Disruption of Cortical Microtubules by Overexpression of Green Fluorescent Protein-Tagged α-Tubulin 6 Causes a Marked Reduction in Cell Wall Synthesis

It has been known that the transverse orientation of cortical microtubules （MTs） along the elongation axis is essential for normal cell morphogenesis, but whether cortical MTs are essential for normal cell wall synthesis is still not clear. In the present study, we have investigated whether cortical MTs affect cell wall synthesis by direct alteration of the cortical MT organization in Arabidopsis thaliana. Disruption of the cortical MT organization by expression of an excess amount of green fluorescent protein-tagged a-tubulin 6 （GFP-TUA6） in transgenic Arabidopsis plants was found to cause a marked reduction in cell wall thickness and a de- crease in the cell wall sugars glucose and xylose. Concomitantly, the stem strength of the GFP-TUA6 overexpressors was markedly reduced compared with the wild type. In addition, expression of excess GFP- TUA6 results in an alteration in cell morphogenesis and a severe effect on plant growth and development. Together, these results suggest that the proper organization of cortical MTs is essential for the normal synthesis of plant cell walls.     

Morphogenesis of the endoplasmic reticulum: beyond active membrane expansion

The endoplasmic reticulum (ER) of higher eukaryotic cells is a dynamic network of interconnected membrane tubules that pervades almost the entire cytoplasm. On the basis of the morphological changes induced by the disruption of the cytoskeleton or molecular motor proteins, the commonly accepted model has emerged that microtubules and conventional kinesin (kinesin-1) are essential determinants in establishing and maintaining the structure of the ER by active membrane expansion. Surprisingly, very similar ER phenotypes have now been observed when the cytoskeleton-linking ER membrane protein of 63 kDa (CLIMP-63) is mutated, revealing stable attachment of ER membranes to the microtubular cytoskeleton as a novel requirement for ER maintenance. Additional recent findings suggest that ER maintenance also requires ongoing homotypic membrane fusion, possibly controlled by the p97/p47/VICP135 protein complex. Work on other proteins proposed to regulate ER structure, including huntingtin, the EF-hand Ca(2+)-binding protein p22, the vesicle-associated membrane protein-associated protein B and kinectin isoforms further contribute to the new emerging concept that ER shape is not only determined by motor driven processes but by a variety of different mechanisms.     

Tubulin must be acetylated in order to form a complex with membrane Na+,K+-ATPase and to inhibit its enzyme activity

In cells of neural and non-neural origin, tubulin forms a complex with plasma membrane Na⁺,K⁺-ATPase, resulting in inhibition of the enzyme activity. When cells are treated with 1 mM L-glutamate, the complex is dissociated and enzyme activity is restored. Now, we found that in CAD cells, ATPase is not activated by L-glutamate and tubulin/ATPase complex is not present in membranes. By investigating the causes for this characteristic, we found that tubulin must be acetylated in order to associate with ATPase and to inhibit its catalytic activity. In CAD cells, the acetylated tubulin isotype is absent. Treatment of CAD cells with deacetylase inhibitors (trichostatin A or tubacin) caused appearance of acetylated tubulin, formation of tubulin/ATPase complex, and reduction of membrane ATPase activity. In these treated cells, addition of 1 mM L-glutamate dissociated the complex and restored the enzyme activity. Cytosolic tubulin from trichostatin A-treated but not from non-treated cells inhibited ATPase activity. These findings indicate that the acetylated isotype of tubulin is required for interaction with membrane Na⁺,K⁺-ATPase and consequent inhibition of enzyme activity.     

Ni2+ induces changes in the morphology of vacuoles, mitochondria and microtubules in Paxillus involutus cells

Organelles of ectomycorrhizal fungi are known to respond to changes in the extracellular environment. The response of vacuoles, mitochondria and microtubules to short-term nickel (Ni2+) exposure were investigated in hyphal tip cells of a Paxillus involutus from a heavy metal-rich soil. Vacuoles, mitochondria and microtubules were labelled with Oregon Green 488 carboxylic acid diacetate, 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and anti-alpha-tubulin antibodies, respectively; hyphae were treated with NiSO4 in the range of 0-1 mmol l(-1) and examined microscopically. Untreated hyphal tip cells contained tubular vacuole and mitochondrial networks. Ni2+ caused loss of organelle tubularity and severe microtubule disruption that were exposure-time and concentration dependent. Fine tubular vacuoles thickened and eventually became spherical in some hyphae, tubular mitochondria fragmented and microtubules shortened and aggregated into patches in most hyphae. Tubular vacuoles reformed on NiSO4 removal and tubular mitochondria in the presence of NiSO4 suggesting cellular detoxification. These results demonstrate that Ni2+ induces changes in organelle and microtubule morphology. Recovery of tubular organelles to pretreatment morphology after Ni2+ exposure suggests cellular detoxification of the metal ion.     

Stochastic computer model of the cell microtubule dynamics

To study the effect of various factors on the microtubule system, one of the main cytoskeletal elements in the cell, which organizes the intracellular transport of different organelles and is necessary for mitosis and meiosis, a computer model of this system is created. Using a stochastic approach, the model describes the microtubule assembly/disassembly as a set of chemical reactions with certain rate constants. Microtubules are visualized in the computer program field, which makes the model vivid. The program imitates the dynamics and structure of the microtubule system with high reliability. The parameters calculated by the model correlate with the corresponding parameters of microtubules in living cells. This approach to modeling microtubules and similar systems continues to be developed so that the models would better describe living systems and the effect of a still broader range of factors could be studied.     

Cytoskeleton-induced alterations of the lectin activity in winter wheat under cold hardening and abscisic acid (ABA)

The roots and leaves of 7-day seedlings of three winter wheat cultivars differing in frost resistant were used to study changes in lectin activity under cytoskeleton modifiers (DMSO-7%; colchicine-1 m m; oryzalin-15 microm; cytochalasin B-15 microm) of non-hardened (23 degrees C) and hardened (2-3 degrees C, 3-7 day) plants. Plants were grown with ABA (30 microm) or without ABA. Pretreatment with colchicine, oryzalin [inhibitors of microtubules (MT) polymerization], cytochalasin B [inhibitor of microfilament (MF) polymerization] increased the activity of cell wall lectins, although pretreatment with DMSO (stabilizer of microtubules) decreased the activity. Both hardening and ABA decreased the effect of the cytoskeletal modifiers. These results could be explained by the appearance of tolerant MTs with less affinity. It is probable that increase in the activity of cell wall lectins may be the compensatory mechanism which stabilizes the cytoskeleton structure in conditions tending to disrupt it. The genotype with low resistance had higher sensitivity of lectin activity to cytoskeleton modifiers than the frost resistant genotype. The results suggest that leaves have more stable MTs and MFs and stronger MT-MF binding than roots.     

Influence of inhibitors of cytoskeleton proteins on water exchange of wheat roots under the after-action of water stress

The dynamics of water molecular state and transport in winter wheat (Triticum aestivum L.) of roots different resistance cultivars was studied by a biophysical method, Nuclear Magnetic Resonance (NMR), and a physiological method, Water-Holding Capacity (WHC). The effective coefficient of water self-diffusion (D(eff)), spin-spin relaxation times (T(2)) and WHC were measured after structural modification of cytoskeleton by colchicine and cytochalasin B after the action of water stress. New information about molecular mechanisms of water state and water transport regulation determined by the influence of dynamic cytoskeleton structure has been obtained. This is very important for the development of a fundamental theory of water exchange in plants, and for the ways of its optimization under conditions of environmental stress.     

From hormone signal, via the cytoskeleton, to cell growth in single cells of tobacco

Cultured mesophyll protoplasts of Nicotiana tabacum L. can be hormonally induced into different developmental pathways. In a medium containing auxins (NAA) and cytokinins (BAP) cells divide and eventually give rise to calli. When only auxins are present cells elongate and finally differentiate into very long tubular cells. We focused on the sequence of events leading to elongation. When cultured in a high (1 mg/l) auxin concentration elongating cells seem to pass a certain threshold and increase their nuclear DNA up to about 16C. Cells cultured in a low (0.065 mg/l) auxin concentration only have C-values up to 4C, are unable to pass this threshold and finally fail to elongate. Besides the concentration dependence of the auxin signal, the efflux of auxin seems to be necessary for elongation since addition of TIBA drastically reduces the amount of elongating cells. Concomitant with the changes in nuclear physiology, auxin-induced axiality is seen as sequential rearrangements of microtubules and actin-filaments and of cell wall cellulose microfibrils from 'randomly' arranged in spherical cells to an orientation perpendicular to the long axis of elongating cells.     

Microfilaments and microtubules: the news from yeast

New evidence that cortical actin patches and the endocytic machinery share components supports the idea that actin patches are in fact transient membrane coats at the initial stage of endocytosis. Recent studies of actin cables have identified formins as the core of a novel actin-filament-assembling machine. Meanwhile, microtubule-binding proteins have been found in the kinetochore, and factors affecting microtubule dynamic instability have been identified.     

Simulation of the effects of microtubules in the cortical rotation of amphibian embryos in normal and zero gravity

This paper reports the results of computer modeling of microtubules that end up in the cortical region of a one-cell amphibian embryo, prior to the first cell division. Microtubules are modeled as initially randomly oriented semi-flexible rods, represented by several lines of point-masses interacting with one another like masses on springs with longitudinal and transverse stiffness. They are also considered to be space-filling rods floating in a viscous fluid (cytoplasm) experiencing drag forces and buoyancy from the fluid under a variable gravity field to test gravitational effects. Their randomly distributed interactions with the surrounding spherical container (the cell membrane) have a statistical nonzero average that creates a torque causing a rotational displacement between the cytoplasm and the rigid cortex. The simulation has been done for zero and normal gravity and it validates the observation that cortical rotation occurs in microgravity as well as on Earth. The speed of rotation depends on gravity, but is still substantial in microgravity.