IQ67 DOMAIN protein 21 is critical for indentation formation in pavement cell morphogenesis

In plants, cortical microtubules anchor to the plasma membrane in arrays and play important roles in cell shape. However, the molecular mechanism of microtubule binding proteins, which connect the plasma membrane and cortical microtubules in cell morphology remains largely unknown. Here, we report that a plasma membrane and microtubule duallocalized IQ67 domain protein, IQD21, is critical for cotyledon pavement cell(PC) morphogenesis in Arabidopsis. iqd21 mutation caused increased indentation wi...     

Analysis of cortical arrays from Tradescantia virginiana at high resolution reveals discrete microtubule subpopulations and demonstrates that confocal images of arrays can be misleading

Cortical microtubule arrays are highly organized networks involved in directing cellulose microfibril deposition within the cell wall. Their organization results from complex interactions between individual microtubules and microtubule-associated proteins. The precise details of these interactions are often not evident using optical microscopy. Using high-resolution scanning electron microscopy, we analyzed extensive regions of cortical arrays and identified two spatially discrete microtubule subpopulations that exhibited different stabilities. Microtubules that lay adjacent to the plasma membrane were often bundled and more stable than the randomly aligned, discordant microtubules that lay deeper in the cytoplasm. Immunolabeling revealed katanin at microtubule ends, on curves, or at sites along microtubules in line with neighboring microtubule ends. End binding 1 protein also localized along microtubules, at microtubule ends or junctions between microtubules, and on the plasma membrane in direct line with microtubule ends. We show fine bands in vivo that traverse and may encircle microtubules. Comparing confocal and electron microscope images of fluorescently tagged arrays, we demonstrate that optical images are misleading, highlighting the fundamental importance of studying cortical microtubule arrays at high resolution.     

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.     

Neuronal Galectin‐4 is required for axon growth and for the organization of axonal membrane L1 delivery and clustering

Axon membrane glycoproteins are essential for neuronal differentiation, although the mechanisms underlying their polarized sorting and organization are poorly understood. We describe here that galectin‐4 (Gal‐4), a lectin highly expressed in gastrointestinal tissues and involved in epithelial glycoprotein transport, is expressed by hippocampal and cortical neurons where it is sorted to discrete segments of the axonal membrane in a microtubule‐ and sulfatide‐dependent manner. Gal‐4 knockdown retards axon growth, an effect that can be rescued by recombinant Gal‐4 addition. This Gal‐4 reduction, as inhibition of sulfatide synthesis does, lowers the presence and clustered organization of axon growth‐promoting molecule NCAM L1 at the axon membrane. Furthermore, we find that Gal‐4 interacts with L1 by specifically binding to LacNAc branch ends of L1 N‐glycans. Impairing the maturation of these N‐glycans precludes Gal‐4/L1 association resulting in a failure of L1 membrane cluster organization. In all, Gal‐4 sorts to axon plasma membrane segments by binding to sulfatide‐containing microtubule‐associated carriers and being bivalent, it organizes the transport of L1, and likely other axonal glycoproteins, by attaching them to the carriers through their LacNAc termini. This mechanism would underlie L1 functional organization on the plasma membrane, required for proper axon growth.     

The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis

It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic microtubule network possesses a novel organization, whose nucleation appears independent of conventional γ-tubulin ring complex containing structures. Our analysis suggests that microtubules are closely associated with the plasma membrane, forming a cortical ring or cellular “cocoon”. Concomitantly other components of the cytoskeleton, such as actin and cytokeratins disassemble. We found that colchicine-mediated disruption of apoptotic microtubule network results in enhanced plasma membrane permeability and secondary necrosis, suggesting that the reformation of a microtubule cytoskeleton plays an important role in preserving plasma membrane integrity during apoptosis. Significantly, cells induced to enter apoptosis in the presence of the pan-caspase inhibitor z-VAD, nevertheless form microtubule-like structures suggesting that microtubule formation is not dependent on caspase activation. In contrast we found that treatment with EGTA-AM, an intracellular calcium chelator, prevents apoptotic microtubule network formation, suggesting that intracellular calcium may play an essential role in the microtubule reformation. We propose that apoptotic microtubule network is required to maintain plasma membrane integrity during the execution phase of apoptosis. Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

Microtubule networks for plant cell division

During cytokinesis the cytoplasm of a cell is divided to form two daughter cells. In animal cells, the existing plasma membrane is first constricted and then abscised to generate two individual plasma membranes. Plant cells on the other hand divide by forming an interior dividing wall, the so-called cell plate, which is constructed by localized deposition of membrane and cell wall material. Construction starts in the centre of the cell at the locus of the mitotic spindle and continues radially towards the existing plasma membrane. Finally the membrane of the cell plate and plasma membrane fuse to form two individual plasma membranes. Two microtubule-based cytoskeletal networks, the phragmoplast and the pre-prophase band (PPB), jointly control cytokinesis in plants. The bipolar microtubule array of the phragmoplast regulates cell plate deposition towards a cortical position that is templated by the ring-shaped microtubule array of the PPB. In contrast to most animal cells, plants do not use centrosomes as foci of microtubule growth initiation. Instead, plant microtubule networks are striking examples of self-organizing systems that emerge from physically constrained interactions of dispersed microtubules. Here we will discuss how microtubule-based activities including growth, shrinkage, severing, sliding, nucleation and bundling interrelate to jointly generate the required ordered structures. Evidence mounts that adapter proteins sense the local geometry of microtubules to locally modulate the activity of proteins involved in microtubule growth regulation and severing. Many of the proteins and mechanisms involved have roles in other microtubule assemblies as well, bestowing broader relevance to insights gained from plants.     

Phospholipase Dδ negatively regulates plant thermotolerance by destabilizing cortical microtubules in Arabidopsis

The pattern of cortical microtubule arrays plays an important role in plant growth and adaptation in response to hormonal and environmental changes. Cortical microtubules are connected with the plasma membrane (PM); however, how the membrane affects cortical microtubule organization is not well understood. Here, we showed that phospholipase Dδ (PLDδ) was associated with the PM and co‐localized with microtubules in cells. In vitro analysis revealed that PLDδ bound to microtubules, resulting in microtubule disorganization. Site‐specific mutations that decreased PLDδ enzymatic activity impaired its effects on destabilizing microtubule organization. Heat shock transiently activated PLDδ, without any change of its PM localization, triggering microtubule dissociation from PM and depolymerization and seedling death in Arabidopsis, but these effects were alleviated in pldδ knockout mutants. Complementation of pldδ with wild‐type PLDδ, but not mutated PLDδ, restored the phenotypes of microtubules and seedling survival to those of wild‐type Arabidopsis. Thus, we conclude that the PM‐associated PLDδ negatively regulates plant thermotolerance via destabilizing cortical microtubules, in an activity‐dependent manner, rather than its subcellular translocation.     

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.     

PP2A interacts with KATANIN to promote microtubule organization and conical cell morphogenesis

The organization of the microtubule cytoskeleton is critical for cell and organ morphogenesis. The evolutionarily conserved microtubule-severing enzyme KATANIN plays critical roles in microtubule organization in the plant and animal kingdoms. We previously used conical cell of Arabidopsis thaliana petals as a model system to investigate cortical microtubule organization and cell morphogenesis and determined that KATANIN promotes the formation of circumferential cortical microtubule arrays in conical cells. Here, we demonstrate that the conserved protein phosphatase PP2A interacts with and dephosphorylates KATANIN to promote the formation of circumferential cortical microtubule arrays in conical cells. KATANIN undergoes cycles of phosphorylation and dephosphorylation. Using co-immunoprecipitation coupled with mass spectrometry, we identified PP2A subunits as KATANIN-interacting proteins. Further biochemical studies showed that PP2A interacts with and dephosphorylates KATANIN to stabilize its cellular abundance. Similar to the katanin mutant, mutants for genes encoding PP2A subunits showed disordered cortical microtubule arrays and defective conical cell shape. Taken together, these findings identify PP2A as a regulator of conical cell shape and suggest that PP2A mediates KATANIN phospho-regulation during plant cell morphogenesis.     

Microtubules do not control orientation of secondary cell wall microfibril deposition inMicrasterias

Summary The influence of the microtubule disorganizing substances amiprophos-methyl (APM) and colchicine on secondary wall formation inMicrasterias denticulata was investigated by the freezeetch technique. The results reveal that neither microtubule inhibitor changes the pattern of microfibril deposition. The application of APM or colchicine also does not cause any structural alterations of the microfibrils or of the protoplasmic (Pf) and the exoplasmic (Ef) fracture face of the plasma membrane, thus indicating that microtubules are not involved in secondary wall formation inM. denticulata. However, since areas of the plasma membrane which collapsed upon freeze-etching are restricted to the Pf-face of cells treated with microtubule inhibitors, cortical microtubules may function as mechanical support during secondary wall formation. In the cortical cytoplasm filamentous structures are found in close spatial relationship and an almost parallel alignment to rosettes of the plasma membrane.     

Extending the Microtubule/Microfibril Paradigm : Cellulose Synthesis Is Required for Normal Cortical Microtubule Alignment in Elongating Cells

The cortical microtubule array provides spatial information to the cellulose-synthesizing machinery within the plasma membrane of elongating cells. Until now data indicated that information is transferred from organized cortical microtubules to the cellulose-synthesizing complex, which results in the deposition of ordered cellulosic walls. How cortical microtubules become aligned is unclear. The literature indicates that biophysical forces, transmitted by the organized cellulose component of the cell wall, provide a spatial cue to orient cortical microtubules. This hypothesis was tested on tobacco (Nicotiana tabacum L.) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben. Isoxaben (0.25–2.5 μm) inhibited the synthesis of cellulose microfibrils (detected by staining with 1 μg mL⁻¹ fluorescent dye and polarized birefringence), the cells failed to elongate, and the cortical microtubules failed to become organized. The affects of isoxaben were reversible, and after its removal microtubules reorganized and cells elongated. Isoxaben did not depolymerize microtubules in vivo or inhibit the polymerization of tubulin in vitro. These data are consistent with the hypothesis that cellulose microfibrils, and hence cell elongation, are involved in providing spatial cues for cortical microtubule organization. These results compel us to extend the microtubule/microfibril paradigm to include the bidirectional flow of information.     

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.     

Microtubule-dependent microtubule nucleation based on recruitment of gamma-tubulin in higher plants

Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array. A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex. Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic gamma-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain gamma-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting gamma-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, gamma-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic gamma-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.     

A ZYG-12–dynein interaction at the nuclear envelope defines cytoskeletal architecture in the C. elegans gonad

Changes in cellular microtubule organization often accompany developmental progression. In the Caenorhabditis elegans embryo, the centrosome, which is attached to the nucleus via ZYG-12, organizes the microtubule network. In this study, we investigate ZYG-12 function and microtubule organization before embryo formation in the gonad. Surprisingly, ZYG-12 is dispensable for centrosome attachment in the germline. However, ZYG-12–mediated recruitment of dynein to the nuclear envelope is required to maintain microtubule organization, membrane architecture, and nuclear positioning within the syncytial gonad. We examined γ-tubulin localization and microtubule regrowth after depolymerization to identify sites of nucleation in germ cells. γ-Tubulin localizes to the plasma membrane in addition to the centrosome, and regrowth initiates at both sites. Because we do not observe organized microtubules around zyg-12(ct350) mutant nuclei with attached centrosomes, we propose that gonad architecture, including membrane and nuclear positioning, is determined by microtubule nucleation at the plasma membrane combined with tension on the microtubules by dynein anchored at the nucleus by ZYG-12.     

MDP25 mediates the fine-tuning of microtubule organization in response to salt stress

Microtubules are dynamic cytoskeleton structures playing fundamental roles in plant responses to salt stress. The precise mechanisms by which microtubule organization is regulated under salt stress are largely unknown. Here, we report that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN 25 (MDP25; also known as PLASMA MEMBRANE-ASSOCIATED CATION-BINDING PROTEIN 1 (PCaP1)) helps regulate microtubule organization. Under salt treatment, elevated cytosolic Ca²⁺ concentration caused MDP25 to partially dissociate from the plasma membrane, promoting microtubule depolymerization. When Ca²⁺ signaling was blocked by BAPTA-AM or LaCl₃, microtubule depolymerization in wild-type and MDP25-overexpressing cells was slower, while there was no obvious change in mdp25 cells. Knockout of MDP25 improved microtubule reassembly and was conducive to microtubule integrity under long-term salt treatment and microtubule recovery after salt stress. Moreover, mdp25 seedlings exhibited a higher survival rate under salt stress. The presence microtubule-disrupting reagent oryzalin or microtubule-stabilizing reagent paclitaxel differentially affected the survival rates of different genotypes under salt stress. MDP25 promoted microtubule instability by affecting the catastrophe and rescue frequencies, shrinkage rate and time in pause phase at the microtubule plus-end and the depolymerization rate at the microtubule minus-end. These findings reveal a role for MDP25 in regulating microtubule organization under salt treatment by affecting microtubule dynamics.     

Cell proliferation,cell shape,and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March–April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.     

Phospholipases may play multiple roles in anisotropic plant cell growth

Both the cortical microtubule cytoskeleton and cellulose microfibrils are important for the anisotropic growth of plant cells. Although the two systems interact, the details of this interaction are far from clear. It has been shown the inhibitors of phospholipase D, phospholipase A₂ and phospholipase C all cause disorganisation of the microtubule cytoskeleton. Since the phospholipases act on the plasma membrane, which links cortical microtubules to cellulose microfibrils in the cell wall, they may play a key role in the communication between the two structures. This communication may take various forms. Microtubule-linked phospholipase activity may cause the organisation of underlying cellulose microfibril liquid crystals. Alternatively, phospholipases may co-operate in the regulation of plasma membrane fluidity, affecting the movement of cellulose synthase complexes in the underlying plasma membrane. GPI-anchored proteins in the plasma membrane, which are cleaved by phospholipases, may possibly play a role.     

Disruption of arabinogalactan proteins disorganizes cortical microtubules in the root of Arabidopsis thaliana

The cortical array of microtubules inside the cell and arabinogalactan proteins on the external surface of the cell are each implicated in plant morphogenesis. To determine whether the cortical array is influenced by arabinogalactan proteins, we first treated Arabidopsis roots with a Yariv reagent that binds arabinogalactan proteins. Cortical microtubules were markedly disorganized by 1 microM beta-D-glucosyl (active) Yariv but not by up to 10 microM beta-D-mannosyl (inactive) Yariv. This was observed for 24-h treatments in wild-type roots, fixed and stained with anti-tubulin antibodies, as well as in living roots expressing a green fluorescent protein (GFP) reporter for microtubules. Using the reporter line, microtubule disorganization was evident within 10 min of treatment with 5 microM active Yariv and extensive by 30 min. Active Yariv (5 microM) disorganized cortical microtubules after gadolinium pre-treatment, suggesting that this effect is independent of calcium influx across the plasma membrane. Similar effects on cortical microtubules, over a similar time scale, were induced by two anti-arabinogalactan-protein antibodies (JIM13 and JIM14) but not by antibodies recognizing pectin or xyloglucan epitopes. Active Yariv, JIM13, and JIM14 caused arabinogalactan proteins to aggregate rapidly, as assessed either in fixed wild-type roots or in the living cells of a line expressing a plasma membrane-anchored arabinogalactan protein from tomato fused to GFP. Finally, electron microscopy of roots prepared by high-pressure freezing showed that treatment with 5 microM active Yariv for 2 h significantly increased the distance between cortical microtubules and the plasma membrane. These findings demonstrate that cell surface arabinogalactan proteins influence the organization of cortical microtubules.     

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.     

Salt stress affects cortical microtubule organization and helical growth in Arabidopsis

Cortical microtubule arrays are critical in determining the growth axis of diffusely growing plant cells, and various environmental and physiological factors are known to affect the array organization. Microtubule organization is partly disrupted in the spiral1 mutant of Arabidopsis thaliana, which displays a right-handed helical growth phenotype in rapidly elongating epidermal cells. We show here that mutations in the plasma membrane Na(+)/H(+) antiporter SOS1 and its regulatory kinase SOS2 efficiently suppressed both microtubule disruption and helical growth phenotypes of spiral1, and that sos1 and sos2 roots in the absence of salt stress exhibited altered helical growth response to microtubule-interacting drugs at low doses. Salt stress also altered root growth response to the drugs in wild-type roots. Suppression of helical growth appeared to be specific to spiral1 since other helical growth mutants were not rescued. The effects of sos1 in suppressing spiral1 defects and in causing abnormal drug responses were nullified in the presence of the hkt1 Na(+) influx carrier mutation in roots but not in hypocotyls. These results suggest that cytoplasmic salt imbalance caused by insufficient SOS1 activity compromises cortical microtubule functions in which microtubule-localized SPIRAL1 is specifically involved.