Spatial organization and isotubulin composition of microtubules in epidermal tendon cells of Artemia franciscana

Epidermally derived tendon cells attach the exoskeleton (cuticle) of the Branchiopod crustacean, Artemia franciscana, to underlying muscle in the hindgut, while the structurally similar transalar tendon (epithelial) cells, which also arise from the epidermis and are polarized, connect dorsal and ventral exopodite surfaces. To establish these latter attachments the transalar tendon cells interact with cuticles on opposite sides of the exopodite by way of their apical surfaces and with one another via basal regions, or the cuticle attachments may be mediated through linkages with phagocytic storage cells found in the hemolymph. In some cases, phyllopod tendon cells attach directly to muscle cells. Tendon cells in the hindgut of Artemia possess microtubule bundles, as do the transalar cells, and they extend from the basal myotendinal junction to the apical domain located near the cuticle. The bundled microtubules intermingle with thin filaments reminiscent of microfilaments, but intermediate filament-like structures are absent. Microtubule bundles converging at apical cell surfaces contact structures termed apical invaginations, composed of cytoplasmic membrane infoldings associated with electron-dense material. Intracuticular rods protrude from apical invaginations, either into the cuticle during intermolt or the molting fluid in premolt. Confocal microscopy of immunofluorescently stained samples revealed tyrosinated, detyrosinated, and acetylated tubulins, the first time posttranslationally modified isoforms of this protein have been demonstrated in crustacean tendon cells. Microfilaments, as shown by staining with phalloidin, coincided spatially with microtubule bundles. Artemia tendon cells clearly represent an interesting system for study of cytoskeleton organization within the context of cytoplasmic polarity and the results in this article indicate functional cooperation of microtubules and microfilaments. These cytoskeletal elements, either acting independently or in concert, may transmit tension from muscle to cuticle in the hindgut and resist compression when connecting exopodite cuticular surfaces.     

Folding, stability and polymerization properties of FtsZ chimeras with inserted tubulin loops involved in the interaction with the cytosolic chaperonin CCT and in microtubule formation

To attain its native conformation, the cytoskeletal protein tubulin needs the concourse of several molecular chaperones, among others the cytosolic chaperonin CCT. It has been previously described that denatured tubulin interacts with CCT in a quasi-folded conformation using several loops located throughout its sequence. These loops are also involved in microtubule formation and are absent in its prokaryote homologue FtsZ, which in vitro folds by itself and does not interact with CCT. Several FtsZ/tubulin chimeric proteins were generated by inserting consecutively one, two or three of the CCT-binding domains of tubulin into the corresponding sequence of FtsZ from Methanococccus jannaschii. The insertion of any of the CCT-binding loops generates in the FtsZ/tubulin chimeras the ability to interact with CCT. The accumulation of CCT-binding loops induces in the FtsZ/tubulin chimeras unfolding and refolding properties that are more similar to tubulin than to its prokaryote counterpart. Finally, the insertion of some of these loops generates in the FtsZ/tubulin chimeras more complex polymeric structures than those found for FtsZ. These results reinforce the notion that CCT has coevolved with tubulin to deal with the folding problems encountered by the eukaryotic protein with the appearance of the new sequences involved in microtubule formation.     

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.     

Model for the yeast cofactor A-beta-tubulin complex based on computational docking and mutagensis

Virtually every biological process involves protein-protein contact but relatively few protein-protein complexes have been solved by X-ray crystallography. As more individual protein structures become available, computational methods are likely to play increasingly important roles in defining these interactions. Tubulin folding and dimer formation are complex processes requiring a variety of protein cofactors. One of these is cofactor A, which interacts with beta-tubulin prior to assembly of the alpha-tubulin-beta-tubulin heterodimer. In the yeast Saccharomyces cerevisiae, beta-tubulin is encoded by TUB2 and cofactor A by RBL2. We have used computational docking and site-directed mutagenesis to generate a model of the Rbl2-Tub2 complex from the solved structures of these two proteins. Residues in the N termini and the loops of the Rbl2 homodimer appear to mediate binding to beta-tubulin. These interact with beta-tubulin residues in the region that contains helices H9 and H10. Rbl2 and alpha-tubulin share overlapping binding sites on the beta-tubulin molecule providing a structural explanation for the mutually exclusive binding of Rbl2 and alpha-tubulin to beta-tubulin.     

Complex formation with kinesin motor domains affects the structure of microtubules

Microtubules are highly dynamic components of the cytoskeleton. They are important for cell movement and they are involved in a variety of transport processes together with motor proteins, such as kinesin. The exact mechanism of these transport processes is not known and so far the focus has been on structural changes within the motor domains, but not within the underlying microtubule structure.Here we investigated the interaction between kinesin and tubulin and our experimental data show that microtubules themselves are changing structure during that process. We studied unstained, vitrified samples of microtubules composed of 15 protofilaments using cryo electron microscopy and helical image analysis. 3D maps of plain microtubules and microtubules decorated with kinesin have been reconstructed to approximately 17A resolution. The alphabeta-tubulin dimer could be identified and, according to our data, alpha- and beta-tubulin adopt different conformations in plain microtubules. Significant differences were detected between maps of plain microtubules and microtubule-kinesin complexes. Most pronounced is the continuous axial inter-dimer contact in the microtubule-kinesin complex, suggesting stabilized protofilaments along the microtubule axis. It seems, that mainly structural changes within alpha-tubulin are responsible for this observation. Lateral effects are less pronounced. Following our data, we believe, that microtubules play an active role in intracellular transport processes through modulations of their core structure.     

Dynamics of outgrowth in a continuum model of neurite elongation

Neurite outgrowth (dendrites and axons) should be a stable, but easily regulated process to enable a neuron to make its appropriate network connections during development. We explore the dynamics of outgrowth in a mathematical continuum model of neurite elongation. The model describes the construction of the internal microtubule cytoskeleton, which results from the production and transport of tubulin dimers and their assembly into microtubules at the growing neurite tip. Tubulin is assumed to be largely synthesised in the cell body from where it is transported by active mechanisms and by diffusion along the neurite. It is argued that this construction process is a fundamental limiting factor in neurite elongation. In the model, elongation is highly stable when tubulin transport is dominated by either active transport or diffusion, but oscillations in length may occur when both active transport and diffusion contribute. Autoregulation of tubulin production can eliminate these oscillations. In all cases a stable steady-state length is reached, provided there is intrinsic decay of tubulin. Small changes in growth parameters, such as the tubulin production rate, can lead to large changes in length. Thus cytoskeleton construction can be both stable and easily regulated, as seems necessary for neurite outgrowth during nervous system development. Action Editor: Upinder Bhalla     

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.     

Macrotubule-dependent protoplast volume regulation in plasmolysed root-tip cells of Triticum turgidum: involvement of phospholipase D

The probable involvement of phospholipase D (PLD)/phosphatidic acid (PA) signalling in the hyperosmotic stress response of Triticum turgidum root cells was investigated by examining the effects of butanol-1, butanol-2, phosphatidylbutanol (PtdBut), N-acylethanolamine (NAE) and PA on the hyperosmotic response, the organization of the tubulin cytoskeleton and the accumulation of a phosphorylated p38-like mitogen-activated protein (MAP) kinase (phospho-p46) in plasmolysed root cells. The effects of all the treatments were assessed by differential interference contrast (DIC) microscopy of living cells, tubulin immunofluorescence, conventional transmission electron microscopy (TEM), tubulin immunogold localization, protoplast volume measurements and western blot analysis. Butanol-1 and NAE compromised the viability of plasmolysed cells, induced a marked reduction in the plasmolysed protoplast volume, and inhibited hyperosmotically induced tubulin macrotubule formation and the accumulation of phospho-p46. Exogenous PA reinforced the hyperosmotic response of T. turgidum root cells and positively affected tubulin macrotubule formation. Additionally, PA reduced the effects of butanol-1 in plasmolysed cells. Taken together, the data suggest that PLD-mediated PA synthesis occurs upstream of the accumulation of phospho-p46 to regulate hyperosmotically induced macrotubule formation in plasmolysed T. turgidum root cells.     

Structural and immunocytochemical characterization of the Ginkgo biloba L. sperm motility apparatus

Summary. Ginkgo biloba and the cycads are the only extant seed plants with motile sperm cells. However, there has been no immunocytochemical characterization of these gametes to determine if they share characteristics with the flagellated sperm found in bryophytes and pteridophytes or might give clues as to the relationships to nonflagellated sperm in all other seed plants. To determine characteristics of proteins associated with the motility apparatus in these motile sperm, we probed thin sections of developing spermatogenous cells of Ginkgo biloba with antibodies to acetylated and tyrosinated tubulin and monoclonal antibodies that recognize mammalian centrosomes and centrin. The blepharoplast that occurs as a precursor to the motility apparatus consists of an amorphous core, pitted with cavities containing microtubules and a surface studded with probasal bodies. The probasal bodies and microtubules within the blepharoplast cavities are labeled with antibodies specific to acetylated tubulin. Positive but weak reactions of the blepharoplast core occur with the centrosomereactive antibodies MPM-2 and C-9. Reactions to centrin antibodies are negative at this developmental stage. From this pre-motility apparatus structure, an assemblage of about 1000 flagella and associated structures arises as the precursor to the motility apparatus for the sperm. The flagellar apparatus consists of a three-layered multilayered structure that subtends a layer of spline microtubules, a zone of amorphous material similar to that in the blepharoplast, and the flagellar band. Centrin antibodies react strongly with the multilayered structure, the transition zone of the flagella, and fibrillar material near the flagellar base at the surface of the amorphous material. Both the spline microtubules and all of the tubules in the flagella react strongly with the antibodies to acetylated tubulin. These localizations are consistent with the localizations of these components in pteridophyte and bryophyte spermatogenous cells, although the blepharoplast material surrounding and connecting flagellar bases does not occur in the seedless (nonseed) land plants. These data indicate that despite the large size of ginkgo gametes and the taxonomic separation between pteridophytes and Ginkgo biloba, similar proteins in gametes of both groups perform similar functions and are therefore homologous among these plants. Moreover, the presence of acetylated tubulin in bands of microtubules may be a characteristic shared with more derived non-flagellated sperm of other conifers and angiosperms. Correspondence and reprints: Southern Weed Science Research Unit, USDA Agricultural Research Service, P.O. Box 350, Stoneville, MS 38776, U.S.A.     

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.