PKC-zeta is required in EGF protection of microtubules and intestinal barrier integrity against oxidant injury

Using monolayers of human intestinal (Caco-2) cells, we showed that epidermal growth factor (EGF) protects intestinal barrier integrity against oxidant injury by protecting the microtubules and that protein kinase C (PKC) is required. Because atypical PKC-zeta isoform is abundant in wild-type (WT) Caco-2 cells, we hypothesized that PKC-zeta mediates, at least in part, EGF protection. Intestinal cells (Caco-2 or HT-29) were transfected to stably over- or underexpress PKC-zeta. These clones were preincubated with low or high doses of EGF or a PKC activator [1-oleoyl-2-acetyl-sn-glycerol (OAG)] before oxidant (0.5 mM H(2)O(2)). Relative to WT cells exposed to oxidant, only monolayers of transfected cells overexpressing PKC-zeta (2.9-fold) were protected against oxidant injury as indicated by increases in polymerized tubulin and decreases in monomeric tubulin, enhancement of architectural stability of the microtubule cytoskeleton, and increases in monolayer barrier integrity toward control levels (62% less leakiness). Overexpression-induced protection was OAG independent and even EGF independent, but EGF significantly potentiated PKC-zeta protection. Most overexpressed PKC-zeta (92%) resided in membrane and cytoskeletal fractions, indicating constitutive activation of PKC-zeta. Stably inhibiting PKC-zeta expression (95%) with antisense transfection substantially attenuated EGF protection as demonstrated by reduced tubulin assembly and increased microtubule disassembly, disruption of the microtubule cytoskeleton, and loss of monolayer barrier integrity. We conclude that 1) activation of PKC-zeta is necessary for EGF-induced protection, 2) PKC-zeta appears to be an endogenous stabilizer of the microtubule cytoskeleton and of intestinal barrier function against oxidative injury, and 3) we have identified a novel biological function (protection) among the atypical isoforms of PKC.     

PKC-zeta prevents oxidant-induced iNOS upregulation and protects the microtubules and gut barrier integrity

Using intestinal (Caco-2) monolayers, we reported that inducible nitric oxide synthase (iNOS) activation is key to oxidant-induced barrier disruption and that EGF protects against this injury. PKC-zeta was required for protection. We thus hypothesized that PKC-zeta activation and iNOS inactivation are key in EGF protection. Wild-type (WT) Caco-2 cells were exposed to H(2)O(2) (0.5 mM) +/- EGF or PKC modulators. Other cells were transfected to overexpress PKC-zeta or to inhibit it and then pretreated with EGF or a PKC activator (OAG) before oxidant. Relative to WT cells exposed to oxidant, pretreatment with EGF protected monolayers by 1) increasing PKC-zeta activity; 2) decreasing iNOS activity and protein, NO levels, oxidative stress, tubulin oxidation, and nitration); 3) increasing polymerized tubulin; 4) maintaining the cytoarchitecture of microtubules; and 5) enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected cells overexpressing PKC-zeta (+2.9-fold) were protected as indicated by decreases in all measures of iNOS-driven pathways and enhanced stability of microtubules and barrier function. Overexpression-induced inhibition of iNOS was OAG independent, but EGF potentiated this protection. Antisense inhibition of PKC-zeta (-95%) prevented all measures of EGF protection against iNOS upregulation. Thus EGF protects against oxidative disruption of the intestinal barrier by stabilizing the cytoskeleton in large part through the activation of PKC-zeta and downregulation of iNOS. Activation of PKC-zeta is by itself required for cellular protection against oxidative stress of iNOS. We have thus discovered novel biologic functions, suppression of the iNOS-driven reactions and cytoskeletal oxidation, among the atypical PKC isoforms.     

OPC-compounds prevent oxidant-induced carbonylation and depolymerization of the F-actin cytoskeleton and intestinal barrier hyperpermeability

Rebamipide (OPC-12759), a quinolone derivative, and OPC-6535, a thiazol-carboxylic acid derivative, are compounds with ability to protect gastrointestinal (GI) mucosal integrity against reactive oxygen metabolites (ROM). The underlying mechanism of OPC-mediated protection remains poorly understood. It is now established that ROM can injure the mucosa by disruption of the cytoskeletal network, a key component of mucosal barrier integrity. We, therefore, investigated whether OPC compounds prevent the oxidation, disassembly, and instability of the cytoskeletal protein actin and, in turn, protect intestinal barrier function against ROM. Human intestinal (Caco-2) cell monolayers were pretreated with OPC (-12759 or -6535) prior to incubation with ROM (H2O2) or HOCl). Effects on cell integrity (ethidium homodimer-1), epithelial barrier function (fluorescein sulfonic acid clearance), and actin cytoskeletal integrity (high-resolution laser confocal) were then determined. Cells were also processed for quantitative immunoblotting of G- and F-actin to measure oxidation (carbonylation) and disassembly of actin. In monolayers exposed to ROM, preincubation with OPC compounds prevented actin oxidation, decreased depolymerized G-actin, and enhanced the stable F-actin. Concomitantly, OPC agents abolished both actin cytoskeletal disruption and monolayer barrier dysfunction. Data suggest for the first time that OPC drugs prevent oxidation of actin and lead to the protection of actin cytoskeleton and intestinal barrier integrity against oxidant insult. Accordingly, these compounds may be used as novel therapeutic agents for the treatment of a variety of oxidative inflammatory intestinal disorders with an abnormal mucosal barrier such as inflammatory bowel disease.     

The sequence of a region of bacteriophage phiX174 DNA coding for parts of genes A and B

The kinetics of microtubule assembly were investigated by monitoring changes in turbidity which result from the scattering of incident light by the polymer. These studies indicated that assembly occurred by a pathway involving a nucleation phase, followed by an elongation phase as evidenced by a lag in the polymerization kinetics, followed by a psuedo-first-order exponential increase in turbidity. Analytical ultracentrifugation of solutions polymerized to equilibrium showed that 6 S tubulin was the only species detectable in equilibrium with microtubules. Investigation of the elongation reaction in mixtures of 6 S tubulin and microtubule fragments demonstrated that: (1) the net rate of assembly was the sum of the rates of polymerization and depolymerization; (2) the rate of polymerization was proportional to the product of the microtubule number concentration and the 6 S tubulin concentration; and (3) the rate of depolymerization was proportional to the number concentration of microtubules. These results demonstrate that microtubule assembly occurs by a condensation polymerization mechanism consisting of distinct nucleation and elongation steps. Microtubules are initiated in a series of protein association reactions in a pathway that has not been fully elucidated. Elongation proceeds by the consecutive association of 6 S tubulin subunits onto the ends of existing microtubules. Similarly, depolymerization occurs by dissociation of 6 S subunits from the ends of microtubules. The rate constants measured for polymerization and depolymerization at 30 °C were 4 × 10⁶m^?1 s^?1 and 7 s^?1, respectively.     

Blockade of hypoxia-inducible factor-1α by YC-1 attenuates interferon-γ and tumor necrosis factor-α-induced intestinal epithelial barrier dysfunction

Proinflammatory cytokines play vital roles in intestinal barrier function disruption. YC-1 has been reported to have potent anti-inflammatory properties, and to be a potential agent for sepsis treatment. Here, we investigated the protective effect of YC-1 against intestinal barrier dysfunction caused by interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). To assess the protective effect of YC-1 on intestinal barrier function, Caco-2 monolayers treated with simultaneous IFN-γ and TNF-α were used to measure transepithelial electrical resistance (TER) and paracellular permeability. To determine the mechanisms involved in the protective action of YC-1, expression and distribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers challenged with simultaneous IFN-γ and TNF-α were analyzed by Western blot and immunofluorescence, respectively. Expressions of phosphorylated myosin light chain (MLC), MLC kinase (MLCK) and hypoxia-inducible factor-1α (HIF-1α) were analyzed by Western blot in IFN-γ and TNF-α-treated Caco-2 monolayers. It was found that YC-1 attenuated barrier dysfunction caused by IFN-γ and TNF-α, and also prevented IFN-γ and TNF-α-induced morphological redistribution of tight junction proteins ZO-1 and occludin in Caco-2 monolayers. In addition, YC-1 suppressed IFN-γ and TNF-α-induced upregulation of MLC phosphorylation and MLCK protein expression. Furthermore, enhanced expression of HIF-1α in Caco-2 monolayers treated with IFN-γ and TNF-α was also suppressed by YC-1. It is suggested that YC-1, by downregulating MLCK expression, attenuates intestinal barrier dysfunction induced by IFN-γ and TNF-α, in which HIF-1α inhibition, at least in part, might by involved. YC-1 may be a potential agent for treatment of intestinal barrier disruption in inflammation.     

Quantitative changes in microtubule distribution correlate with guard cell function in Arabidopsis

Radially arranged cortical microtubules are a prominent feature of guard cells. We observed guard cells expressing GFP-tubulin (GFP-TUA6) with confocal microscopy and found recognizable changes in the appearance of microtubules when stomata open or close (Eisinger et al., 2012). In the present study, analysis of fluorescence distribution showed a dramatic increase in peak intensities of microtubule bundles within guard cells as stomata open. This increase was correlated with an increase in the total fluorescence that could be attributed to polymerized tubulin. Adjacent pavement cells did not show similar changes in peak intensities or integrated fluorescence when stomatal apertures changed. Imaging of RFP-tagged end binding protein 1 (EB1) and YFP-tagged α-tubulin expressed in the same cell revealed that the number of microtubules with growing ends remained constant, although the total amount of polymerized tubulin was higher in open than in closed guard cells. Taken together, these results indicate that the changes in microtubule array organization that are correlated with and required for normal guard cell function are characterized by changes in microtubule clustering or bundling.     

Biochemical and electron microscopy analysis of the endotoxin binding to microtubulesin vitro

The mechanisms involved in cellular activation and damage by bacterial endotoxins are not completely defined. In particular, there is little information about possible intracellular targets of endotoxins. Recently, the participation of a microtubule associated protein in endotoxin actions on macrophages has been suggested. In the present work, we have studied the effect ofE. coli lipopolysaccharide on the polymerization of microtubular proteinin vitro. Electrophoretic analysis of the polymerization mixtures showed that the endotoxin inhibited the polymerization when present at high concentrations. At lower concentrations, LPS selectively displaced the microtubule associated protein MAP-2 from the polymerized microtubules. Electron microscopy showed that LPS binds to microtubules of tubulin+MAPs and to microtubules of purified tubulin (without MAPs) polymerized with taxol. Gel filtration experiments confirmed the binding of LPS to tubulin, and by ligand blot assays an interaction LPS — MAP-2 was detected. The ability of LPS to interact with microtubular proteins suggests a possible participation of microtubules on the cellular effects of endotoxins.     

Over-expression of betaI tubulin in MDCK cells and incorporation of exogenous betaI tubulin into microtubules interferes with adhesion and spreading.

Little is known about the presence and distribution of tubulin isotypes in MDCK cells although essential epithelial functions in these monolayers are regulated by dynamic changes in the microtubule architecture. Using specific antibodies, we show here that the betaI, betaII, and betaIV isotypes are differentially distributed in the microtubules of these cells. Microtubules in subconfluent cells radiating from the perinuclear region contain betaI and betaII tubulins, while those extending to the cell edges are enriched in betaII. Confluent cells contain similar proportions of betaI and betaII along the entire microtubule length. betaIV is the less abundant isotype and shows a similar distribution to betaII. The effect of modifying tubulin isotype ratios in the microtubules that could affect their dynamics and function was analyzed by stably expressing in MDCK cells betaI tubulin from CHO cells. Three recombinant clones expressing different levels of the exogenous betaI tubulin were selected and subcloned. Clone 17-2 showed the highest expression of CHO beta1 tubulin. Total betaI tubulin levels (MDCK+CHO) in the clones were approximately 1.8 to 1.1-fold higher than in mock-transfected cells only expressing MDCK beta1 tubulin. In all the cells, betaII tubulin levels remained unchanged. The cells expressing CHO beta1 tubulin showed defective attachment, spreading, and delayed formation of adhesion sites at short times after plating, whereas mock-transfected cells attached and spread normally. Analysis of cytoskeletal fractions from clone 17-2 showed a MDCK betaI/CHO betaI ratio of 1.89 at 2 h that gradually decreased to 1.0 by 24 h. The ratio of the two isotypes in the soluble fraction remained unchanged, although with higher values than those found for the polymerized betaI tubulin. By 24 h, the transfected cells had regained normal spreading and formed a confluent monolayer. Our results show that excess levels of total betaI tubulin, resulting from the expression of the exogenous beta1 isotype, and incorporation of it into microtubules affect their stability and some cellular functions. As the levels return to normal, the cells recover their normal phenotype. Regulation of betaI tubulin levels implies the release of the MDCK betaI isotype from the microtubules into the soluble fraction where it would be degraded.     

Enhancement of tubulin assembly as monitored by a rapid filtration assay

The early kinetics of microtubule formation from lamb brain tubulin isolated by affinity chromatography can be followed by a newly developed filter assay. The rapid collection of microtubules on glass fiber filters permits the calculation of the moles of tubulin polymerized. The filter assay gives both a rate and extent of polymerization that are identical to those obtained by turbidity or sedimentation analysis, respectively. The microtubules trapped by the filter are readily depolymerized by cold (t12= 3 min) and slowly by colchicine (t1/2= 32min). Tubulin purified by affinity chromatography requires a high protein concentration (>4 mg/ml) for polymerization. Although 5m glycerol allows polymerization to occur at tubulin concentrations below 2 mg/ml, the maximum amount of microtubule formation is observed at low tubulin concentration when microtubule-associated proteins are present. These proteins are not retained by the affinity resin; however, they can be eluted from diethylaminoethyl-Sephadex by solutions containing 0.3m KCl. Microtubule-associated proteins enhance both the rate of polymerization and the total amount of tubulin polymerized as assessed by the filter assay, suggesting that they are involved in both initiation and elongation of microtubules.     

Key role of PKC and Ca2+ in EGF protection of microtubules and intestinal barrier against oxidants

Using monolayers of human intestinal (Caco-2) cells, we showed that growth factors (GFs) protect microtubules and barrier integrity against oxidative injury. Studies in nongastrointestinal cell models suggest that protein kinase C (PKC) signaling is key in GF-induced effects and that cytosolic calcium concentration ([Ca2+](i)) is essential in cell integrity. We hypothesized that GF protection involves activating PKC and maintaining normal ([Ca2+](i)) Monolayers were pretreated with epidermal growth factor (EGF) or PKC or Ca2+ modulators before exposure to oxidants (H2O2 or HOCl). Oxidants disrupted microtubules and barrier integrity, and EGF protected from this damage. EGF caused rapid distribution of PKC-alpha, PKC-betaI, and PKC-zeta isoforms to cell membranes, enhancing PKC activity of membrane fractions while reducing PKC activity of cytosolic fractions. EGF enhanced (45)Ca2+ efflux and prevented oxidant-induced (sustained) rises in ([Ca2+](i)). PKC inhibitors abolished and PKC activators mimicked EGF protection. Oxidant damage was mimicked by and potentiated by a Ca2+ ionophore (A-23187), exacerbated by high-Ca2+ media, and prevented by calcium removal or chelation or by Ca2+ channel antagonists. PKC activators mimicked EGF on both (45)Ca2+ efflux and ([Ca2+](i)). Membrane Ca2+-ATPase pump inhibitors prevented protection by EGF or PKC activators. In conclusion, EGF protection of microtubules and the intestinal epithelial barrier requires activation of PKC signal transduction and normalization of ([Ca2+](i)).     

Postpolymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules

Tyrosinated (Tyr) and detyrosinated (Glu) alpha-tubulin, species interconverted by posttranslational modification, are largely segregated in separate populations of microtubules in interphase cultured cells. We sought to understand how distinct Tyr and Glu microtubules are generated in vivo, by examining time-dependent alterations in Tyr and Glu tubulin levels (by immunoblots probed with antibodies specific for each species) and distributions (by immunofluorescence) after microtubule regrowth and stabilization. When microtubules were allowed to regrow after complete depolymerization by microtubule antagonists, Glu microtubules reappeared with a delay of approximately 25 min after the complete array of Tyr microtubules had regrown. In these experiments, Tyr tubulin immunofluorescence first appeared as an aster of distinct microtubules, while Glu tubulin staining first appeared as a grainy pattern that was not altered by detergent extraction, suggesting that Glu microtubules were created by detyrosination of Tyr microtubules. Treatments with taxol, azide, or vinblastine, to stabilize polymeric tubulin, all resulted in time-dependent increases in polymeric Glu tubulin levels, further supporting the hypothesis of postpolymerization detyrosination. Analysis of monomer and polymer fractions during microtubule regrowth and in microtubule stabilization experiments were also consistent with postpolymerization detyrosination; in each case, Glu polymer levels increased in the absence of detectable Glu monomer. The low level of Glu monomer in untreated or nocodazole-treated cells (we estimate that Glu tubulin comprises less than 2% of the monomer pool) also suggested that Glu tubulin entering the monomer pool is efficiently retyrosinated. Taken together these results demonstrate that microtubules are polymerized from Tyr tubulin and are then rapidly converted to Glu microtubules. When Glu microtubules depolymerize, the resulting Glu monomer is retyrosinated. This cycle generates structurally, and perhaps functionally, distinct microtubules.     

Flexural rigidity of individual microtubules measured by a buckling force with optical traps

We used direct buckling force measurements with optical traps to determine the flexural rigidity of individual microtubules bound to polystyrene beads. To optimize the accuracy of the measurement, we used two optical traps and antibody-coated beads to manipulate each microtubule. We then applied a new analytical model assuming nonaxial buckling. Paclitaxel-stabilized microtubules were polymerized from purified tubulin, and the average microtubule rigidity was calculated as 2.0 x 10(-24) Nm2 using this novel microtubule buckling system. This value was not dependent on microtubule length. We also measured the rigidity of paclitaxel-free microtubules, and obtained the value of 7.9 x 10(-24) Nm2, which is nearly four times that measured for paclitaxel-stabilized microtubules.     

Enteropathogenic E. coli effectors EspG1/G2 disrupt microtubules,contribute to tight junction perturbation and inhibit restoration

Enteropathogenic Escherichia coli (EPEC) uses a type 3 secretion system to transfer effector proteins into the host intestinal epithelial cell. Several effector molecules contribute to tight junction disruption including EspG1 and its homologue EspG2 via a mechanism thought to involve microtubule destruction. The aim of this study was to investigate the contribution of EspG‐mediated microtubule disruption to TJ perturbation. We demonstrate that wild type EPEC infection disassembles microtubules and induces the progressive movement of occludin away from the membrane and into the cytosol. Deletion of espG1/G2 attenuates both of these phenotypes. In addition, EPEC infection impedes barrier recovery from calcium switch, suggesting that inhibition of TJ restoration, not merely disruption, prolongs barrier loss. TJs recover more rapidly following infection with ΔespG1/G2 than with wild type EPEC, demonstrating that EspG1/G2 perpetuate barrier loss. Although EspG regulates ADP‐ribosylation factor (ARF) and p21‐activated kinase (PAK), these activities are not necessary for microtubule destruction or perturbation of TJ structure and function. These data strongly support a role for EspG1/G2 and its associated effects on microtubules in delaying the recovery of damaged tight junctions caused by EPEC infection.     

Bull sperm 19S dynein polymerizes brain tubulin into microtubules

Crude dynein extracted from bull sperm flagella polymerized pure phosphocellulose tubulin isolated from brain tissues into microtubules. This effect was predominantly due to the 19S dynein particle in the extract. ATP stimulated up to five fold the polymerization of brain tubulin by bull sperm dynein. Hydrolysis of ATP was not required since vanadate at a concentration sufficient to block dynein ATPase activity did not interfere with ATP stimulation and because the non hydrolyzable ATP analogue adenylyl (beta-gamma-methylene) diphosphate (AMPPCP) had effects similar to those of ATP. These results suggest that, in addition to hydrolyzing ATP to generate the driving force necessary for microtubule sliding within the axoneme, dynein may also interact with ATP to polymerize tubulin into microtubules.     

Theta-isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-theta

Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the -isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC- is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC- (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC- showed monolayer injury as indicated by decreased native PKC- activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC- was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC- was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-/tubulin complex. Second, DN transfection to inhibit the endogenous PKC- led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC- led to a mostly cytosolic distribution of -isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1) PKC- isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2) the molecular event underlying this novel biological effect of PKC- involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-. microtubules; tubulin; occludin; epithelial barrier permeability; protein kinase C isoform     

Effect of quinones on microtubule polymerization: a link between oxidative stress and cytoskeletal alterations in Alzheimer's disease

Increases in the concentration of quinones, such as benzoquinone, in pathological processes mediated by oxidative imbalance play a role in the disorganization and disassembly of the microtubule network in both non-neural and neural cells. In this study, we show that the effects on microtubules appear to be a direct result of the action of the quinones on tubulin, the main component of microtubules, since tubulin modification by quinones, including benzoquinone and juglone, leads to aggregation into dimers and other oligomers. Therefore, quinones and quinone-mediated effects provide a mechanistic link between oxidative stress, microtubule disruption, neuronal dysfunction and death, i.e., key salient feature of Alzheimer's disease.     

An ATPase activity associated with brain microtubules.

A persistent ATPase/GTPase activity has been found to be associated with highly recycled bovine brain microtubules. A GTP regeneration system was introduced to minimize the inhibitory effects of this hydrolase on microtubule polymerization. The characteristics of the ATPase indicate that it is not involved in GTP-induced mictrotubule polymerization, but is directly involved in ATP-induced polymerization. ATP-induced polymerization was also shown to require stoichiometric amounts of GDP, but higher levels of GDP inhibited both microtubule formation and the ATPase activity. An ammonium sulfate fractionation procedure was devised to separate microtubule protein into an ATPase-rich fraction and a pure tubulin fraction. The pure tubulin fraction polymerized in the presence of GTP, but not in the presence of ATP and GDP. In contrast, the ATPase-rich fraction polymerized with either ATP or GTP. It is still not known whether the microtubule associated ATPase plays a significant role in cellular microtubule function.     

PKC-beta1 mediates EGF protection of microtubules and barrier of intestinal monolayers against oxidants

Using monolayers of human intestinal (Caco-2) cells, we found that oxidants and ethanol damage the cytoskeleton and disrupt barrier integrity; epidermal growth factor (EGF) prevents damage by enhancement of protein kinase C (PKC) activity and translocation of the PKC-beta1 isoform. To see if PKC-beta1 mediates EGF protection, cells were transfected to stably over- or underexpress PKC-beta1. Transfected monolayers were preincubated with low or high doses of EGF (1 or 10 ng/ml) or 1-oleoyl-2-acetyl-sn-glycerol [OAG; a PKC activator (0.01 or 50 microM)] before treatment with oxidant (0.5 mM H(2)O(2)). Only in monolayers overexpressing PKC-beta1 (3.1-fold) did low doses of EGF or OAG initiate protection, increase tubulin polymerization (assessed by quantitative immunoblotting) and microtubule architectural integrity (laser scanning confocal microscopy), maintain normal barrier permeability (fluorescein sulfonic acid clearance), and cause redistribution of PKC-beta1 from cytosolic pools into membrane and/or cytoskeletal fractions (assessed by immunoblotting), thus indicating PKC-beta1 activation. Antisense inhibition of PKC-beta1 expression (-90%) prevented these changes and abolished EGF protection. We conclude that EGF protection against oxidants requires PKC-beta1 isoform activation. This mechanism may be useful for development of novel therapies for the treatment of inflammatory gastrointestinal disorders including inflammatory bowel disease.     

The mechanism of anaphase spindle elongation: uncoupling of tubulin incorporation and microtubule sliding during in vitro spindle reactivation

To study tubulin polymerization and microtubule sliding during spindle elongation in vitro, we developed a method of uncoupling the two processes. When isolated diatom spindles were incubated with biotinylated tubulin (biot-tb) without ATP, biot-tb was incorporated into two regions flanking the zone of microtubule overlap, but the spindles did not elongate. After biot-tb was removed, spindle elongation was initiated by addition of ATP. The incorporated biot-tb was found in the midzone between the original half-spindles. The extent and rate of elongation were increased by preincubation in biot-tb. Serial section reconstruction of spindles elongating in tubulin and ATP showed that the average length of half-spindle microtubules increased due to growth of microtubules from the ends of native microtubules. The characteristic packing pattern between antiparallel microtubules was retained even in the "new" overlap region. Our results suggest that the forces required for spindle elongation are generated by enzymes in the overlap zone that mediate the sliding apart of antiparallel microtubules, and that tubulin polymerization does not contribute to force generation. Changes in the extent of microtubule overlap during spindle elongation were affected by tubulin and ATP concentration in the incubation medium. Spindles continued to elongate even after the overlap zone was composed entirely of newly polymerized microtubules, suggesting that the enzyme responsible for microtubule translocation either is bound to a matrix in the spindle midzone, or else can move on one microtubule toward the spindle midzone and push another microtubule of opposite polarity toward the pole.     

Taxol binds to polymerized tubulin in vitro

Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5’-guanylyl-α,β-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.