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.     

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.     

Mechanics of Microtubules: Effects of Protofilament Orientation

Microtubules are hollow cylindrical polymers of the protein tubulin that play a number of important dynamic and structural roles in eukaryotic cells. Both in vivo and in vitro microtubules can exist in several possible configurations, differing in the number of protofilaments, helical rise of tubulin dimers, and protofilament skew angle with respect to the main tube axis. Here, finite element modeling is applied to examine the mechanical response of several known microtubule types when subjected to radial deformation. The data presented here provide an important insight into microtubule stiffness and reveal that protofilament orientation does not affect radial stiffness. Rather, stiffness is primarily dependent on the effective Young's modulus of the polymerized material and the effective radius of the microtubule. These results are also directly correlated to atomic force microscopy nanoindentation measurements to allow a more detailed interpretation of previous experiments. When combined with experimental data that show a significant difference between microtubules stabilized with a slowly hydrolyzable GTP analog and microtubules stabilized with paclitaxel, the finite element data suggest that paclitaxel increases the overall radial flexibility of the microtubule wall.     

Toward understanding the structure and interactions of microtubules and motor proteins

To obtain an overall three-dimensional picture of the interaction between microtubules and the motor proteins of the kinesin family it will be necessary to take account of both atomic resolution structures obtained by X-ray crystallography and medium resolution reconstructions obtained by electron cryomicroscopy. We examine the problems associated with obtaining the required structural information from electron micrographs of vitreous ice-embedded microtubules decorated with motor domains. We find that the minus-end directed motor, ncd, decorates microtubules with an 80 Å periodicity as for kinesin. Our theoretical analysis and experiments with ncd illustrate the difficulty in determining unambiguously the surface lattice organization by diffraction analysis of micrographs. 3D reconstructions of decorated microtubules are required to accurately locate the motor domains. Helical diffraction theory is not usually applicable because microtubules are cylindrical structures that rarely have complete helical symmetry. We propose using a back-projection method based on the long pitch helices formed by individual protofilaments. Model reconstructions show that this approach is feasible. © 1995 Wiley-Liss, Inc.     

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.     

A structural analysis of the interaction between ncd tail and tubulin protofilaments

ncd is a minus-end directed, kinesin-like motor, which binds to microtubules with its motor domain and its cargo domain as well. Typical of retrograde motors, the motor domain of ncd locates to the C-terminal end of the polypeptide chain, and hence, the cargo domain constitutes the N-terminal region. To date, several studies have investigated the interaction properties of the motor domain with microtubules, but very few structural data are available about the tail itself or its interaction with microtubules as cargo. Here, we applied cryo-electron microscopy and helical 3D image reconstruction to 15 protofilament microtubules decorated with an ncd tail fragment (N-terminal residues 83-187, named NT6). In our study, the ncd tail shows a behaviour resembling filamentous MAPs such as tau protein, exhibiting a highly flexible structure with no large globular domains. NT6 binds to four different sites on the outer side of microtubules within the proximity of the kinesin motor-binding site. Two of these sites locate within the groove between two neighbouring protofilaments, and appear as strong binding sites, while the other two sites, located at the outer rim, appear to play a secondary role. In addition, the ncd tail fragment induces the formation of large protofilament sheets, suggesting a tail-induced modification of lateral protofilament contacts.     

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.     

Arrangement of high molecular weight associated proteins on purified mammalian brain microtubules

The arrangement of the high molecular weight proteins associated with the walls of reconstituted mammalian brain microtubules has been investigated by electron microscopy of negatively stained preparations. The images are found to be consistent with an arrangement whereby the high molecular weight molecules are spaced 12 tubulin dimers apart, i.e., 960 A, along each protofilament of the microtubule, in agreement with the relative stoichiometry of tubulin and high molecular weight protein. Molecules on neighbouring protofilaments seem to be staggered so that they give rise to a helical superlattice, which can be superimposed on the underlying tubulin lattice. In micrographs of disintegrating tubules there is some indication of lateral interactions between neighbouring high molecular weight molecules. When the microtubules are depolymerized into a mixture of short spirals and rings, the high molecular weight proteins appear to remain attached to their respective protofilaments.     

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.     

Direct visualization of the microtubule lattice seam both in vitro and in vivo

Microtubules are constructed from alpha- and beta-tubulin heterodimers that are arranged into protofilaments. Most commonly there are 13 or 14 protofilaments. A series of structural investigations using both electron microscopy and x-ray diffraction have indicated that there are two potential lattices (A and B) in which the tubulin subunits can be arranged. Electron microscopy has shown that kinesin heads, which bind only to beta-tubulin, follow a helical path with a 12-nm pitch in which subunits repeat every 8-nm axially, implying a primarily B-type lattice. However, these helical symmetry parameters are not consistent with a closed lattice and imply that there must be a discontinuity or "seam" along the microtubule. We have used quick-freeze deep-etch electron microscopy to obtain the first direct evidence for the presence of this seam in microtubules formed either in vivo or in vitro. In addition to a conventional single seam, we have also rarely found microtubules in which there is more than one seam. Overall our data indicates that microtubules have a predominantly B lattice, but that A lattice bonds between tubulin subunits are found at the seam. The cytoplasmic microtubules in mouse nerve cells also have predominantly B lattice structure and A lattice bonds at the seam. These observations have important implications for the interaction of microtubules with MAPs and with motor proteins, and for example, suggest that kinesin motors may follow a single protofilament track.     

Behavior of C-shaped microtubule endings in the cell

The association of incomplete microtubule assemblies with either another incomplete structure or complete microtubules was studied in two organisms, the phytoflagellate Polytoma papillatum and the phorid fly Megaselia scalaris, using transmission electron microscopy. In the alga, hook-shaped appendages on cytoplasmic and spindle microtubules were detected. These resulted from the lateral association of a curved ribbon of protofilaments with the surface of a complete microtubular wall. In the fly, an S-shaped protofilament sheet was found embedded in the kinetochore plate of a prometaphase I spermatocyte. Tracing of the S-shaped element towards the spindle pole revealed that it was formed by the lateral junction of two curved protofilament sheets. In all cases, the C-shaped protofilament sheets represented the endings of complete microtubules. Incomplete microtubules are generally considered as representing intermediates of microtubule assembly and disassembly. Since high molecular weight proteins are believed to be responsible for maintaining microtubule-microtubule spacing, it is hypothesized that the endings of growing and shrinking microtubules are sparsely studded with these proteins; their depletion allows lateral microtubule contacts. In addition, the microtubule-microtubule contacts may be rendered possible by the flexibility of the slender elongated microtubule-associated molecules. Relatively long C-shaped protofilament appendages (0.6-1.4 microns) were detected in this study. Therefore, it is plausible to assume that the protofilament sheets are stabilized by contact with one another or with an intact tubule wall.     

Structural rearrangements in tubulin following microtubule formation

Microtubules are essential cytoskeletal structures that mediate several dynamic processes in a cell. To shed light on the structural processes relating to microtubule formation and dynamic instability, we investigated microtubules composed of 15 protofilaments using cryo-electron microscopy, helical image reconstruction and computational modelling. Analysis of the configuration of the alpha beta-tubulin heterodimer shows distinct structural differences in both subunits, and illustrates that the tubulin subunits have different roles in the microtubule lattice. Our modelling data suggest that after GTP hydrolysis microtubules, adopt a conformational state somewhere between a straight protofilament conformation--as found in zinc-induced tubulin sheets--and an outward curved conformation--as found in tubulin-stathmin complexes. The tendency towards a curved conformation seems to be mediated mostly by beta-tubulin, whereas alpha-tubulin resembles a state more related to the straight structure. Our data suggest a possible explanation of dynamic instability of microtubules, and for nucleotide-sensitive microtubule-binding properties of microtubule-associated proteins and molecular motors.     

Effect of microtubule-associated proteins on the protofilament number of microtubules assembled in vitro

In order to demonstrate the effect of microtubule-associated proteins on the protofilament number of microtubules, we used different systems of microtubule formation in vitro in which these proteins are either functionally eliminated (by DNA or glycerol) or absent (purified tubulin). The results obtained by electron microscopy of ultrathin-sectioned material indicate that under standard conditions in the presence of microtubule-associated proteins microtubules are formed consisting predominantly of 14 protofilaments. In cases of deficiency of microtubule-associated proteins, the mean value of the protofilament number is lower, and the protofilament number within the microtubule population varies remarkably. On the other hand, the action of microtubule-associated proteins is enhanced by histones resulting in increased protofilament numbers. A model is proposed illustrating that the quality and the quantity of microtubule-associated proteins bound to microtubules determine the curvature between the protofilaments and restrict the variety of their binding angles. In this way the microtubule-associated proteins may be regarded as an important factor in determining the structural fidelity of microtubules.     

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.     

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.     

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.     

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.     

Orientation and structure of the Ndc80 complex on the microtubule lattice

The four-subunit Ndc80 complex, comprised of Ndc80/Nuf2 and Spc24/Spc25 dimers, directly connects kinetochores to spindle microtubules. The complex is anchored to the kinetochore at the Spc24/25 end, and the Ndc80/Nuf2 dimer projects outward to bind to microtubules. Here, we use cryoelectron microscopy and helical image analysis to visualize the interaction of the Ndc80/Nuf2 dimer with microtubules. Our results, when combined with crystallography data, suggest that the globular domain of the Ndc80 subunit binds strongly at the interface between tubulin dimers and weakly at the adjacent intradimer interface along the protofilament axis. Such a binding mode, in which the Ndc80 complex interacts with sequential α/β-tubulin heterodimers, may be important for stabilizing kinetochore-bound microtubules. Additionally, we define the binding of the Ndc80 complex relative to microtubule polarity, which reveals that the microtubule interaction surface is at a considerable distance from the opposite kinetochore-anchored end; this binding geometry may facilitate polymerization and depolymerization at kinetochore-attached microtubule ends.     

Characteristics of the sperm tail of the velvet worm, Euperipatoides leuckarti (Onychophora)

The onychophoran sperm tail contains several kinds of microtubulcs; probably more than that of any other animal group. There are thus a peripheral manchette consisting of many tightly spaced microtubules, a ring of nine 'peripheral singlets' and a central axoneme of the classical 9 + 2 type (nine doublets and two central singlets). The protofilament organization of these various microtubules was examined and compared to the structure and mode of formation of the peripheral singlets with that of its analogues in other animal groups. The onychophoran peripheral singlets were found to differ in two respects from those in insects: they are formed from the manchette rather than from the axonemal doublets and their transient connection to the axoneme is to the A-subtubules of the doublet rather than to the B-subtubules. The manchette microtubules as well as the peripheral singlets consist of 13 protoh'laments. The manchette may serve a mechanical function (to strengthen the unusually thick sperm tail) hut the role of the peripheral singlets remains unknown.     

Posttranslational acetylation of α-tubulin constrains protofilament number in native microtubules

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Highlights? α-TAT mutants have short microtubules and variable protofilament number ? α-tubulin K40 acetylation promotes interprotofilament salt bridges ? α-tubulin K40 acetylation is a key constraint on protofilament number in vivo