MICROTUBULES: EVIDENCE FOR 13 PROTOFILAMENTS

When microtubules are fixed in glutaraldehyde in the presence of tannic acid and thin sections cut, the subunit structure of the microtubule is readily observed without the need of image reinforcement. Seven types of microtubules were analyzed: those in the heliozoan axoneme, the mitotic apparatus, the contractile axostyle, repolymerized microtubules derived from the chick brain, the central pair in flagella, and the A tubules of flagella and the basal body. In all cases microtubules were composed of 13 equally spaced protofilaments. The B tubules in flagella and the basal body appear to be composed of 11 subunits. The connections of the B to the A and the C to the B are described. A model of a microtubule is presented.     

Axostyle structure in the termite protozoon Pyrsonympha vertens

The axostyle of Pyrsonympha vertens is a cellular organelle composed of interconnected microtubules. In living organisms the axostyle has waves which originate at the anterior end of the protozoon and traverse the length to the posterior end of the protozoon so that an average of 3–4 waves are present in the organelle at any given point in time. The part of the axostyle between the waves is straight. In sections through the middle of the straight part, the microtubules are hexagonally packed, with predominant connections between tubules in rows across the width of the axostyle, but the microtubules are rectilinearly packed through the wave. The wave appears to involve changes in orientation and arrangement of the microtubules. The general structure of the microtubules, cross-bridges and axostyle in the straight and bent portions are described and related to the wave propagation by this organelle.     

Structural basis of motility in the microtubular axostyle: implications for cytoplasmic microtubule structure and function

The gross morphology of the protozoan microtubule axostyle of Saccinobaculus ambloaxostylus can now be described in macromolecular detail. The left-handed coil of the axostyle is seen to be dependent upon the asymmetry inherent in the constituent microtubules as expressed by the specific array of linkages between microtubules and by a possible tendency for microtubules to coil into left-handed helices. The laminated sheets of microtubules are not aligned parallel to the long axis of the organelle, but become increasingly tilted off-axis as one descends through the sheets of microtubules from the convex to the concave surface of the axostyle. Fine-structural analysis of the axostyle indicates similarities of the linkages to dynein. The potential loci of the force-generating protein(s) are discussed as well as implications of the axostyle's structure on general microtubule function.     

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.     

Ultrastructure and Organization of the Cytoskeleton in Oxymonas, an Intestinal Flagellate of Termites

ABSTRACT. Oxymonas has the characteristic structures and organization of other oxymonads including two separated pairs of basal bodies/flagella, a preaxostylar lamina, a paracrystalline axostyle, and an absence of mitochondria and Golgi. Like other Oxymonadinae genera it possesses a long proboscis, the rostellum which is terminated by the holdfast. Like the genera Pyrsonympha and Streblomastix, Oxymonas possesses a holdfast which permits it to attach to the cuticle of the termite hind-gut. This holdfast is subdivided into rhizoids and is filled with microfilaments. The rostellum is variable in length and contains two distinct microtubular bundles. One bundle is composed of convoluted microtubular ribbons which originate at the base of the holdfast and extend posteriorly along the rostellum and before penetrating into the cell body. The second bundle is composed of flexuous free microtubules which originate at different levels of the rostellum, increasing in number from top to base. They occupy the axial part of the rostellum and incorporate into the axostylar rows at the basal body/flagellar level. Microtubules of the paracrystalline axostyle are cross-linked by bridges forming parallel rows like in the contractile axostyles of other oxymonads such as Pyrsonympha and Saccinobaculus . Most of the microtubules of the axostyle originate at the flagellar/preaxostylar level but some originate from the axial flexous free microtubules of the rostellum, as indicated above. The possibility of an extension/retraction of the rostellum, suggested by other authors, is discussed.     

Lattice defects in microtubules: protofilament numbers vary within individual microtubules

We have used cryo-electron microscopy of vitrified specimens to study microtubules assembled both from three cycle purified tubulin (3x-tubulin) and in cell free extracts of Xenopus eggs. In vitro assembled 3x-tubulin samples have a majority of microtubules with 14 protofilaments whereas in cell extracts most microtubules have 13 protofilaments. Microtubule polymorphism was observed in both cases. The number of protofilaments can change abruptly along individual microtubules usually by single increments but double increments also occur. For 3x-tubulin, increasing the magnesium concentration decreases the proportion of 14 protofilament microtubules and decreases the average separation between transitions in these microtubules. Protofilament discontinuities may correspond to dislocation-like defects in the microtubule surface lattice.     

Protofilament number in microtubules in cells of two parasitic nematodes

The parasitic nematodes, Ascaridia galli and Trichostrongylus colubriformis, were prepared for electron microscopy with fixatives containing tannic acid, which allowed their microtubule protofilament number to be examined. In contrast to many mammalian tissues, the nematodes did not contain microtubules with 13 protofilaments. Ascaridia galli contained microtubules with 11 protofilaments in all tissues examined, including nerve, intestinal, pharyngeal, and hypodermal cells. Trichostrongylus colubriformis contained nerve cells, known as microtubule cells, with bundles of larger microtubules (approximately 30 nm in diameter) with 14 protofilaments. The microtubules in these cells did not appear to be continuous for the entire length of the axon. Other cells examined in T. colubriformis, including nerve, intestinal and pharyngeal cells, contained two distinct types of microtubules, one with 11 protofilaments and an approximate diameter of 25 nm, and one with 12 protofilaments and an approximate diameter of 27 nm. All cell types examined contained both types of microtubules.     

THE FLAGELLAR APPARATUS OF CHILOMONAS PARAMECIUM (CRYPTOPHYCEAE) AND ITS COMPARISON WITH CERTAIN ZOOFLAGELLATES1

The major components of the internal flagellar apparatus of Chilomonas paramecium Ehr. are two large microtubular roots and a striated root paralleled by three microtubules. The two microtubular roots overlap at the basal bodies. One microtubular root follows a curved path in the anterior of the cell, and the other extends straight to the posterior passing through a groove in the nucleus. The striated root extends laterally from the basal bodies. Except that it is smaller, the posteriorly directed root bears a strong resemblance to the axostyle of oxymonads. The overall arrangement and structure of the flagellar roots is similar to the pelta, axostyle and costa of trichomonads and the pelta and axostyle of oxymonads, groups of mitochondrion-less, largely parasitic or symbiotic protozoans. An affinity between cryptomonads and oxymonads or trichomonads would have many phylogenetic implications, some of which are discussed.     

ISOLATION AND REACTIVATION OF THE AXOSTYLE : Evidence for a Dynein-like ATPase in the Axostyle

The contractile axostyle is a ribbon-shaped organelle present in certain species of flagellates found in the hindgut of wood eating insects. This organelle propagates an undulatory wave whose motion, like flagella and cilia, is related to microtubules. Unlike the axoneme of cilia and flagella, however, the axostyle is composed of singlet microtubules linked together in parallel rows. Axostyles were isolated from Cryptocercus gut protozoa with Triton X-100. Normal motility of the isolated axostyle could be restored with adenosine triphosphate (ATP); the specific conditions necessary for this reactivation were essentially identical with those reported for the reactivation of isolated flagella or whole sperm. ATPase activity of the isolated axostyle was comparable to the values reported for ciliary or flagellar axonemes. The axostyle was reasonably specific for ATP. Most of the proteins of the isolated axostyle comigrated with proteins of the ciliary axoneme on sodium dodecyl sulfate (SDS) polyacrylamide gels (i e. equivalent molecular weights). These included the following: the higher molecular weight component of dynein, tubulin, linkage protein (nexin), and various secondary proteins. Evidence for dynein in the axostyle is presented and a model proposed to explain how repeated propagated waves can be generated.     

Characterization of microtubule protofilament numbers. How does the surface lattice accommodate?

Frozen-hydrated specimens of microtubules assembled in vitro were observed by cryoelectron microscopy. Specimens were of both pure tubulin, and of microtubule protein isolated by three cycles of assembly and disassembly. It is shown that the characteristic image contrast of individual microtubules allows the microtubule protofilament number to be determined unambiguously. Microtubules with 13, 14 and 15 protofilaments are observed to coexist in specimens prepared under various assembly conditions. Confirmation of these results is obtained by observations of thin sections of pelleted samples fixed and stained using the glutaraldehyde/tannic acid technique. Images of individual microtubules show both characteristic contrast profiles across their width and typical variations of these profiles along their length. The profiles across the images indicate the protofilament number of the microtubule. The lengthwise variations indicate how the protofilaments are aligned with respect to the microtubule axis giving what has previously been called a supertwist. In 13 protofilament microtubules the protofilaments are paraxial. In 14 and 15 protofilament microtubules, the protofilaments are skewed with respect to the microtubule axis. The skew is greater for the 15 protofilament case than for 14 protofilaments. The skew allows the extra protofilaments to be accommodated by the surface lattice. These results should also be relevant to situations in vivo.     

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.     

Different structural states of a microtubule cross-linking molecule, captured by quick-freezing motile axostyles in protozoa

Freeze-etch preparation of the laminated bundles of microtubules in motile axostyles demonstrates that the cross-bridges populating individual layers or laminae are structurally similar to the dynein arms of cilia and flagellae. Also, like dynein, they are extracted by high salt and undergo a change in tilt upon removal of endogenous ATP (while the axostyle as a whole straightens and becomes stiff). On the other hand, the bridges running between adjacent microtubule laminae in the axostyle turn out to be much more delicate and wispy in appearance, and display no similarity to dynein arms. Thus we propose that the internal or "intra-laminar" cross-bridges are the active force- generating ATPases in this system, and that they generate overall bends or changes in the helical pitch of the axostyle by altering the longitudinal and lateral register of microtubules in each lamina individually; e.g., by "warping" each lamina and creating longitudinal shear forces within it. The cross-links between adjacent laminae, on the other hand, would then simply be force-transmitting elements that serve to translate the shearing forces generated within individual laminae into overall helical shape changes. (This hypothesis differs from the views of earlier workers who considered a more active role for the later cross-links, postulating that they cause an active sliding between adjacent layers that somehow leads to axostyle movement.) Also described here are physical connections between adjacent intra-laminar cross-bridges, structurally analogous to the overlapping components of the outer dynein arms of cilia and flagella. As with dynein, these may represent a mechanism for propagating local changes from cross-bridge to cross-bridge down the axostyle, as occurs during the passage of bends down the length of the organelle.     

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.     

THE AXOSTYLE OF SACCINOBACULUS : I. Structure of the Organism and Its Microtubule Bundle

The axostyle of the flagellate Saccinobaculus is a motile ribbon composed of microtubules, cross-bridged to form interconnected rows. We find a centriole-related row of dark-staining tubules near the nucleus at the anterior end of the axostyle. Other tubule rows bind parallel to this primary row, acquire ordered relationships, and become the tubules of the axostyle proper. The number of tubule rows is constant in Saccinobaculus lata from the region near the nucleus to within a few micrometers of the posterior tip of the cell. In Saccinobaculus ambloaxostylus a few tubule rows are added to the axostyle posterior to the nucleus, giving this axostyle a leaf spring construction. The tubules of S. lata are held in rows by links with a 140 Å periodicity along the tubule axis; bridges between rows of tubules are also seen but are not apparently periodic. Each tubule in S. ambloaxostylus shows an axial periodicity of 150 Å due to pairs of arms, one of which is always part of the intrarow link. Interrow bridges in this species run either from tubule to tubule or from tubule to the free arm, but as in S. lata they do not display an obvious axial periodicity. An average unit cell is presented for the axostyle of each species, and the relation of the intertubule links to the microtubule substructure is discussed.     

Deuterium oxide promotes assembly and bundling of FtsZ protofilaments

The assembly and bundling of FtsZ protofilaments play an important role during bacterial cell division. Deuterium oxide (D2O) is known to have strong stabilization effects on the assembly dynamics of several proteins including tubulin, a homologue of FtsZ. Here, we found that D2O enhanced the light-scattering intensity of the assembly reaction, increased sedimentable polymer mass, and induced bundling of FtsZ protofilaments. D2O also increased the stability of FtsZ polymers under challenged GTP conditions and suppressed dilution-induced disassembly of protofilaments. D2O enhances the assembly parameters of FtsZ and microtubules albeit differently. For example, D2O induced bundling of FtsZ protofilaments, whereas it did not induce bundling of microtubules in vitro. In addition, D2O strongly suppressed the GTP hydrolysis rate of microtubules, but it had no effect on the initial rate of GTP hydrolysis of the FtsZ assembly. D2O (80%) also increased the helical content of FtsZ by 25% compared to the helical content of FtsZ in aqueous buffer. D2O was shown to reduce the binding of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to tubulin. In contrast, we found that D2O strongly enhanced the binding of bis-ANS to FtsZ. The results indicated that D2O promotes assembly and bundling of FtsZ protofilaments by increasing hydrophobic interactions between the protofilaments. The results also suggest that the phosphate release rather than the on-site GTP hydrolysis is the rate-limiting step of the GTP turnover reaction.     

Microtubule structure at improved resolution.

Microtubule architecture can vary with eukaryotic species, with different cell types, and with the presence of stabilizing agents. For in vitro assembled microtubules, the average number of protofilaments is reduced by the presence of sarcodictyin A, epothilone B, and eleutherobin (similarly to taxol) but increased by taxotere. Assembly with a slowly hydrolyzable GTP analogue GMPCPP is known to give 96% 14 protofilament microtubules. We have used electron cryomicroscopy and helical reconstruction techniques to obtain three-dimensional maps of taxotere and GMPCPP microtubules incorporating data to 14 A resolution. The dimer packing within the microtubule wall is examined by docking the tubulin crystal structure into these improved microtubule maps. The docked tubulin and simulated images calculated from "atomic resolution" microtubule models show tubulin heterodimers are aligned head to tail along the protofilaments with the beta subunit capping the microtubule plus end. The relative positions of tubulin dimers in neighboring protofilaments are the same for both types of microtubule, confirming that conserved lateral interactions between tubulin subunits are responsible for the surface lattice accommodation observed for different microtubule architectures. Microtubules with unconventional protofilament numbers that exist in vivo are likely to have the same surface lattice organizations found in vitro. A curved "GDP" tubulin conformation induced by stathmin-like proteins appears to weaken lateral contacts between tubulin subunits and could block microtubule assembly or favor disassembly. We conclude that lateral contacts between tubulin subunits in neighboring protofilaments have a decisive role for microtubule stability, rigidity, and architecture.     

Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.     

Sperm Axoneme in Some Apterygote Insects Examined by Computer-aided Image Analysis

Sperm tail axonemes from three insect species, representing the three apterygote orders Diplura, Archeognatha and Zygentoma, have been examined by high resolution electron microscopy. The samples were fixed in a mixture of glutaraldehyde and tannic acid followed by uranyl acetate post-fixation and the electron micrographs were subjected to computer analysis in order better to characterize the minute details of the axonemal microtubules. The dipluran Campodea was seen to have a row of accessory microtubules composed of 13 protofilaments, the archeognath Machilinus to have two rows of accessory microtubules composed of 16 protofilaments. and the zygentoman Lepismodes to have accessory microtubules in contact with the flagellar doublets and with a wall containing 16 protofilaments and an electron-dense lumen. The axonemal structure in Zygentoma is the one most similar to those of the pterygote insects. The protofilaments of the axonemal doublets were seen to have a somewhat widened interspace between some pairs of protofilaments and these ‘gaps’ coincided with the location of the basal parts of the outer dynein arm, the inner dynein arm, the spoke, and the intertubular material. It hence seems that these microtubule-associated structures influence the architecture of the microtubular wall.     

Alterations in number of protofilaments in microtubules assembled in vitro

Tubulin from bovine brain was polymerized in vitro using a variety of assembly conditions. Many of the formed microtubules were shown to contain 14 wall protofilaments. The number of microtubules containing 14 protofilaments increased with consecutive repetitions of cold-dissociation followed by reassembly in vitro.