Effect of neocarzinostatin on the nucleus, kinetoplast and microtubules of Trypanosoma gambiense and Trypanosoma evansi.

The antibiotic neocarzinostatin (NCS) induces the anucleate form, not the dyskinetoplastic form, of Trypanosoma gambiense and Trypanosoma evansi. Light and electron microscopic studies indicated that production of the anucleate form is due to delay or inhibition of nuclear division. Excess pellicular microtubules are formed after treatment of trypanosomes with NCS, suggesting that in trypanosomes the microtubules replicate by induction, not by division. NCS also causes deformation of the axonemal and spindle microtubules. The K clone of T. evansi is more sensitive than the AK clone to the effects of NCS in inhibiting nuclear division and inducing the anucleate form.     

Evidence for nucleating microtubules in microtubular associations and for an opening polarity under colchicine action.

Bundles of microtubular structures appear in the cytoplasm of germinal cells of the African frog Dicroglossus occipitalis. They are made of several associated microtubules. Every bundle contains one normal singlet and numerous arch-shaped microtubular structures growing in all directions from the singlet wall. The walls of these microtubules are shown to contain 10 to 13 protofilaments. Attempts made with colchicine point out their susceptibility to this antimitotic drug. The formation and opening of these microtubular structures give evidence of complex organization.     

Abnormal spore cleavage: Abnormal spores of Allomyces macrogynus

In the aquatic phycomycete Allomyces macrogynus abnormal spore cleavage takes place in the presence of colchicine or benomyl resulting in multinucleate–multiflagellate spores due to failure in the formation of cytoplasmic microtubules after the induction of zoosporogenesis. The 27 cytoplasmic microtubules which normally surround the nucleus and nuclear cap of the mature spore are not formed in the presence of colchicine or benomyl. At high concentrations of colchicine (4–8 mg/ml) the spores do not have a flagellum. Colchicine or benomyl inhibit microtubule formation during zoosporogenesis and also appear to perturb the mobilization of the gamma bodies which are believed to be the source of the vesicles which form the axonemal membrane and cleavage furrows. These observations are discussed in relation to the hypothesis of Heath that cytoplasmic microtubules formed during zoosporogenesis determine cytoplasmic domains which will delimit the spore initials at cleavage. The observations presented here appear to confirm this hypothesis.     

Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium

Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of approximately 24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60% of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered.     

The initiation of neurite outgrowth by sympathetic neurons grown in vitro does not depend on assembly of microtubules [published erratum appears in J Cell Biol 1995 Feb;128(3):443]

Neurite formation by dissociated chick sympathetic neurons in vitro begins when one of the many filopodia that emanate from the cell body of a neuron is invaded by cytoplasm containing microtubules and other components of axoplasm (Smith, 1994). This study was undertaken to determine whether this process depends on assembly of microtubules. To inhibit microtubule assembly, neurons were grown in medium containing nocodazole or colchicine. In one series of experiments, neurons first were exposed to the microtubule-stabilizing drug, taxol, so that existing microtubules would remain intact while assembly of new microtubules was inhibited. The ability of neurons to form neurites was assessed by time-lapse video microscopy. Neurons subsequently were stained with antibodies against the tyrosinated and acetylated forms of alpha-tubulin and examined by laser confocal microscopy to visualize microtubules. Neurons were able to form short processes despite inhibition of microtubule assembly and they did so in a way that closely resembled process formation in control medium. Processes formed by neurons that had not been pretreated with taxol were devoid of microtubules. However, microtubules were present in processes of taxol- pretreated neurons. These microtubules contained acetylated alpha- tubulin, as is typical of stable microtubules, but not tyrosinated alpha-tubulin, the form present in recently assembled microtubules. These findings show that the initial steps in neurite formation do not depend on microtubule assembly and suggest that microtubules assembled in the cell body can be translocated into developing neurites as they emerge. The results are compatible with models of neurite formation which postulate that cytoplasm from the cell body is transported into filopodia by actomyosin-based motility mechanisms.     

Polymerization of tubulin in the presence of colchicine or podophyllotoxin. Formation of a ribbon structure induced by guanylyl-5'-methylene diphosphonate

GTP-dependent in vitro polymerization of rat brain microtubular protein is inhibited to 50% by substoichiometric concentrations of the antimitotic drugs colchicine (0.12 mol/mol of tubulin) and podophyllotoxin (0.14 mol/mol of tubulin). Substitution of pp(CH₂)pG² for GTP, however, results in an extensive microtubular protein polymerization at such concentrations. In the presence of pp(CH₂)pG, suprastoichiometric concentrations of podophyllotoxin (19 mol/mol of tubulin) are required to inhibit the polymerization process by 50%. Colchicine is very ineffective since 3 × 10⁵ moles/mole of tubulin are required to give a 50% inhibition. Electron microscopical analysis shows that the polymers formed by microtubular protein in the presence of suprastoichiometric concentrations of drugs are not the normal short microtubules typical of pp(CH₂)pG-driven polymerization, but are ribbons with three or four protofilaments. The colchicine content of the harvested ribbons has been measured directly and found to be approximately 0.8 moles colchicine/mole of tubulin. Treatment of microtubular protein with substoichiometric concentrations of drugs results in an increase in the number of protofilaments forming the ribbons. Many of the ribbons can close into morphologically normal microtubules when microtubular protein is treated with only 0.05 moles of either colchicine or podophyllotoxin per mole of tubulin.     

Effects of organic acids on tubulin polymerization and associated guanosine 5'-triphosphate hydrolysis

We have examined the effects of a number of organic anions, which stabilize tubulin, on tubulin polymerization, associated GTP hydrolysis, and polymer morphology. While microtubule-associated proteins, as well as glycerol, induced formation of typical microtubules in a reaction coupled to GTP hydrolysis at an initial 1:1 stoichiometry, the organic anions had varying effects. Only 2-(N-morpholino)ethanesulfonate induced formation of structures with the morphology of microtubules. With glutamate, fructose 1,6-bisphosphate, piperazine-N-N'-bis(2-ethanesulfonate), glutarate, and glucose 1-phosphate, the predominant structures formed were sheets of parallel protofilaments rather than microtubules. Creatine phosphate induced the formation of clusters of rings. GTP hydrolysis was closely coupled to polymerization only with glutamate. With creatine phosphate, there was minimal GTP hydrolysis. With all other organic anions, GTP hydrolysis substantially exceeded polymerization at all time points, with the onset of hydrolysis significantly preceding the onset of turbidity development. Nevertheless, the rate of GTP hydrolysis was a sigmoidal function of tubulin concentration under all conditions examined, suggesting that tubulin-tubulin interactions are required for hydrolysis. All anion-induced reactions were temperature dependent and cold reversible, but only the creatine phosphate induced reaction was not inhibited by GDP, CA2+, or colchicine and did not require GTP.     

Genetic analysis of microtubule structure: a beta-tubulin mutation causes the formation of aberrant microtubules in vivo and in vitro

A recessive male sterile mutation (B2t8) that encodes a stable variant of the testis-specific beta 2-tubulin of Drosophila causes the assembly of aberrant microtubules both in vivo and in vitro. The B2t8 mutation appears to cause defects in the formation of interprotofilament bonds. In testes from homozygous mutant males, the most commonly observed aberrant structures were sheets of protofilaments curved to form an S in cross section rather than a normal, closed microtubule. These characteristic S-shaped structures appear in the meiotic spindle, in place of axonemes in differentiating spermatids, and in cytoplasmic microtubules, including those that lie next to the nucleus during nuclear elongation. Homozygous mutant males exhibit defects in chromosome movement and cytokinesis during meiosis, flagellar elongation, and nuclear shaping, indicating that the ability to form normal closed microtubules is required for each of these events. The presence of the aberrant microtubules in three architecturally different microtubule arrays demonstrates conclusively the multifunctional nature of the beta 2-tubulin gene product. Although the mutant beta 2-tubulin subunit causes assembly of aberrant microtubules in vitro and in homozygous males, in the presence of wild-type beta 2-tubulin in heterozygous males, the variant subunit coassembles with the wild-type subunit into functional sperm.     

Mechanism of inhibition of microtubule polymerization by colchicine: inhibitory potencies of unliganded colchicine and tubulin-colchicine complexes.

The tubulin-colchicine binding reaction appears to involve a number of intermediate steps beginning with rapid formation of a transient preequilibrium complex that is followed by one or more slow steps in which conformational changes in tubulin and colchicine lead to formation of a poorly reversible final-state complex. In the present study, we investigated the relative ability of unliganded colchicine and preformed final-stage tubulin-colchicine complex to incorporate at microtubule ends and to inhibit addition of tubulin at the net assembly ends of bovine brain microtubules in vitro. Addition of 0.1 microM final-stage tubulin-colchicine complex to suspensions of microtubules at polymer-mass steady-state resulted in rapid incorporation of one to two molecules of tubulin-colchicine complex per microtubule net assembly end concomitant with approximately 50-60% inhibition of tubulin addition. Incorporation of colchicine-tubulin complex continued slowly with time, without significant additional change in the rate of tubulin addition. In contrast, addition of unliganded colchicine to microtubule suspensions resulted in incorporation of small numbers of colchicine molecules at microtubule ends and inhibition of tubulin addition only after periods of time that varied from several minutes to approximately 20 min depending upon the concentration of colchicine. Inhibition of tubulin addition beginning with unliganded colchicine increased slowly with time, concomitant with increases in the concentration of final-state tubulin-colchicine complex and the amount of colchicine bound per microtubule end. The results indicate that inhibition of tubulin incorporation at microtubule ends is caused by colchicine-liganded tubulin in the form of a final-state complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Kinase activities in the non-histone chromosomal proteins of resting and proliferating BHK21 C13 cells.

The in vitro polymerization of bovine brain tubulin in the presence of Zn²⁺ has been studied. Zn²⁺, at concentrations higher than 5 × 10^?5 M caused the formation of sheets, sometimes consisting of up to 50–60 protofilaments oriented in parallel. The sheets were stable towards colchicine, Ca²⁺ and cold treatment. The induction of sheets cannot solely be ascribed to the sulfhydryl blocking properties of Zn²⁺, since other SH reagents (NEM, PCMBS, Cd²⁺ and Hg²⁺) failed to cause similar effects. There are reasons to believe that Zn²⁺ interferes with the closing process of microtubules so that more protofilaments are added in the assembly process than during normal conditions. The system described herein offers a favourable preparation for revealing the ultrastructural organization of microtubules.     

Tannic acid-stained microtubules with 12, 13, and 15 protofilaments

Subunit structure in the walls of sectioned microtubules was first noted by Ledbetter and Porter (6), who clearly showed that certain microtubules of plant meristematic cells have 13 wall protofilaments when seen in cross section. Earlier, protofilaments of microtubular elements had been described in negatively stained material, although exact counts of their number were difficult to obtain. In microtubular elements of axonemes, some success has been achieved in visualizing protofilaments in conventionally fixed and sectioned material (8, 10); much less success has been achieved in identifying and counting protofilaments of singlet cytoplasmic microtubules. By using glutaraldehyde-tannic acid fixation, as described by Misuhira and Futaesaku (7), Tilney et al. (12) studied microtubules from a number of sources and found that all have 13 protofilaments comprising their walls. These authors note that "...the number of subunits and their arrangement as protofilaments appear universal...". Preliminary studies of ventral nerve cord of crayfish fixed in glutaraldehyde-tannic acid indicated that axonal microtubules in this material possess only 12 protofilaments (4). On the basis of this observation, tannic acid preparations of several other neuronal and non-neuronal systems were examined. Protofilaments in microtubules from these several cell types are clearly demonstrated, and counts have been made which show that some kinds of microtubules have more or fewer protofilaments than the usual 13 and that at least one kind of microtubule has an even rather than an odd number.     

New data on the microtubule surface lattice

The in vitro polymerisation of tubulin is a remarkable example of protein self-assembly in that several closely related microtubule structures coexist on the polymerisation plateau. Unfixed and unstained in vitro assembled microtubules were observed in vitreous ice by cryo-electron microscopy. New results are reported that considerably extend previous observations [47]. In ice, microtubule images have a distinctive contrast related to the number and skew of the protofilaments. The microtubules observed have from twelve to seventeen protofilaments. Comparison with thin sections of pelleted material allows a direct identification of images from microtubules with thirteen, fourteen and fifteen protofilaments. A surface lattice accommodation mechanism, previously proposed to explain how variable numbers of protofilaments can be incorporated into the basic thirteen protofilament structure, is described in detail. Our new experimental results are shown to be in overall agreement with the theoretical predictions. Only thirteen protofilament microtubules have unskewed protofilaments, this was confirmed by observations on axoneme fragments. The results imply that the microtubule surface lattice is based on a mixed packing which combines features of the standard A and B lattices.     

Electron microscope studies of pH effects on assembly of tubulin free of associated proteins. Delineation of substructure by tannic acid staining

Bovine brain tubulin, purified by phosphocellulose chromatography (PC), was assembled in the presence of 10% dimethyl sulfoxide (DMSO), and the reaction was monitored turbidimetrically. Samples were fixed in glutaraldehyde-tannic acid after completion of polymerization, as indicated by no further change in absorbance, and then sectioned and studied electron microscopy, with special attention being given to the arrangement of protofilaments in the walls of formed elements. Samples of PC-tubulin were polymerized in buffer having various pH values from 6.0 to 7.7. At the lower pH values, only branched and flattened ribbons of protofilaments are formed. At intermediate values, the ribbons are unbranched, narrower, and more curved in cross section; complete microtubules are also seen. At the higher pH values, the predominate formed elements are complete microtubules. Most of the complete microtubules examined in this study had 14 wall protofilaments. The effect of pH on tubulin assembly was shown not to be an effect of DMSO. The dimers of associated protofilaments in ribbons and microtubules are conceptually viewed as having trapezoidal profiles in cross section, and, as additional dimers are added, the "C"-shaped ribbon closes to form a tube. The tilt angle of the lateral surfaces of the "trapezoidal" dimers will determine the number of wall protofilaments in the microtubules. At low pH, it is theorized that the trapezoidal profile of the dimer is shifted to a more rectangular configuration such that flat ribbons are formed by the lateral association of dimers. Also, variously shaped ribbon structures are formed at intermediate pH values, including "S"- and "W"-shaped structures, and elements shaped like a figure "6," all representing ribbons viewed in cross section. By visualizing the trapezoidal dimer in three-dimensions, and by arbitrarily indexing its six binding surfaces, it is possible to discuss interdimer binding in terms of preferred and possible binding interactions.     

Microtubules, colchicine, and lymphocyte blastogenesis.

We have studied the time course of disassembly of microtubules of resting and stimulated mouse lymphocytes caused by the drug colchicine, as well as the effect of this compound on DNA and RNA synthesis of human and mouse lymphocytes. Fine-structure studies with the electron microscope showed a great increase in number of microtubules resulting from stimulation of mouse lymphocytes by the mitogenic lectin Con A. The presence of a network of microtubules was demonstrated in resting lymphocytes by use of the technique of immunofluorescence; this technique was not effective for the study of the microtubules of stimulated lymphocytes in the blast stage. The disappearance of microtubular networks in some cells (approximately 25%) was caused by the protocol of colchicine treatment used in many laboratories (30 min at 10(6) M); a 6- to 8-h treatment was required to cause all cells to lose their microtubules. It is indicated in these findings that there is need for extreme caution in implicating microtubule disruption as the cause of certain colchicine effects, such as that on the Con A-induced inhibition of receptor-ligand migration. The addition of colchicine to stimulated cells at varying times of culture caused marked inhibition of DNA synthesis provided that sufficient time (approximately 20 h for maximum inhibition) elapsed between addition of the drug to the stimulated culture and assay of DNA synthesis. Our data on the time course of inhibition of DNA synthesis by alpha-methyl mannoside (alpha MM) and by colchicine do not exclude the possibility that the latter compound may act partially by affecting the commitment of stimulated lymphocytes to DNA synthesis but they show that it can inhibit well after commitment is complete. The later the time of assay of thymidine incorporation, the more disparate were the curves relating the effects of alpha MM and colchicine to DNA synthesis of human cells. In the case of mouse splenic lymphocytes, there was no resemblance between the time course of the alpha MM and of the colchicine effects. Synthesis of RNA after 12 h of culture of stimulated human lymphocytes was also sensitive to colchicine.     

Microtubules in bacteria: Ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as "bacterial microtubules" (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening.     

BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin

BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.     

Structural and functional diversity in the neuronal microtubules of Caenorhabditis elegans

Tannic acid fixation reveals differences in the number of protofilaments between microtubules (MTs) in the nematode Caenorhabditis elegans. Most cells have MTs with 11 protofilaments but the six touch receptor neurons (the microtubule cells) have MTs with 15 protofilaments. No 13-protofilament (13-p) MT has been seen. The modified cilia of sensory neurons also possess unusual structures. The cilia contain nine outer doublets with A subfibers of 13 protofilaments and B subfibers of 11 protofilaments and a variable number of inner singlet MTs containing 11 protofilaments. The 15-p MTs but not the 11-p MTs are eliminated by colchicine-treatment or by mutation of the gene mec-7. Concomitantly, touch sensitivity is also lost. However, whereas colchicine treatment leads to the loss of all MTs from the microtubule cells, mutations in mec-7 result in the partial replacement of the 15-p MTs with 11-p MTs. Benzimidazoles (benomyl and nocodazole) have more general effects on C. elegans (slow growth, severe uncoordination, and loss of processes from the ventral cord) but do not affect the 15-p MTs. Benomyl will, however, disrupt the replacement 11-p MTs found in the microtubule cells of mec-7 mutants. The 11-p and 15-p MTs also respond differently to temperature and fixation conditions. It is likely that either type of MT will suffice for the proper outgrowth of the microtubule cell process, but only the 15-p MT can function in the specialized role of sensory transduction of the microtubule cells.     

Insight into the GTPase activity of tubulin from complexes with stathmin-like domains

Microtubules are dynamically unstable tubulin polymers that interconvert stochastically between growing and shrinking states, a property central to their cellular functions. Following its incorporation in microtubules, tubulin hydrolyzes one GTP molecule. Microtubule dynamic instability depends on GTP hydrolysis so that this activity is crucial to the regulation of microtubule assembly. Tubulin also has a much lower GTPase activity in solution. We have used ternary complexes made of two tubulin molecules and one stathmin-like domain to investigate the mechanism of the tubulin GTPase activity in solution. We show that whereas stathmin-like domains and colchicine enhance this activity, it is inhibited by vinblastine and by the N-terminal part of stathmin-like domains. Taken together with the structures of the tubulin-colchicine-stathmin-like domain-vinblastine complex and of microtubules, our results lead to the conclusions that the tubulin-colchicine GTPase activity in solution is caused by tubulin-tubulin associations and that the residues involved in catalysis comprise the beta tubulin GTP binding site and alpha tubulin residues that participate in intermolecular interactions in protofilaments. This site resembles the one that has been proposed to give rise to GTP hydrolysis in microtubules. The widely different hydrolysis rates in these two sites result at least in part from the curved and straight tubulin assemblies in solution and in microtubules, respectively.