Diameters of microtubules change during rotation of the lipotubuloids of Ornithogalum umbellatum stipule epidermis as a result of varying protofilament monomers sizes and distance between them

Microtubules in lipotubuloids of the Ornithogalum umbellatum stipule epidermis cells change their diameters depending on the motion of the cytoplasmic domains rich in microtubules and lipid bodies. Microtubules fixed during rotary and progressive motion of the lipotubuloids composed of the same number of protofilaments fall into two populations – wide (43–58 nm) and narrow (24–39 nm) in size. Following blockage of the motion with 2,4-dinitrophenol (DNP), the range of this diversity is smaller, microtubules become a medium-sized population (34–48 nm). When DNP is removed and the motion reactivated, 2 populations of microtubules reappear. Analysis of the structure of the microtubule wall revealed that changes in the microtubule diameters resulted from varying distances between the adjacent protofilaments, and stretching and compression of tubulin subunits in the protofilaments.A supposition has been put forward that the changes in the sizes of O. umbellatum microtubule diameters: 1) are connected with the interactions between microtubules and actin microfilaments lying along these microtubules; 2) can be the driving force of the rotary motion of lipotubuloids.     

Kinesin follows the microtubule's protofilament axis

We tested the hypothesis that kinesin moves parallel to the microtubule's protofilament axis. We polymerized microtubules with protofilaments that ran either parallel to the microtubule's long axis or that ran along shallow helical paths around the cylindrical surface of the microtubule. When gliding across a kinesin-coated surface, the former microtubules did not rotate. The latter microtubules, those with supertwisted protofilaments, did rotate; the pitch and handedness of the rotation accorded with the supertwist measured by electron cryo- microscopy. The results show that kinesin follows a path parallel to the protofilaments with high fidelity. This implies that the distance between consecutive kinesin-binding sites along the microtubule must be an integral multiple of 4.1 nm, the tubulin monomer spacing along the protofilament, or a multiple of 8.2 nm, the dimer spacing.     

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.     

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.     

Microtubules switch occasionally into unfavorable configurations during elongation

Tubulin assembles to form a range of structures that differ by their protofilament and monomer helix-start numbers. The microtubule lattice is believed to accommodate these different configurations by skewing the protofilaments so that the lateral interactions between tubulin subunits are maintained. Here, we present the characterization of 14 types of microtubules, including six novel ones, through an extensive analysis of microtubules assembled in vitro from pure tubulin. Although the six new types represented only 1 % of the total length of the population examined ( approximately 17 mm), they define the limits of microtubule structure and assembly. Protofilament skewing is restricted to within +/-2 degrees. Outside this range, the restoring force induced by the skewed protofilaments is compensated by a longitudinal shift (less than +/-0.2 nm) between adjacent protofilaments. Configurations with theoretical protofilament skew angles larger than +/-4 degrees or that necessitate larger modifications of the microtubule surface lattice were not observed. Analysis of the microtubule types distribution reveals that it is sharply peaked around the less skewed conformations. These results indicate that both the flexibility of the protofilaments and the strength of their lateral interactions restrict the range of structures assembled. They also demonstrate that growing microtubules can occasionally switch into energetically unfavorable configurations, a behavior that may account for the stochastic nature of catastrophes.     

Arrangement of tubulin subunits and microtubule-associated proteins in the central-pair microtubule apparatus of squid (Loligo pealei) sperm flagella

This study provides a comprehensive, high-resolution structural analysis of the central-pair microtubule apparatus of sperm flagella. It describes the arrangement of several microtubule-associated "sheath" components and suggests, contrary to previous thinking, that microtubules are structurally asymmetric. The two microtubules of the central pair are different in several respects: the C2 tubule bears a single row of 18-nm-long sheath projections with an axial periodicity of 16 nm, whereas the C1 tubule possesses rows of 9-nm globular sheath components with an axial repeat of 32 nm. The lumen of the C2 tubule always appears completely filled with electron-dense material; that of the C1 tubule is frequently hollow. The C2 tubule also possesses a series of beaded chains arranged around the microtubule; the beaded chains are composed of globular subunits 7.5-10 nm in diameter and appear to function in the pairing of the C1 and C2 tubules. These findings indicate: that the beaded chains are not helical, but assume the form of lock washers arranged with a 16-nm axial periodicity on the microtubule; and that the lattice of tubulin dimers in the C2 tubule is not helically symmetric, but that there are seams between certain pairs of protofilaments. Proposed lattice models predict that, because of these seams, central pair and perhaps all singlet microtubules may contain a ribbon of 2-5 protofilaments that are resistant to solubilization; these models are supported by the results of the accompanying paper (R. W. Linck, and G. L. Langevin. 1981. J. Cell Biol. 89: 323-337.     

Electron microscopic observations of an unusual cytoplasmic inclusion in the calcium-starved white mutant (LU887 × LU897) of Physarum polycephalum

Electron micrographs of Physarum polycephalum microplasmodia (LU887 × LU897) reveal cytoplasmic inclusions that appear “striated” at low magnifications; at higher magnifications these exhibit a structure that we have interpreted as microtubule bundles. The light and dark regions in the inclusions are due to the affinity of some microtubules for osmic acid; these appear to have dense regions while other microtubules remain electron lucent. The diameters of the microtubules are about 32–33nm; the subunits forming the tubule walls measure about 8–9nm in diameter. The diameter measurements are slightly larger than the dimensions assigned to vertebrate microtubules (28nm); however, the diameter of the subunits in the microtubule wall measures about 8–9nm which is essentially the same measurement reported for vertebrate tubulin dimers.     

On the surface lattice of microtubules: helix starts, protofilament number, seam, and handedness

The tubulin monomers of brain microtubules reassembled in vitro are arranged on a 3-start helix, irrespective of whether the number of protofilaments is 13 or 14. The dimer packing is that of the B-lattice described for flagellar microtubules. This implies that the tubulin core of microtubules contains at least one helical discontinuity. Neither 5-start nor 8-start helices have a physical significance and thus cannot be implicated in models of microtubule elongation, but the structure is compatible with elongation of protofilaments by dimers or protofilamentous oligomers. The inner and outer surfaces of the microtubule wall can be visualized by propane jet freezing, freeze fracturing, and metal replication, at a resolution of at least 4 nm. The 3-start helix is left-handed, in contrast to a previous study based on negative staining and shadowing. The reasons for this discrepancy are 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.     

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.     

Assembly and three-dimensional image reconstruction of tubulin hoops

The three-dimensional structure of tubulin hoops has been determined by image reconstruction. The surface lattice of hoops is similar to that of microtubules, but in addition hoops possess a superstructure of protofilament triplets. The protofilaments differ mainly in their apparent volumes and lateral spacings. The volumes depend strongly on the orientation on the carbon support, while the spacings do not. The differences of appearance do not reflect changes of intrinsic subunit structure. They are explained by differential staining related to the orientation and packing of protofilament. Microtubule-associated proteins do not contribute to the average subunit structure. All apparent protofilament structures differ from that expected from X-ray patterns of microtubules in terms of subunit tilt and distribution of contrast. It is concluded that the negatively stained structure is a reliable representation of the arrangement of protein subunits, but not of their shape. Tubulin hoops occur in conditions of microtubule assembly near the critical concentration in a stabilizing buffer. Their formation depends on microtubule-associated proteins and on the initial presence of tubulin oligomers, which may associate into short protofilament triplets. If their elongation is rapid compared to lateral aggregation, they form closed hoops. The growth phase is followed by a redistribution phase, during which hoops disappear in favour of microtubules. This behaviour is explained by kinetic overshoot assembly. Each triplet resembles an incomplete microtubule wall so that the junction between two triplets may be compared to a junction between microtubule walls. Such junctions are formed by a closely spaced pair of protofilaments. They are analogous to junctions between microtubules and incomplete microtubule walls, and they have the same clockwise curvature when viewed at the growing end.     

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.     

Limited flexibility of the inter-protofilament bonds in microtubules assembled from pure tubulin

The superposition of the regular arrangement of tubulin subunits in microtubules gives rise to moiré patterns in cryo-electron micrographs. The moiré period can be predicted from the dimensions of the tubulin subunits and their arrangement in the surface lattice. Although the average experimental moiré period is usually in good agreement with the theoretical one, there is variation both within and between microtubules. In this study, we addressed the origin of this variability. We examined different possibilities, including artefacts induced by the preparation of the vitrified samples, and variations of the parameters that describe the microtubule surface lattice. We show that neither flattening nor bending of the microtubules, nor changes in the subunit dimensions, can account for the moiré period variations observed in 12 and 14 protofilament microtubules. These can be interpreted as slight variations, in the range –0.5 Å to +0.9 Å, of the lateral interactions between tubulin subunits in adjacent protofilaments. These results indicate that the inter-protofilament bonds are precisely maintained in microtubules assembled in vitro from pure tubulin. The fact that the moiré period is not affected by bending indicates that the local interactions between tubulin subunits are sufficiently stiff to accommodate large deformations of the microtubule wall.     

A single protofilament is sufficient to support unidirectional walking of Dynein and Kinesin

Cytoplasmic dynein and kinesin are two-headed microtubule motor proteins that move in opposite directions on microtubules. It is known that kinesin steps by a 'hand-over-hand' mechanism, but it is unclear by which mechanism dynein steps. Because dynein has a completely different structure from that of kinesin and its head is massive, it is suspected that dynein uses multiple protofilaments of microtubules for walking. One way to test this is to ask whether dynein can step along a single protofilament. Here, we examined dynein and kinesin motility on zinc-induced tubulin sheets (zinc-sheets) which have only one protofilament available as a track for motor proteins. Single molecules of both dynein and kinesin moved at similar velocities on zinc-sheets compared to microtubules, clearly demonstrating that dynein and kinesin can walk on a single protofilament and multiple rows of parallel protofilaments are not essential for their motility. Considering the size and the motile properties of dynein, we suggest that dynein may step by an inchworm mechanism rather than a hand-over-hand mechanism.     

Microtubule dynamics and microtubule caps: a time-resolved cryo- electron microscopy study

Microtubules display the unique property of dynamic instability characterized by phase changes between growth and shrinkage, even in constant environmental conditions. The phases can be synchronized, leading to bulk oscillations of microtubules. To study the structural basis of dynamic instability we have examined growing, shrinking, and oscillating microtubules by time-resolved cryo-EM. In particular we have addressed three questions which are currently a matter of debate: (a) What is the relationship between microtubules, tubulin subunits, and tubulin oligomers in microtubule dynamics?; (b) How do microtubules shrink? By release of subunits or via oligomers?; and (c) Is there a conformational change at microtubule ends during the transitions from growth to shrinkage and vice versa? The results show that (a) oscillating microtubules coexist with a substantial fraction of oligomers, even at a maximum of microtubule assembly; (b) microtubules disassemble primarily into oligomers; and (c) the ends of growing microtubules have straight protofilaments, shrinking microtubules have protofilaments coiled inside out. This is interpreted as a transition from a tense to a relaxed conformation which could be used to perform work, as suggested by some models of poleward chromosome movement during anaphase.     

Structural dynamics within mixed populations of microtubules and protofilament ribbons

In the presence of glycerol, microtubule proteins reassemble into both microtubules and protofilament ribbons with C- and S-shaped cross-section profiles. By means of electron micrographs of cross-sectioned assemblies, we have demonstrated that, during the steady state, the percentage of ribbons, especially of C-shaped ones, decreases in favour of the formation of microtubules. The following conversion modes are discussed: A, closure of the protofilament wall by increasing its curvature; B, lateral association of C-ribbons; C, completion of C-ribbons to microtubules by lateral association of tubulin; D, disassembly of ribbons and elongation of microtubules. We conclude that ribbon disassembly proceeding in an end-wise fashion and microtubule elongation is the favoured mode of conversion. Microtubule-associated proteins were found to be required for the steady-state conversions of ribbons into microtubules. In the absence of microtubule-associated proteins, C-ribbons associate laterally, forming S-ribbons. It is shown that the protofilaments of the counter-curved parts of S-ribbons have the same polarity.     

Evidence for a mixed lattice in microtubules reassembled in vitro

An extensive structural analysis of microtubules assembled in vitro has been carried out using electron microscopy in conjunction with computer analysis based on Fourier transforms and helical diffraction theory. Microtubules assembled in vitro displayed a range of numbers of protofilaments from 12 to 16, with 14 the most abundant (84% in one large sampling). In almost all structures observed protofilaments are staggered to form a characteristic 3-start shallow helix. The presence of the 3-start helix was confirmed by fiber tilting experiments to correct the effects of microtubule flattening. Since α and β tubulin subunits alternate along the protofilaments, continuous helical lattices can be constructed with interactions between adjacent protofilaments involving unlike subunits (type A lattice) or like subunits (type B lattice). However, the 14-protofilament, 3-start microtubules are incompatible with either the A or B-type continuous helical lattice. Evidence is presented which indicates that lattice discontinuities are present which generate features of both the A and B-types, with the latter predominating.     

Biochemical and Structural Analyses of Microtubules in the Pellicular Membrane of Leishmania tropica1

The structure of the major protein of the pellicular membrane of Leishmania tropica was investigated. This protein is composed of two polypeptides, of ca. 50,000 d molecular weight, that were found to cross-react immunologically with the α and β subunits of pig brain tubulin. The polypeptides and pig brain tubulin subunits were partially digested with S. aureus V8 protease, and the peptides obtained analysed by SDS-polyacrylamide gel electrophoresis. A comparison of the patterns showed that the β subunits of Leishmania and pig tubulin have very similar primary structures, while the α subunits have evolved divergently. These experiments demonstrate that the major polypeptides found in the pellicular membrane of L. tropica are α and β subunits of tubulin. Immuno-electron microscopy indicates that the tubulin is located in the microtubules associated with the pellicular membrane of Leishmania. Arrays of microtubules were prepared by nonionic detergent treatment of the cells and observed by electron microscopy after negative staining. Optical diffraction reveals a 5 nm spacing between protofilaments in the microtubule and a 4 nm axial periodicity corresponding to the tubulin subunits. The pitch of the shallow left-hand three-start helix is 12°. A distance of 47 nm separates each microtubule from the next. These data show that the dimensions and supramolecular organization of the tubulin subunits in the microtubules are identical in the pellicular membrane of L. tropica and in mammalian brain.     

Tubulin protofilaments and kinesin-dependent motility

Microtubules are built of tubulin subunits assembled into hollow cylinders which consist of parallel protofilaments. Thus, motor molecules interacting with a microtubule could do so either with one or several tubulin subunits. This makes it difficult to determine the structural requirements for the interaction. One way to approach the problem is to alter the surface lattice. This can be done in several ways. Proto-filaments can be exposed on their inside (C-tubules or "sheets"), they can be made antiparallel (zinc sheets), or they can be rolled up (duplex tubules). We have exploited this polymorphism to study how the motor protein kinesin attached to a glass surface interacts and moves the various tubulin assemblies. Microtubules glide over the surface along straight paths and with uniform velocities. In the case of C-tubules, approximately 40% glide similarly to microtubules, but a major fraction do not glide at all. This indicates (a) that a full cylindrical closure is not necessary for movement, and (b) that the inside surface of microtubules does not support gliding. With zinc sheets, up to 70% of the polymers move, but the movement is discontinuous, has a reduced speed, and follows along a curved path. Since zinc sheets have protofilaments alternating in orientation and polarity, this result suggests that in principle a single protofilament can produce movement, even when its neighbors cannot. Duplex microtubules do not move because they are covered with protofilaments coiled inside out, thus preventing the interaction with kinesin. The data can be explained by assuming that the outside of one protofilament represents the minimal track for kinesin, but smooth gliding requires several parallel protofilaments. Finally, we followed the motion of kinesin-coated microbeads on sea-urchin sperm flagella, from the flagellar outer doublet microtubules to the singlet microtubule tips extending from the A-tubules. No change in behavior was detected during the transition. This indicates that even if these microtubules differ in surface lattice, this does not affect the motility.     

MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments

MAP2 and tau exhibit microtubule-stabilizing activities that are implicated in the development and maintenance of neuronal axons and dendrites. The proteins share a homologous COOH-terminal domain, composed of three or four microtubule binding repeats separated by inter-repeats (IRs). To investigate how MAP2 and tau stabilize microtubules, we calculated 3D maps of microtubules fully decorated with MAP2c or tau using cryo-EM and helical image analysis. Comparing these maps with an undecorated microtubule map revealed additional densities along protofilament ridges on the microtubule exterior, indicating that MAP2c and tau form an ordered structure when they bind microtubules. Localization of undecagold attached to the second IR of MAP2c showed that IRs also lie along the ridges, not between protofilaments. The densities attributable to the microtubule-associated proteins lie in close proximity to helices 11 and 12 and the COOH terminus of tubulin. Our data further suggest that the evolutionarily maintained differences observed in the repeat domain may be important for the specific targeting of different repeats to either alpha or beta tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces.