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.     

Hydrostatic pressure induced changes in the cytoarchitecture of pheochromocytoma (PC-12) cells.

Confocal microscopy, in association with three-dimensional reconstruction, revealed that microtubules and microfilaments in differentiating PC-12 cells were disrupted in a dose-dependent manner following pressure treatment. Hydrostatic pressure caused cell rounding, microtubule and microfilament disorganization, neurite retraction and the formation of a microtubule ring adjacent to the cell surface. Volume analysis from computer-generated reconstructed cells, at atmospheric pressure, showed that the apparent volume of microtubules and microfilaments, normalized to 100 units, was 22 and 11 respectively. At 4000 and 8000 psi, the apparent microtubule volume was reduced to 16 and 12 units, respectively, and the apparent microfilament volume was reduced to 8 and 5 units, respectively. Thus, the apparent microtubule and microfilament volumes in PC-12 cells decreased as pressure increased. In the presence of taxol and phalloidin which stabilize the cytoarchitecture, cells resist the effects of hydrostatic pressure. In the presence of colchicine and cytochalasin D compounds which destabilize the cytoarchitecture, cells are more susceptible to the disrupting effects of hydrostatic pressure. The effects of hydrostatic pressure on cell morphology were reversible.     

The effects of hydrostatic pressure-induced changes on the cytoskeleton and on the regulation of gene expression in pheochromocytoma (PC-12) cells.

The purpose of this investigation was to determine the relationship of hydrostatic pressure-induced changes in the cytoarchitecture to regulation of gene expression in PC-12 cells. Hydrostatic pressure disrupts the cytoskeleton, decreases tubulin and actin mRNA levels and causes changes in the localization of tubulin and actin mRNA. Actin mRNA levels, at 6000 and 10,000 psi for 20 min, were reduced to 78% and 64%, respectively, in undifferentiated cells and to 81% and 72%, respectively, in 4-day differentiating cells, relative to untreated controls. Tubulin mRNA levels, at 6000 and 10,000 psi for 20 min, were reduced to 75% and 67%, respectively, in undifferentiated cells and to 84% and 74%, respectively, in 4-day differentiating cells. Changes in the localization of mRNA in the soluble and cytoskeletal fractions were determined by measuring the pressure level where the mRNA level in the cytoskeletal fraction equals the mRNA level in the soluble fraction. This measurement was designated the cytoskeletal/soluble fraction index (CSFI(50)). CSFI(50)measurements indicated that following hydrostatic pressure, actin mRNA cytoskeletal association was more stable than tubulin mRNA cytoskeletal association. The addition of chemicals which stabilize or destabilize microtubules and microfilaments to pressure treatment resulted in additional changes in the CSFI(50).     

Molecular determinants for α-tubulin methylation by SETD2

Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. It was recently shown that methylation of tubulin by the histone methyltransferase SETD2 occurs on mitotic spindle microtubules during cell division, with its absence resulting in mitotic defects. However, the catalytic mechanism of methyl addition to tubulin is unclear. We used a truncated version of human wild type SETD2 (tSETD2) containing the catalytic SET and C-terminal Set2–Rpb1–interacting (SRI) domains to investigate the biochemical mechanism of tubulin methylation. We found that recombinant tSETD2 had a higher activity toward tubulin dimers than polymerized microtubules. Using recombinant single-isotype tubulin, we demonstrated that methylation was restricted to lysine 40 of α-tubulin. We then introduced pathogenic mutations into tSETD2 to probe the recognition of histone and tubulin substrates. A mutation in the catalytic domain (R1625C) allowed tSETD2 to bind to tubulin but not methylate it, whereas a mutation in the SRI domain (R2510H) caused loss of both tubulin binding and methylation. Further investigation of the role of the SRI domain in substrate binding found that mutations within this region had differential effects on the ability of tSETD2 to bind to tubulin versus the binding partner RNA polymerase II for methylating histones in vivo, suggesting distinct mechanisms for tubulin and histone methylation by SETD2. Finally, we found that substrate recognition also requires the negatively charged C-terminal tail of α-tubulin. Together, this study provides a framework for understanding how SETD2 serves as a dual methyltransferase for both histone and tubulin methylation.     

TRPV1-tubulin complex: involvement of membrane tubulin in the regulation of chemotherapy-induced peripheral neuropathy

Existence of microtubule cytoskeleton at the membrane and submembranous regions, referred as 'membrane tubulin' has remained controversial for a long time. Since we reported physical and functional interaction of Transient Receptor Potential Vanilloid Sub Type 1 (TRPV1) with microtubules and linked the importance of TRPV1-tubulin complex in the context of chemotherapy-induced peripheral neuropathy, a few more reports have characterized this interaction in in vitro and in in vivo condition. However, the cross-talk between TRPs with microtubule cytoskeleton, and the complex feedback regulations are not well understood. Sequence analysis suggests that other than TRPV1, few TRPs can potentially interact with microtubules. The microtubule interaction with TRPs has evolutionary origin and has a functional significance. Biochemical evidence, Fluorescence Resonance Energy Transfer analysis along with correlation spectroscopy and fluorescence anisotropy measurements have confirmed that TRPV1 interacts with microtubules in live cell and this interaction has regulatory roles. Apart from the transport of TRPs and maintaining the cellular structure, microtubules regulate signaling and functionality of TRPs at the single channel level. Thus, TRPV1-tubulin interaction sets a stage where concept and parameters of 'membrane tubulin' can be tested in more details. In this review, I critically analyze the advancements made in biochemical, pharmacological, behavioral as well as cell-biological observations and summarize the limitations that need to be overcome in the future.     

Effects of extrusion and glycerol content on properties of oxidized and acetylated corn starch-based films

Oxidized and acetylated corn starch-based films were prepared by casting with glycerol as a plasticizer. The present study investigated the effects of extrusion prior to film-making and glycerol content on the properties of starch films. The films with extrusion exhibited lower tensile strength, higher elongation at break, higher water vapor permeability and higher oil permeability than those without extrusion. Extrusion reduced heat sealability of the films. With the increase of glycerol content, the films became more flexible with higher elongation at break and lower tensile strength. Water vapor permeability, oil permeability and the range between the onset temperature and the melt peak temperature rose as glycerol content increased. The thermograms indicated that plasticizers and biopolymers were compatible. These results suggested that extrusion did no good to starch films while glycerol content had apparent effect on the mechanical and barrier properties of the films.     

Interfacing protein lysine acetylation and protein phosphorylation: ?Ancient modifications meet on ancient proteins

Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature.