Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium

Summary— Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the α N-terminal domain, one in the β C-terminal one, and two in α and β central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of α-tubulin whereas the epitopes of three other ones (TAP 952°, AXO 58 and AXO 49°) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both α- and β-tubulins. The AXO 49° specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.     

Human serum reacting specifically with a subset of beta-tubulin isoforms

The serum of a patient suffering from myeloma was found to decorate microtubules and mitotic spindles of cultured cells. Immunoblots performed after one- and two-dimensional electrophoresis showed a reaction with a certain subset of beta-tubulin isoforms, but not with beta'- and alpha-tubulins. The tubulin subset contained both ubiquitous (beta-3) and neurospecific (beta-4,5,6) isoforms. An IgM lambda and an IgA kappa myeloma protein were found in this serum. Immunoblots performed with specific anti-isotype second antibodies showed that the tubulin subset could be evidenced using anti-mu, alpha, lambda, and kappa-specific antisera. Moreover, the tubulin subset was also evidenced using an anti-gamma second antibody. These results, which do not exclude a participation of the myeloma proteins in the anti-tubulin reactivity, indicate, however, that the antibody response was polyclonal. The same restricted specificity of all classes of anti-tubulin antibodies of this serum favours the hypothesis that the immune response of the patient was directed against an antigen sharing epitopes with tubulin rather than with tubulin itself.     

Microtubular Systems of Paramecium in Division: Pattern of Cytospindle Assembly

The pattern of cytospindle assembly and the modifications of the microtubular cytoplasmic network during division of Paramecium are studied by means of indirect immunofluorescence. The assembly of cytospindle starts at two independent areas placed respectively around the proter's and opisthe's buccal overture. The moment of the microtubule bundles’appearance depends on their distance from the buccal opening, with those closest appearing 1st. The existence of microtubule organizing centers that act transiently during division of Paramecium is discussed.     

Interaction of captan with mammalian microtubules

Using turbidometry, electron microscopy and immunofluorescent microscopy experiments we studied the effect of captan, a widely used pesticide on mammalian microtubules and microfilaments. Turbidometry at 350 nm showed a dose-dependent inhibition of tubulin assembly incubated with captan. The pesticide, given at equimolar concentration with tubulin (30 microM), caused the total inhibition of microtubule formation, while at lower concentrations (5-20 microM) the inhibition of tubulin polymerization was less extensive. At the same concentration range (5-30 microM), captan also promoted the disassembly of performed microtubules. The results of the in vitro effects of captan with microtubules were confirmed in parallel by electron microscopic studies. In vivo, captan caused also depolymerization of microtubules in cultured mouse fibroblasts as shown by indirect immunofluorescent staining of tubulin. The extent of microtubules disassembly was concentration- and time-dependent. While incubation of the cells with 10 microM captan for 3 h disturbs totally the microtubular structures, incubation with 5 microM captan needs 12 h for the same effect. Recovery of microtubules was observed, when preincubated cells were extensively washed. No interaction of this drug with equimolar concentration of G- or F-actin could be observed in vitro, as shown by polymerization experiments. In line with this, the fluorescent actin pattern in mouse fibroblasts incubated with 10 mM captan for up to 12 h did not seem to be altered. From these results it is concluded that captan interacts in equimolar concentrations with tubulin affecting the assembly and disassembly of microtubules in vitro and in cultures of mammalian cells.     

Effect of phalloidin on liver actin distribution,content, and turnover

Phalloidin increases F-actin microfilament content and actin-directed immunofluorescence in hepatocytes in vivo and also increases actin polymerization and the stability of F-actin in vitro. We studied the sensitivity of immunofluorescent staining of actin to an actin depolymerizing factor (ADF) as well as actin content, degree of polymerization, and turnover in livers of in vivo phalloidin-treated rats. Pretreatment with ADF abolished anti-actin antibody (AAA) staining of normal liver but did not modify staining of livers from phalloidin-treated animals. Plani-metric analyses of SDS-polyacrylamide gels snowed the percent actin of total protein was increased by approximately 40% and the absolute amount of actin by approximately 43%, ten days after daily phalloidin treatment (50 μg/100 gm body weight). Similar but smaller changes could be seen after one day of treatment. Ultracentrifugational analyses of liver extracts indicated no change in the amount or proportion of G-actin but a 194% increase in the proportion of F-actin in ten-day treated animals, changes also apparent in one day animals. Neither the relative fractional rate of actin synthesis nor its synthesis as a percent of total protein synthesis was altered either at one-day or ten-day post-phalloidin treatment. Dual-isotope experiments indicated that the rate of actin degradation was decreased selectively in the one- to three-day period -following drug treatment. Thus, phalloidin appears to stabilize actin against the depolymerizing actions of ADF, increases the proportion of F-actin without altering the size of the G-actin pool, and causes accumulation of actin by decreasing its relative rate of degradation.     

JVG9, a benzimidazole derivative,alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes

Trypanosoma cruzi has a particular cytoskeleton that consists of asubpellicular network of microtubules and actin microfilaments. Therefore, it is anexcellent target for the development of new anti-parasitic drugs. Benzimidazole2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown toinhibit the in vitro growth of many protozoa. Therefore, to find efficientanti-trypanosomal (trypanocidal) drugs, our group has designed and synthesisedseveral benzimidazole derivatives. One, named JVG9(5-chloro-1H-benzimidazole-2-thiol), has been found to be effectiveagainst T. cruzi bloodstream trypomastigotes under both in vitroand in vivo conditions. Here, we present the in vitro effects observed by laserscanning confocal and scanning electron microscopy on T. cruzitrypomastigotes. Changes in the surface and the distribution of thecytoskeletal proteins are consistent with the hypothesis that the trypanocidalactivity of JVG9 involves the cytoskeleton as a target.     

Crystal structure of the CCTgamma apical domain: implications for substrate binding to the eukaryotic cytosolic chaperonin

The chaperonin containing TCP-1 (CCT, also known as TRiC) is the only member of the chaperonin family found in the cytosol of eukaryotes. Like other chaperonins, it assists the folding of newly synthesised proteins. It is, however, unique in its specificity towards only a small subset of non-native proteins. We determined two crystal structures of mouse CCTgamma apical domain at 2.2 A and 2.8 A resolution. They reveal a surface patch facing the inside of the torus that is highly evolutionarily conserved and specific for the CCTgamma apical domain. This putative substrate-binding region consists of predominantly positively charged side-chains. It suggests that the specificity of this apical domain towards its substrate, partially folded tubulin, is conferred by polar and electrostatic interactions. The site and nature of substrate interaction are thus profoundly different between CCT and its eubacterial homologue GroEL, consistent with their different functions in general versus specific protein folding assistance.     

Isolation and characterization of cytoskeletons from cotton fiber cytoplasts

Summary Over the last 25 yr, success in characterizing the individual protein components of animal cytoskeletons was possible, in part, due to technical advances in the isolation and purification of anucleate cytoskeletons from animal cells. As a step towards characterizing protein components of the plant cytoskeleton, we have isolated cytoskeletons from cytoplasts (anucleate protoplasts) prepared from cotton fiber cells grown in ovule culture. Cytoplasts isolated into a hypertonic, Ca²⁺-free medium at pH 6.8 retained internal structures after extraction with the detergent, Triton X-100. These structures were shown to include microtubule and microfilament arrays by immunofluorescence and electron microscopy. Actin and tubulin were the only abundant proteins in these preparations, suggesting that microfilaments and microtubules were the major cytoskeleta elements in the isolated cytoskeletons. The absence of additional, relatively abundant proteins suggests that (a) other cytoskeletal arrays potentially present in fiber cells (e.g., intermediate filaments) were either lost during detergent extraction or were minor components of the fiber cell cytoskeleton; and (b) high ratios of individual cytoskeletal-associated proteins relative to actin and tubulin were not required to maintain microtubules and microfilaments in organized structures.     

The regional association of actin and myosin with sites of particle phagocytosis

Contractile proteins are thought to play a causative role in motile processes such as phagocytosis. In order to investigate their role in phagocytosis further, simultaneous immunofluorescence localization of F-actin and myosin was carried out in resident mouse peritoneal macrophages after phagocytosis of opsonized zymosan particles. Both actin and myosin appeared to concentrate rapidly at sites of particle phagocytosis. The observed concentration of both proteins at such sites preceded ultimate particle engulfment. Cytochalasin B, a drug which was shown to block pseudopod extensions around the particle, did not prevent the concentration of the two contractile proteins at cell-particle binding sites. This result ruled out path-length effects as an explanation for the observed concentration of actin and myosin at phagocytic sites. Kinetic analysis showed that actin rapidly concentrates at particle-cell binding sites within minutes (or less) of contact with cell surface. The two proteins are present throughout the engulfment phase until and after ingestion is complete. Finally, at later times the particles become clustered over the cell nucleus and the particle-associated actin-myosin seen earlier is no longer evident.     

Paraquat induces irreversible actin cytoskeleton disruption in cultured human lung cells

The herbicide paraquat (PQ) induces the selective necrosis of type I and type II alveolar pneumocytes. We investigated the effect of PQ on human lung A549 cells to determine the possible role of cytoskeleton in lung cytotoxicity. At 80 mol/L PQ, a concentration that did not affect cell viability, the organization of actin cytoskeleton network depended on incubation time with the herbicide. Microfilaments appeared less numerous in 30% of the cells treated for 1 h. After 24 h, all the treated cells displayed only short filaments in the periphery. The effect of PQ on actin cytoskeleton was irreversible. Moreover, no modification of microtubule network was observed in PQ-treated cells. Next, we studied the effect of PQ on Chang Liver, an epithelial cell line from human liver. These cells appeared less sensitive to the herbicide than A549, and no cytoskeletal alteration was observed. To verify whether actin filament modifications in A549 cells were related to intracellular alterations of ATP concentrations, nucleotide levels during incubation with PQ were determined. The intracellular levels of ATP were not different in control and treated cells. Our results indicate that PQ induces specifically an irreversible actin filament disorganization on A549 cells and that the observed effect is independent of intracellular concentration of ATP.Abbreviations BSA bovine serum albumin - IC₅₀ concentration that produces 50% inhibitiition - PBS phosphate-buffered saline - PQ paraquat, 1,1-dimethyl-4,4-bipyridinium dichloride - SE standard error of the mean     

A novel agent, methylophiopogonanone B, promotes Rho activation and tubulin depolymerization

Cytoskeletal reorganization, including reconstruction of actin fibers and microtubules, is essential for various biological processes, such as cell migration, proliferation and dendrite formation. We show here that methylophiopogonanone B (MOPB) induces cell morphological change via melanocyte dendrite retraction and stress fiber formation. Since members of the Rho family of small GTP-binding proteins act as master regulators of dendrite formation and actin cytoskeletal reorganization, and activated Rho promotes dendrite retraction and stress fiber formation, we studied the effects of MOPB on the small GTPases using normal human epidermal melanocytes and HeLa cells. In in vitro binding assay, MOPB significantly increased GTP-Rho, but not GTP-Rac or GTP-CDC42. Furthermore, a Rho inhibitor, a Rho kinase inhibitor and a small GTPase inhibitor each blocked MOPB-induced stress fiber formation. The effect of MOPB on actin reorganization was blocked in a Rho dominant negative mutant. These results suggest MOPB acts via the Rho signaling pathway, and it may directly or indirectly activate Rho. Quantitative Western blot analysis indicated that MOPB also induced microtubule destabilization and tubulin depolymerization. Thus, MOPB appears to induce Rho activation, resulting in actin cytoskeletal reorganization, including dendrite retraction and stress fiber formation.     

蝶蛹金小蜂寄生对菜粉蝶血细胞骨架相关蛋白基因的转录调控

为揭示蝶蛹金小蜂Pteromalus puparum抑制寄主菜粉蝶Pieris rapae血细胞免疫反应的分子机制, 克隆了菜粉蝶肌动蛋白、肌动蛋白解聚因子及微管蛋白cDNA, 并研究了蝶蛹金小蜂寄生对其转录水平的影响。结果显示： 菜粉蝶肌动蛋白cDNA ORF为1 131 bp, 编码377 aa, 预测分子量为41.78 kDa, pI为5.29, 与其他昆虫肌动蛋白的相似性非常高, 在90%以上。氨基酸组成特点和系统进化分析表明, 克隆到的菜粉蝶肌动蛋白基因属细胞质型肌动蛋白。菜粉蝶肌动蛋白解聚因子cDNA全长为1 243 bp, ORF为 447 bp, 编码149 aa, 预测分子量为16.97 kDa, pI为7.11,与家蚕Bombyx mori、赤拟谷盗Tribolium castaneum、意大利蜜蜂Apis mellifera和桑粉介壳虫Maconellicoccus hirsutus肌动蛋白解聚因子的相似性分别为97%, 87%, 89%和72%。菜粉蝶微管蛋白cDNA全长为1 757 bp, ORF为1 344 bp, 编码448 aa, 预测分子量为50.38 kDa, pI为4.86, 与家蚕β型微管蛋白1, 2, 3和4的相似性分别为97%, 97%, 87%和93%。系统进化分析表明, 克隆到的菜粉蝶微管蛋白基因属β型微管蛋白。RT-PCR分析表明, 蝶蛹金小蜂寄生能抑制肌动蛋白、肌动蛋白解聚因子及微管蛋白基因在菜粉蝶蛹血细胞中的转录水平。由此推断蝶蛹金小蜂调控寄主血细胞骨架相关蛋白基因的转录是该蜂抑制寄主血细胞免疫反应的分子机制之一。     

Tubulin and Neurofilament Proteins Are Transported Differently in Axons of Chicken Motoneurons

SUMMARY1. We previously showed that actin is transported in an unassembled form with its associated proteins actin depolymerizing factor, cofilin, and profilin. Here we examine the specific activities of radioactively labeled tubulin and neurofilament proteins in subcellular fractions of the chicken sciatic nerve following injection of L-[³⁵S]methionine into the lumbar spinal cord.2. At intervals of 12 and 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Analysis of the fractions by high-resolution two-dimensional gel electrophoresis, immunoblotting, fluorography, and computer densitometry showed that tubulin was transported as a unimodal wave at a slower average rate (2–2.5 mm/day) than actin (4–5 mm/day). Moreover, the specific activity of soluble tubulin was five times that of its particulate form, indicating that tubulin is transported in a dimeric or small oligomeric form and is assembled into stationary microtubules.3. Neurofilament triplet proteins were detected only in the particulate fractions and transported at a slower average rate (1 mm/day) than either actin or tubulin.4. Our results indicate that the tubulin was transported in an unpolymerized form and that the neurofilament proteins were transported in an insoluble, presumably polymerized form.     

Organization and slow axonal transport of cytoskeletal proteins under normal and regenerating conditions

The organization of the axonal cytoskeleton was investigated by analyzing the solubility and transport profile of the major cytoskeletal proteins in motor axons of the rat sciatic nerve under normal and regenerating conditions. When extracted with the Triton-containing buffer at low temperature, 50% of tubulin and 30% of actin were recovered in the insoluble form resistant to further depolymerizing treatments. Most of this cold-insoluble form was transported in slow component a (SCa), the slower of the two subcomponents of slow axonal transport, whereas the cold-soluble form showed a biphasic distribution between SCa and SCb (slow component b). Changes in slow transport during regeneration were studied by injuring the nerve either prior to (experiment I) or after (experiment II) radioactive labeling. In experiment I where the transport of proteins synthesized in response to injury was examined, selective acceleration of SCb was detected together with an increase in the relative proportion of this component. In experiment II where the response of the preexisting cytoskeleton was examined, a shift from SCa to SCb of the cold-soluble form was observed. The differential distribution and response of the two forms of tubulin and actin suggest that the cold-soluble form may be more directly involved in axonal transport.