membrane. For efficient passage of nutrients between the bacteroids and the plant, it seems likely that infected cell structure is highly organised with clear diffusion pathways through the cell (i. e. between symbiosomes). We have investigated the cytoskeleton of infected cells using immunocytochemical microscopy. In mature nodules, both cortical and nuclear associated microtubule (MT) arrays were observed in infected cells. The cortical MT’s appeared to occur in bundles and were organised so that strands were roughly parallel and oriented transverse to the long axis of the cell. They did not appear to be associated with symbiosomes. Three different arrays of actin filaments could be distinguished in infected cells: cortical, cytoplasmic and nuclear-associated. The cortical array was limited to a few prominent strands with similar orientation as the microtubules. The nuclear-associated actin array radiated from the nucleus to the cell cortex. The most striking aspect of infected cell actin was the cytoplasmic array which extended throughout the cytoplasm of the cell in a honeycomb structure, probably inserted between and interacting with the symbiosomes. In contrast, uninfected cells had only cortical actin which was oriented randomly. Cells in the nodule primordia (which contain no symbiosomes) had only nuclear associated and cortical actin arrays, the latter in randomly organised, circular patterns. In developing nodules, infected cells showed a cytoplasmic array of actin filaments but it was randomly organised without the distinctive honeycomb structure seen in mature nodule cells.

Putting a bifunctional motor to work: insights into the role of plant KCH kinesins

The subfamily II catalytic subunits of protein phosphatase 2A (PP2A) regulate the cortical microtubule dynamics in Arabidopsis, through interaction with TONNEAU2 (TON2)/FASS and modulation of α-tubulin dephosphorylation. Protein phosphatase 2A is a major protein phosphatase in eukaryotes that dephosphorylates many different substrates to regulate their function. PP2A is assembled into a heterotrimeric complex of scaffolding A subunit, regulatory B subunit, and catalytic C subunit. Plant PP2A catalytic C subunit (PP2AC) isoforms are classified into two subfamilies. In this study, we investigated the cellular functions of the Arabidopsis PP2AC subfamily II genes PP2AC-3 and PP2AC-4, particularly regarding the cortical microtubule (MT) organization. PP2AC-3 and PP2AC-4 strongly interacted with the B'' regulatory subunit TON2. Simultaneous silencing of PP2AC-3 and PP2AC-4 by virus-induced gene silencing (PP2AC-3,4 VIGS) significantly altered plant morphology in Arabidopsis, increasing cell numbers in leaves and stems. The leaf epidermis of PP2AC-3,4 VIGS plants largely lost its jigsaw-puzzle shape and exhibited reduced trichome branch numbers. VIGS of PP2AC-3,4 in Arabidopsis transgenic plants that expressed GFP-fused β-tubulin 6 isoform (GFP-TUB6) for the visualization of MTs caused a reduction in the cortical MT array density in the pavement cells. VIGS of TON2 also led to similar cellular phenotypes and cortical MT patterns compared with those after VIGS of PP2AC-3,4, suggesting that PP2AC-3,4 and their interaction partner TON2 play a role in cortical MT organization in leaf epidermal cells. Furthermore, silencing of PP2AC-3,4 did not affect salt-induced phosphorylation of α-tubulin but delayed its dephosphorylation after salt removal. The reappearance of cortical MT arrays after salt removal was impaired in PP2AC-3,4 VIGS plants. These results suggest an involvement of PP2AC subfamily II in the regulation of cortical MT dynamics under normal and salt-stress conditions in Arabidopsis.

Contents Summary I. Introduction II. MT arrays in plant cells III. γ-Tubulin and MT nucleation IV. MT nucleation sites or flexible MTOCs in plant cells V. MT-dependent MT nucleation VI. Generating new MTs for spindle assembly VII. Generation of MTs for phragmoplast expansion during cytokinesis VIII. MT generation for the cortical MT array IX. MT nucleation: looking forward Acknowledgements References SUMMARY: Cytoskeletal microtubules (MTs) have a multitude of functions including intracellular distribution of molecules and organelles, cell morphogenesis, as well as segregation of the genetic material and separation of the cytoplasm during cell division among eukaryotic organisms. In response to internal and external cues, eukaryotic cells remodel their MT network in a regulated manner in order to assemble physiologically important arrays for cell growth, cell proliferation, or for cells to cope with biotic or abiotic stresses. Nucleation of new MTs is a critical step for MT remodeling. Although many key factors contributing to MT nucleation and organization are well conserved in different kingdoms, the centrosome, representing the most prominent microtubule organizing centers (MTOCs), disappeared during plant evolution as angiosperms lack the structure. Instead, flexible MTOCs may emerge on the plasma membrane, the nuclear envelope, and even organelles depending on types of cells and organisms and/or physiological conditions. MT-dependent MT nucleation is particularly noticeable in plant cells because it accounts for the primary source of MT generation for assembling spindle, phragmoplast, and cortical arrays when the γ-tubulin ring complex is anchored and activated by the augmin complex. It is intriguing what proteins are associated with plant-specific MTOCs and how plant cells activate or inactivate MT nucleation activities in spatiotemporally regulated manners.

α- and β-tubulins, the building blocks of the microtubule (MT) polymer, are encoded by multiple genes that are largely functionally redundant in plants. Null tubulin mutants are thus phenotypically indistinguishable from the wild type, but miss-sense or deletion mutations of critical amino acid residues that are important for the assembly, stability, or dynamics of the polymer disrupt the proper organization and function of the resultant MT arrays. Mutant tubulins co-assemble with wild-type tubulins into mutant MTs with compromised functions, and thus mechanistically act as dominant-negative MT poisons. Cortical MT arrays in interphase plant cells are most sensitive to tubulin mutations, and are transformed into helical structures or random orientation, which produce twisted or radially swollen cells. Mutant plants resistant to MT-targeted herbicides may possess tubulin mutations at the binding sites of the herbicides. Tubulin mutants are valuable tools for investigating how individual MTs are organized into particular patterns in cortical arrays, and for defining the functional contribution of MTs to various MT-dependent or -assisted cellular processes in plant cells.

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.

The spatial organization of microtubule (MT) arrays in the surface cell layer of the shoot apical meristem ofHedera was investigated by using a newly developed method. The method facilitates isolation of the intact surface cell layer in its original curved shape and allows subsequent processing for tubulin immunofluorescence microscopy. By optical sectioning of the preparation, MTs of interphase cortical arrays, preprophase bands, mitotic spindles and phragmoplasts were visualized in the entire cell layer. Interphase cortical MTs form circumferential, approximately cylindrical arrays, with the axes of the cylinders lying periclinally. Strands of MTs in individual cells may be oriented in various directions along the inner periclinal wall, appearing as V-shaped or crossed cylindrical arrays. Due to extensive intercellular alignment these arrays form a complex network that occupies most of the central region of the meristem. With increasing distance from the central region, the complex network becomes polarized into an ordered pattern of parallel cylindrical arrays, oriented in accord with topographical contours of the inclined flanks of the meristem and emerging leaf primordia. The process of polarization appears to be promoted by gibberellic acid and may involve a decline in the occurrence of postcytokinetic cells that orient their cortical MT arrays perpendicular to the new cell wall.

The morphogenesis of lobed plant cells has been considered to be controlled by microtubule (MT) and/or actin filament (AF) organization. In this article, a comprehensive mechanism is proposed, in which distinct roles are played by these cytoskeletal components. First, cortical MT bundles and, in the case of pavement cells, radial MT arrays combined with MT bundles determine the deposition of local cell wall thickenings, the cellulose microfibrils of which copy the orientation of underlying MTs. Cell growth is thus locally prevented and, consequently, lobes and constrictions are formed. Arch-like tangential expansion is locally imposed at the external periclinal wall of pavement cells by the radial arrangement of cellulose microfibrils at every wall thickening. Whenever further elongation of the original cell lobes occurs, AF patches assemble at the tips of growing lobes. Intercellular space formation is promoted or prevented by the opposite or alternate, respectively, arrangement of cortical MT arrays between neighboring cells. The genes that are possibly involved in the molecular regulation of the above morphogenetic procedure by MT and AF array organization are reviewed.

Fucus zygotes polarise and germinate a rhizoid before their first asymmetrical division. The role of microtubules (MTs) in orienting the first division plane has been extensively studied by immunofluorescence approaches. In the present study, the re-organisation of MT arrays during the development of Fucus zygotes and embryos was followed in vivo after microinjection of fluorescent tubulin. A dynamic cortical MT array that shows dramatic reorganization during zygote polarization was detected for the first time. Randomly distributed cortical MTs were redistributed to the presumptive rhizoid site by the time of polarisation and well before rhizoid germination. The cortical MT re-organisation occurs independently of centrosome separation and nucleation. By the time of mitosis the cortical array depolymerised to cortical foci in regions from which it also reformed following mitosis, suggesting that it is nucleated from cortical sites. We confirm previous indications from immunodetection studies that centrosomal alignment and nuclear rotation occur via MT connexions to stabilised cortical sites and that definitive alignment is post-metaphasic. Finally, we show that cortical MTs align parallel to the growth axis during rhizoid tip growth and our results suggest that they may be involved in regulating rhizoid growth by shaping the rhizoid and containing turgor pressure.

The cortical microtubule (MT) array and its organization is important in defining the growth axes of plant cells. In roots, the MT array exhibits a net-like configuration in the division zone, and a densely-packed transverse alignment in the elongation zone. This transition is essential for anisotropic cell expansion and consequently has been the subject of intense study. Cotyledons exhibit a net-like array in pavement cells and a predominantly aligned array in the petioles, and provide an excellent system for determining the basis of plant MT organization. We show that in both kinds of MT array, growing MTs frequently encounter existing MTs. Although some steep-angled encounters result in catastrophes, the most frequent outcome of these encounters is successful negotiation of the existing MT by the growing MT to form an MT crossover. Surprisingly, the outcome of such encounters is similar in both aligned and net-like arrays. In contrast, aligned arrays exhibit a much higher frequency of MT severing events compared with net-like arrays. Severing events occur almost exclusively at sites where MTs cross over one another. This process of severing at sites of MT crossover results in the removal of unaligned MTs, and is likely to form the basis for the difference between a net-like and an aligned MT array.

BackgroundPlants can perceive and respond to mechanical signals. For instance, cortical microtubule (CMT) arrays usually reorganize following the predicted maximal tensile stress orientation at the cell and tissue level. While research in the last few years has started to uncover some of the mechanisms mediating these responses, much remains to be discovered, including in most cases the actual nature of the mechanosensors. Such discovery is hampered by the absence of adequate quantification tools that allow the accurate and sensitive detection of phenotypes, along with high throughput and automated handling of large datasets that can be generated with recent imaging devices.ResultsHere we describe an image processing workflow specifically designed to quantify CMT arrays response to tensile stress in time-lapse datasets following an ablation in the epidermis — a simple and robust method to change mechanical stress pattern. Our Fiji-based workflow puts together several plugins and algorithms under the form of user-friendly macros that automate the analysis process and remove user bias in the quantification. One of the key aspects is also the implementation of a simple geometry-based proxy to estimate stress patterns around the ablation site and compare it with the actual CMT arrays orientation. Testing our workflow on well-established reporter lines and mutants revealed subtle differences in the response over time, as well as the possibility to uncouple the anisotropic and orientational response.ConclusionThis new workflow opens the way to dissect with unprecedented detail the mechanisms controlling microtubule arrays re-organization, and potentially uncover the still largely elusive plant mechanosensors.