Detection of the microtubule cytoskeleton of the coccidian Toxoplasma gondii and the hemoflagellate Leishmania donovani by monoclonal antibodies specific for beta-tubulin

Seven monoclonal antibodies specific for mammalian beta-tubulin demonstrate the microtubule cytoskeleton of Toxoplasma gondii and Leishmania donovani by indirect immunofluorescence microscopy. Immunoblots of T. gondii and L. donovani proteins separated by SDS polyacrylamide gel electrophoresis confirm the specificity of the monoclonal antibodies for tubulin. Differential staining of flagellar and subpellicular microtubule populations was not seen in L. donovani with these antibodies. All seven antibodies also detected the subpellicular microtubules of T. gondii, but the polar ring and conoid of this organism was not visualized by any of them. This technique provides a rapid and specific way to assess microtubular organization in whole organisms.     

TgICMAP1 Is a Novel Microtubule Binding Protein in Toxoplasma gondii

The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction.     

Overexpression,purification, and functional analysis of recombinant human tubulin dimer

Microtubules consisting of tubulin dimers play essential roles in various cellular functions. Investigating the structure–function relationship of tubulin dimers requires a method to prepare sufficient quantities of recombinant tubulin. To this end, we simultaneously expressed human α1- and β3-tubulin using a baculovirus-insect cell expression system that enabled the purification of 5 mg recombinant tubulin per litre of cell culture. The purified recombinant human tubulin could be polymerized into microtubules that glide on a kinesin-coated glass surface. The method provides a powerful tool for in vitro functional analyses of microtubules.     

Ultrastructure of Sarcocystis tenella. I. Endozo?te (after negative staining]

The employment of negative staining technics for the endozoites (cyst stages) of Sarcocystis tenella allowed the elucidation of certain aspects of their fine structure. The conoid consists of similar to 20 oblique fibers and is surmounted by a ring with regular ornamentation. In the conoid's interior there are 2 excentric parallel microtubules which extend posteriorly for a considerable distance into the adjacent cytoplasm. The fibers of the conoid, intraconoid microtubules, appear to have the same diameter and structure as the 22 subpellicular microtubules. They are "cemented" anteriorly into a periconoidal ring which surrounds the conoid. The "reticulated" pellicle has certain differentiations: the micropore, surrounded by a "fibrillar" element, similar to 10 subcircular structures arranged into an anterior crown, and 11 rows of granules converging toward the posterior end. The sarconemes look like rice grains which, contrary to previous statements, are independent of one another. It is established that there are only 2 rhoptries.     

Cytoskeleton of Toxoplasma gondii

The cytoskeleton of Toxoplasma gondii was studied by electron microscopy using whole mounts of detergent-extracted parasites and thin sections of routine preparations, tannic acid-stained organisms, and detergent-extracted parasites. In whole mounts, the spiral arrangement of the 22 pellicular microtubules closely corresponded to the pattern of surface ridges seen previously by scanning electron microscopy and reflected the torsion of the parasite body during locomotion. The microtubules had free posterior ends and were anchored anteriorly in the polar ring, presumed to be a microtubule organizing center (MTOC). The insertions of the microtubules were supported by blunt projections of the polar ring, forming a cogwheel pattern in transverse view. The internal microtubules had 13 protofilaments and were twice the length of the conoid. They extended through the conoid and ended at the anterior preconoidal ring, presumably a second MTOC. The subunits of the conoid were arranged in a counterclockwise spiral when traced from base to tip, as were the pellicular microtubules. We postulate that as the conoid moves, the polar ring complex moves along the spiral pathway of the conoid subunits. Retraction of the conoid would then rotate the polar ring, producing the torsion of the body we observed by SEM.     

A Comparative Electron Microscopic Study of the Morphology of Toxoplasma gondii by Freeze-Etch Replication and Thin Sectioning Technic

SYNOPSIS. Freeze-etch preparations of Toxoplasma gondii reveal details of structure and organelles in 3-dimensional relationships. The subpellicular microtubules and their relationship to the polar ring, the tripartite pellicle, the pellicle constituents, and the spatial relationship of the rhoptries to the conoid and conoid canal are clearly demarcated.     

Influence of calcium ion on host cell invasion and intracellular replication by Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite, which invades a wide range of hosts including humans. The exact mechanisms involved in its invasion are not fully understood. This study focused on the roles of Ca2+ in host cell invasion and in T. gondii replication. We examined the invasion and replication of T. gondii pretreated with several calcium modulators, the conoid extrusion of tachyzoites. Calmodulin localization in T. gondii were observed using the immunogold method, and Ca2+ levels in tachyzoites by confocal microscopy. In light microscopic observation, tachyzoites co-treated with A23187 and EGTA showed that host cell invasion and intracellular replication were decreased. The invasion of tachyzoites was slightly inhibited by the Ca2+ channel blockers, bepridil and verapamil, and by the calmodulin antagonist, calmidazolium. We observed that calcium saline containing A23187 induced the extrusion of tachyzoite conoid. By immunoelectron microscopy, gold particles bound to anti-calmodulin or anti-actin mAb, were found to be localized on the anterior portion of tachyzoites. Remarkably reduced intracellular Ca2+ was observed in tachyzoites treated with BAPTA/AM by confocal microscopy. These results suggest that host cell invasion and the intracellular replication of T. gondii tachyzoites are inhibited by the calcium ionophore, A23187, and by the extracellular calcium chelator, EGTA.     

Sites of Tubulin Polymerization in PC 12 Cells

The site at which tubulin enters into polymer in the neuritic process is a very important datum in terms of our understanding of the mechanism of transport of the microtubular cytoskeleton out the axon. If the form of tubulin being transported out the axon is the microtubule, then assembly of tubulin into microtubules should occur at or near the cell body; if, however, the form of tubulin transported is free tubulin dimer, then assembly can occur at any free microtubule end out the neurite. We have injected a fluorescent analog of tubulin into differentiated PC 12 cells and used differential extraction protocols to extract free dimer but not microtubules. We have imaged these cells before and after extraction by low-light-level video fluorescence microscopy and have used image analysis to examine the sites of tubulin incorporation into polymer or other unextracted components as a function of time. We find that tubulin in the distal reaches of the neurite is found initially as monomer and that its appearance in the unextracted component occurs later. This pattern of appearance of fluorescent tubulin initially in the soluble fraction and later in the unextractable component is qualitatively similar to that reported by other workers for biotinylated tubulin, but we see a larger gap between the rates of appearance in soluble fraction and in polymer. Quantitative analysis of fluorescence intensities in the two compartments with distance out the neurite reveals substantial variation between different neurites: In some neurites, the pattern of variation of unextracted/total tubulin suggests that tubulin enters into the unextracted component primarily near the cell body and that this unextracted component moves out the neurite with time, and in other neurites it suggest that monomer adds into microtubule ends staggered out the neurite. In no case do we see a pattern suggesting that distal addition predominates. These analyses of fluorescence intensities in extracted and unextracted neurites suggest that both transport of polymerized microtubules and monomer addition onto staggered microtubule ends occur in PC12 neurites and that in individual neurites one or the other of these two behaviors may predominate.     

Microtubule dynamics in the chromosomal spindle fiber: analysis by fluorescence and high-resolution polarization microscopy

We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24 degrees C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24 degrees C. In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into "rods" composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.     

Comparative studies on biological activity of certain microtubule-interacting taxanes

We have compared the nature of interaction of certain taxanes with microtubular protein, and the mechanism of action underlying cytotoxic activity. Taxanes induced tubulin assembly in vitro, but only taxanes bearing side chain were capable of inducing the formation of stable tubulin polymers. Electron microscopy detections showed that taxane-induced polymers are structurally similar to microtubules formed by paclitaxel, with differences in length. Otherwise, light microscopy views have shown that intracellular microtubule network is deeply reorganized by taxanes into short fibers, unlike paclitaxel-bundled microtubules. Taxanes inhibited the growth of various human tumor cell lines, but cell cycle analysis did not always indicate a block in the G2/M phase. These agents alter some apoptotic signal transduction pathways, probably by a mechanism distinct from microtubule interaction. Briefly, the effectiveness of taxanes is closely related to their chemical structure, and depends on their interaction with microtubular protein. By virtue of this mechanism, some of these taxanes may provide usefulness for therapeutic improvements.     

Monoclonal antibody directed against Neospora caninum tachyzoite carbohydrate epitope reacts specifically with apical complex-associated sialylated beta tubulin

Monoclonal antibodies (mabs) were generated against whole sonicated Neospora caninum tachyzoites as immunogen. Initial ELISA screening of the reactivity of hybridoma culture supernatants using the same antigen and antigen treated with sodium periodate prior to antibody binding resulted in the identification of 8 supernatants with reactivity against putative carbohydrate epitopes. Following immunoblotting, mab6D12 (IgG1), binding a 52/48-kDa doublet, and mab6C6 (IgM), binding a 190/180-kDa doublet, were selected for further studies. Immunofluorescence of tachyzoite-infected cultures localized the corresponding epitopes not to the surface, but to interior epitopes at the apical part of N. caninum tachyzoites. During in vitro tachyzoite to bradyzoite stage conversion, mab6C6 labeling translocated toward the cyst periphery, while for mab6D12 no changes in localization were noted. Upon extraction of tachyzoites with the nonionic detergent Triton-X-100, the 52-kDa band recognized by mab6D12 was present exclusively in the insoluble, cytoskeletal fraction of both N. caninum and Toxoplasma gondii tachyzoites. Tandem mass spectrometry analysis identified this protein as N. caninum beta tubulin. The 48-kDa band labeled by mab6D12 was a Vero cell protein contamination. The protein(s) reacting with mab6C6 could not be conclusively identified by mass spectrometry. Immunofluorescence consistently failed to label T. gondii tachyzoites, indicating that beta tubulin in T. gondii and N. caninum could be differentially modified or that the reactive epitope in T. gondii is masked. Immunogold TEM of isolated apical cytoskeletal preparations and dual immunofluorescence with antibody to tubulin confirmed that mab6D12 binds to the anterior part of apical complex-associated microtubules. The sodium periodate sensitivity of the beta tubulin associated epitope was confirmed by immunoblotting and ELISA, and treatment of N. caninum cytoskeletal proteins with sialidase prior to mab6D12 labeling resulted in a profound loss of antibody binding, suggesting that mab6D12 reacts with sialylated beta tubulin.     

Posttranslational modifications of alpha tubulin: detyrosination and acetylation differentiate populations of interphase microtubules in cultured cells

Subsets of microtubules enriched in posttranslationally detyrosinated (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski. 1984. Cell. 38:779) or acetylated (Piperno, G., M. Le Dizet, and X. Chang. 1987. J. Cell Biol. 104:298), alpha tubulin have previously been described in interphase cultured cells. In this study an immunofluorescence comparison of these minor populations of microtubules revealed that, in African green monkey kidney epithelial cells (TC-7 line), the population of microtubules enriched in detyrosinated tubulin was virtually coincident with the population enriched in acetylated alpha tubulin. In some cell types, however, such as human HeLa or marsupial PtK-2 cells, only one posttranslationally modified form of tubulin, i.e., acetylated or detyrosinated, respectively, was detectable in microtubules. In TC-7 cells, although both modifications were present, dissimilar patterns and kinetics of reappearance of microtubules enriched in detyrosinated and acetylated tubulin were observed after recovery of cells from microtubule-depolymerizing treatments or from mitosis. Thus, a minor population of microtubules exists in cultured cells that contains an elevated level of tubulin modified in either one or two ways. While these two modifications occur primarily on the same subset of microtubules, they differ in their patterns of formation in vivo.     

Quantitative analysis of tubulin and microtubule compartments in isolated rat hepatocytes

A combined morphometric and biochemical approach has been used to identify and quantitate microtubules and tubulin in isolated hepatocytes. The total soluble pool of microtubule protein was estimated by specific high affinity binding to radiolabeled colchicine. Scatchard analysis of the data identified two populations of binding sites: high affinity-low capacity sites resembling tubulin and low affinity-high capacity sites believed to represent nonspecific colchicine-binding sites. Data from these studies indicate that tubulin represents 1% of the soluble protein of the cell, that 9.0 X 10(-14) dimers of tubulin are present per microgram soluble hepatocyte protein, and that the average hepatocyte contains 3.1 X 10(7) tubulin dimers. Our calculations suggest that this amount of tubulin would form a microtubule 1.9 cm in length if totally assembled. However, stereological measurements indicate that the actual length of microtubules in the cytosolic compartment of the average hepatocyte is only 0.28 cm. Thus, these experiments suggest that only 15% of the available tubulin in hepatocytes of postabsorptive rats is assembled in the form of microtubules.     

Local anesthetic-induced inhibition of collagen secretion in cultured cells under conditions where microtubules are not depolymerized by these agents

Tertiary amine local anesthetics previously have been shown to influence some microtubule-dependent cellular functions. Since several cell secretion processes, including secretion of collagen, have been shown to be inhibited by microtubule-disrupting drugs such as colchicine, we determined whether local anesthetics affect collagen secretion. Six local anesthetics inhibited collagen and non-collagen protein secretion (up to 98%) into the extracellular medium of 3T3 cells and human fibroblasts, an effect apparently independent of influences on proline transport and total protein synthesis. A combination of colchicine and cytochalasin B did not duplicate the effects of local anesthetics. The effects of subsaturating concentrations of colchicine and procaine on secretion were additive, suggesting that both drugs act on the secretory pathway at the level of microtubules, but other effects of the two types of drugs were strikingly different. In comparing the mechanisms of action of colchicine and local anesthetics, it was seen that, in contrast to colchicine, radioactive procaine and lidocaine were slowly transported into 3T3 cells, did not bind to the tubulin-containing TCA-insoluble fraction, and did not bind to purified tubulin in vitro. The fraction of cellular tubulin present as microtubules (47% in normal cells) was determined by measuring tubulin in stabilized, sedimentable microtubules compared to total tubulin, using a [3H]colchicine binding assay. Pretreatment of cells in the cold or with colchicine led to depolymerization of microtubules, but pretreatment with five local anesthetics tested did not. Therefore, in contrast to colchicine, local anesthetics in concentrations that inhibit secretion do not directly interact with or depolymerize microtubules. These drugs, however, do affect a microtubule-dependent process and may do so by detaching the microtubular system from the cell membrane.     

Detection of microtubules of the flagellate Trichomonas vaginalis by monoclonal antibodies specific for beta-tubulin

Monoclonal antibodies specific for mammalian beta-tubulin recognized the microtubule cytoskeleton of the flagellated protozoon Trichomonas vaginalis. Of seven antibodies, two demonstrated the axostyle, costa, recurrent flagellum, and anterior flagella by indirect immunofluorescence microscopy. The remaining five stained a hazy reticular pattern in the cytoplasm of formaldehydefixed, detergent-extracted organisms. Western immunoblots of whole T. vaginalis extracts treated with protease inhibitors and electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate showed a major band at molecular weight 50,000 when probed with only one of the antibodies which stained the axial cytoskeleton. The antibodies which stained only the cytoplasm showed a different western blot pattern with a major doublet band at MW 58,000-60,000. Another antibody, which stained both the axial cytoskeleton and the reticular cytoplasmic pattern showed major bands at MW 58,000-60,000 and also at MW 40,000-42,000. The recognition of microtubule populations in T. vaginalis by these monoclonal antibodies was different than we found earlier with Leishmania donovani and Toxoplasma gondii, where all seven antibodies recognize cytoskeletal microtubules and produce western blots characteristic of tubulin. Only one of these seven antibodies recognizes tubulin in T. vaginalis by immunoblot. The microtubules of T. vaginalis do not demonstrate all epitopes recognized by monoclonal antibodies specific for mammalian beta-tubulin; one of the antibodies appears to recognize an epitope which is morphologically associated with microtubules but does not have the characteristic MW of tubulin.     

Induction and regulation of conoid extrusion in Toxoplasma gondii

Cell invasion by the intracellular parasite Toxoplasma gondii occurs through an active process that involves dynamic events, such as gliding motility and conoid extrusion, followed by a sequential secretion from specialized secretory organelles. Increase of intracellular Ca²⁺ by ionophores induces conoid extrusion, although in an irreversible way, thus limiting the characterization of the regulatory pathways. In this report we studied the effect of different activating conoid conditions to characterize the regulatory mechanisms involved. Exposure of tachyzoites to ethanol, a well-known activator of microneme secretion through the increase of intracellular Ca²⁺, induced conoid extrusion without affecting parasite viability nor its in vitro invasive capability, in a process that could be completely reverted and repeatedly reactivated. A temporal relationship between conoid extrusion and microneme secretion was here studied. Under this condition, signal transduction pathways and the precise role of the parasite cytoskeleton were characterized. Our results indicate that phospholipase C, Ca²⁺ released through channels sensitive to inositol-3-phosphate and ryanodine, as well as myosin together with actin filaments, but not microtubules, all participate in conoid extrusion. Specific inhibitors for serine-threonine kinases blocked conoid extrusion; in contrast, calmodulin inhibitors did not affect the induction. A regulatory model for conoid activation is here proposed.     

Formation of double-walled microtubules and multilayered tubulin sheets by basic proteins

Some basic proteins enable microtubule protein to form special assembly products in vitro, known as double-walled microtubules. Using histones (H1, core histones) as well as the human encephalitogenic protein to induce the formation of double-walled microtubules, we made the following electron microscopic observations: (1) Double-walled microtubules consist of an "inner" microtubule which is covered by electron-dense material, apparently formed from the basic protein, and by a second tubulin wall. (2) The tubulin of the second wall seems to be arranged as protofilaments, surrounding the inner microtubule in a helical or ring-like manner. (3) The surface of double-walled microtubules lacks the projections of microtubule-associated proteins, usually found on microtubules. (4) In the case of protofilament ribbons (incomplete microtubules), H1 binds exclusively to their convex sides that correspond to the surface of microtubules. Zn2+-induced tubulin sheets, consisting in contrast to microtubules of alternately arranged protofilaments, are covered by H1 on both surfaces. Furthermore, multilayered sheet aggregates appeared. The results indicate that the basic proteins used interact only with that protofilament side which represents the microtubule surface. In accordance with this general principle, models on the structure of double-walled microtubules and multilayered tubulin sheets were derived.     

Organization of the flagellar apparatus and associate cytoplasmic microtubules in the quadriflagellate alga Polytomella agilis

The organization of microtubular systems in the quadriflagellate unicell Polytomella agilis has been reconstructed by electron microscopy of serial sections, and the overall arrangement confirmed by immunofluorescent staining using antiserum directed against chick brain tubulin. The basal bodies of the four flagella are shown to be linked in two pairs of short fibers. Light microscopy of swimming cells indicates that the flagella beat in two synchronous pairs, with each pair exhibiting a breast-stroke-like motion. Two structurally distinct flagellar rootlets, one consisting of four microtubules in a 3 over 1 pattern and the other of a striated fiber over two microtubules, terminate between adjacent basal bodies. These rootlets diverge from the basal body region and extend toward the cell posterior, passing just beneath the plasma membrane. Near the anterior part of the cell, all eight rootlets serve as attachment sites for large numbers of cytoplasmic microtubules which occur in a single row around the circumference of the cell and closely parallel the cell shape. It is suggested that the flagellar rootless may function in controlling the patterning and the direction of cytoplasmic microtubule assembly. The occurrence of similar rootlet structures in other flagellates is briefly reviewed.     

Reformation of the marginal band of avian erythrocytes in vitro using calf-brain tubulin: peripheral determinants of microtubule form

The microtubules of nucleated erythrocytes form an extraordinary structure: they are organized into a marginal band at the periphery of the cell. This unusual organelle, recurring in detail in each cell, provides an excellent opportunity to study the determinants of microtubule form. We have been able to reform the marginal band, using detergent-extracted erythrocytes that have been depleted of microtubules in vivo and phosphocellulose-purified tubulin from calf brain. We find that detergent-extracted cytoskeletons incubated under these conditions again have microtubules, and that the pattern of these microtubules recapitulates several features of the intact marginal band. In particular, most of the microtubules after regrowth are located in a band at the periphery of the cell, and curve to form an ellipse. These results support the hypothesis that the specification of microtubule location and shape in these cells is governed by determinants that reside at the periphery of the cell.