- Email: [email protected]
Tubulin and microtubule structure
16
Tubulin and microtubule structure Kenneth H Downing* and Eva Nogales Our knowledge of microtubule structure and its relationship to microtubule function continues to grow. Cryo-electron microscopy has given us new images of the microtubule polymerization and depolymerization processes and of the interaction of these polymers with motor proteins. We now know more about the effect of nucleotide state on the structure and dynamic instability of microtubules. The atomic model of tubulin, very recently obtained by electron crystallography, is bringing new insight into the properties of this protein and its self-assembly into microtubules, and promises to inspire new experimental efforts that should lead us to an understanding of the microtubule system at the
system is maintained by a cap of tubulin.GTP at the ends, and when this cap is lost the microtubule can come apart. This property is the basis for the dynamic instability of microtubules. It has been shown that a cap of one single tubulin.GTP per protofilament is sufficient to stabilize the microtubule end [1°]. Recent experiments have added a new twist to the dynamic instability problem by showing that microtubules may not immediately begin shortening after being cut, even though there could be no G T P cap at the cut site [2°]. This observation indicates that there is a third kinetic state in addition to the generally recognized growing and shortening states.
molecular level.
Addresses Lawrence Berkeley National Laboratory, Donner Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA *e-maih [email protected]
Current Opinion in Cell Biology 1998, 10:16-22 http :/Ibiomednet.comlelecreflO95506 7401000016 © Current Biology Ltd ISSN 0955-0674
Introduction Microtubules are ubiquitous cytoskeletal structures that are formed by the self-assembly of a13 tubulin heterodimers. Around thirteen parallel protofilaments, each of which is a linear arrangement of dimers, form the microtubule wall to which a variety of microtubule-associated proteins and motor proteins bind. Microtubules are involved in diverse functions that include cell movement, vesicle transport, and chromosome segregation during mitosis. Essential to microtubule functions are both their polarity and their dynamic nature. Numerous ligands also bind to tubulin, affecting its assembly properties, among them several drugs that have proven to have anticancer properties. T h e present review is dedicated to recent progress in the structural and functional characterization of microtubules and their building block, tubulin. (x a n d 13 t u b u l i n o~ and 13 tubulin (proteins o f - 4 5 0 amino acids each) are highly homologous. Each monomer binds a guanine nucleotide, which is nonexchangeable in et tubulin (the nucleotide binding site in ct tubulin is known as the N-site) and exchangeable in 13 tubulin (the binding site in 13 tubulin is known as the E-site). G T P at the E-site is required for microtubule assembly, and its hydrolysis follows addition of a dimer to the microtubule end, upon which it becomes non-exchangeable. T h e stability of the
Both c~ and 13 subunits exist in several isotypic forms and undergo a variety of post-translational modifications. T h e catboxy-terminal residues of both ~t and 13 monomers are highly varied among isotypes, and thus have been proposed to be involved in discrimination among isotypes. T h e significance of the various tubulin isotypes is receiving increasing attention. Luduefia [3 °°] has reviewed isotype diversity and several hypotheses about the evolution and function of isotypes. It has long been recognized that different isotypes are often found with different distributions in different cell types, or in different sets of microtubules in a single cell, and yet they can often co-polymerize. Recent experiments continue to demonstrate that isotypes are partially but not completely interchangeable. An isotype expressed in an unnatural context can usually form microtubules, but these may have a different character (e.g. number of protofilaments) or lack the function of the normal isotype in that context (see [4] for a review). Experiments with transgenic Drosophila, incorporating a moth 13tubulin [5°°], have shown that when expressed alone in spermatids the moth tubulin failed to form microtubules, when expressed as a small fraction of the total [3 tubulin, it caused formation of aberrant axonemes, some with moth-like accessory tubules. Studies of microtubule structure Structural information on microtubules came originally from electron microscopy studies of negatively stained samples. These studies showed the general arrangement of subunits in the microtubule, but were limited in resolution and suffered from artifacts of negative stain and dehydration. T h e development of cryoelectron microscopy of vitrified biological specimens has provided a very reliable image of the undisturbed microtubule structure in the last six or seven years. Furthermore, rapid freezing has allowed the capturing of different structural states, leading to important insights into the structural intermediates in both the the assembly and the disassembly of microtubules. Of particular interest are the changes in the curvature of protofilaments that have been shown to
Tubulin and microtubule structure Downing and Nogales
accompany the assembly and disassembly processes (see below). The study of the interaction between microtubules and motor proteins using cryoelectron microscopy has been an especially hot topic for the past three years [6,7,8,9°',10",11"]. This flurry of activity in the study of microtubule-motor complexes in at least five different labs is a result of the convergence of maturing cryoelectron microscopy methodology and molecular biology that has made it possible to obtain good quantities of well-characterized motor constructs. In a recent article the atomic model of the ncd (non-claret disjunctional) motor domain was ftted into a reconstruction of an ncd-decorated microtubule [12"°]. Reconstructions of vitrified microtubules decorated with motor protein constructs, at a resolution of about 30.~, have revealed interesting facts not only about the interaction of tubulin with kinesin and ncd, but also about the arrangement of tubulin subunits within the microtubule [13,14]. These studies have confirmed that tubulin forms a B-lattice, where the main lateral contacts across protofilaments are between subunits of the same type (i.e. (x-c~ and 13-13). Microtubules with 13 protofilaments, the most common type in vivo, have a 'seam' along their length in which the lateral contacts are reversed (i.e. ~-~ and ~--oc contacts occur), resulting in loss of helical symmetry. Although the relevance of this 'seam' is still being debated, it could be related to the microtubule polymerization process, as indicated by time-resolved cryoelectron microscopy which shows that microtubules grow by elongation of open sheets that later close into a cylinder [15]. The study of microtubules using helical reconstruction methods (a set of techniques used in electron microscopy to obtain 3-D maps of objects with helical symmetry) forces the selection of certain microtubules within a population for which the right combination of protofilament number and star helices results in true helical symmetry (a star helix of 3, the most common in microtubules, indicates that 3 helices are required to cover the entire surface of the microtubule). Reconstructions have been obtained with 10-2 (i.e. 10 protofilament, 2 star) microtubules [6], 15-4 [10°,16], or 16--4 [8,11"] naturally occurring microtubules from crickets. Figure 1 illustrates the features seen in these reconstructions. A different kind of approach, the back-projection method, takes advantage of the supertwist in microtubule protofilaments to do 'tomography without tilt' [9"',17]. While reconstructions by helical and back-projection methods give very similar results [16,17], the latter has the advantage that it can resolve the seam in non-helical structures. The helical approach has the advantage that it is less computationally demanding, but caution has to be taken as seams have been found unexpectedly in microtubules that seemed to obey the 'perfect helix' rules and thus had been assumed to be helical [16]. Finally, studies of open microtubules in negative stain using tilt series reconstructions have
17
Figure 1
(a)
(b)
(¢)
Current Opinion in Cell Biology
Three-dimensional view of a microtubule. The microtubule structure was obtained by cryoelectron microscopy of vitrified samples and helical reconstruction of 15-4 microtubules (15 protofilaments, 4 star). Different sections through the density are shown. (a) Section showing the outside microtubule surface. (b) Inside surface of the microtubule. The cross section corresponding to one dimer (80 A in length) is marked in light grey. The orientation of the microtubule in (a) and (b) is such that the plus end corresponds to the top of the figure. (c) End-on view of the microtubule from the minus end showing the clockwise slew of the protofilaments. This figure was made with a reconstruction provided by Ronald Milligan and Michael Whittaker.
also been used to study the interaction of tubulin with kinesin and ncd. These studies have suggested a possible
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Cytoskeleton
conformational change in tubulin upon binding of motor proteins [18"]. While caution has to be taken due to possible negative stain artifacts, these reconstructions have the highest resolution of these complexes to date. Determination of microtubule polarity by kinesin decoration of microtubules growing from axonemes resulted originally in opposite conclusions from different research groups, although a consensus now seems to have been found [13,19]. New methods have been developed to determine the polarity of microtubules more directly. Images of vitrified microtubules obtained by cryoelectron microscopy display a lengthwise arrowhead pattern that arises from the superposition of the front and back views of the microtubule wall. It has been shown that these arrowhead moir6 patterns point toward the plus end when the protofilament skew is right-handed, and toward the minus end when it is left-handed [20"] (see Figure 2). The hand of the skew can be obtained by comparing views of the microtubule tilted to different angles, or by examination of the diffraction patterns from microtubule images (see Figure 2b,e). Microtubule polarity has now also been related to three-dimensional reconstructions of both motor-decorated and undecorated microtubules [9"']. In an end-on view of the microtubule from the minus end, there is a clockwise slew of the tubulin monomers (see Figure lc), while there is an anticlockwise slew of the motor.
Further information on microtubules and other tubulin polymers has come from X-ray solution scattering. This technique has the advantage of good sample preservation and continuous time resolution. Interpretation of the data relies on electron microscopy information and/or on computational models that simulate the scattering profiles for different polymer forms. T h e technique has been recently used to characterize the assembly of tubulin.GDP in the presence of taxoids [21"]. T h e scattering profiles have been interpreted by reference to the known scattering from tubulin dimers, double rings [22] and microtubules [23]. Double rings or microtubules were formed from tubulin.GDP depending on the divalent ion concentration, the presence of taxoid and the temperature. Both assemblies require the initial presence of small linear tubulin oligomers, whose formation is magnesium induced. Systematic proteolysis studies on tubulin dimers and microtubules have been conducted in an effort to gain information both on the surface structure of tubulin and on the sequence segments that could be involved in microtubule polymerization [24°]. T h e studies show five major proteolytic regions within each monomer. Two of the nicking points found in (x tubulin and one of the points found in 13tubulin become protected upon polymerization, indicating their possible involvement in subunit contacts within the microtubule.
Visualization of tubulin structure Electron crystallography of zinc-induced tubulin sheets has been used during the past few years to gain higher and higher resolution reconstructions of tubulin [25,26]. An atomic model of tubulin has recently been obtained using this technique [27"']. T h e model shows that the (x and 13tubulin monomers have basically identical structures (Figure 3). Each monomer is formed by two interacting beta sheets surrounded by alpha helices. T h e monomer structure is very compact, but can be divided into three functional domains. T h e amino-terminal domain forms a Rossmann fold (five alpha helices and six parallel beta strands), at the base of which sits the nucleotide. T h e intermediate domain is formed by three sequential alpha helices followed by a mixed beta sheet and two more helices, and contains the taxol-binding site. T h e carboxy-terminal domain is all alpha helical and overlaps the two previous domains, making the 'crest' of the protofilament on the outside surface of the microtubule where microtubule-associated proteins and motor proteins bind. Although the last 10 residues in (x tubulin and the last 18 residues in ~ tubulin are not seen in the map, presumably because of disorder resulting from their high charge density, it is clear that they extend from a point that is near the ridge of the protofilament. These residues are the most highly variable part of the tubulin molecule, and the main determinants of isotype variety. T h e fact that they are exposed on the outside of the microtubule is certain to promote the idea that there are isotype-selective proteins interacting with the microtubule that may provide the functional basis for microtubule isotype variety and which would be crucial to constructing specific types of microtubules and determining their particular function.
In the atomic model, the two monomers have remarkably similar structures, even though they are in different nucleotide-bound states ( G T P is bound to (~ tubulin and GDP and taxol are bound to 13 tubulin). Researchers in the tubulin field like to talk about two tubulin conformations; a 'straight' conformation of tubulin with bound G T P at the E-site in 13 tubulin (tubulin.GTP), and a 'curved' conformation containing tubulin.GDP. Within the microtubule, tubulin.GDP is kept in a 'straight' conformation by the subunit contacts within the lattice. Electron microscopy studies have shown that, upon loss of the G T P cap, protofilaments curl and peel off [28,29], revealing the 'curved' conformation of the tubulin.GDP and often forming closed rings. Such images are in agreement with studies showing that the free energy of hydrolysis following addition of a new dimer to a microtubule end is stored in the microtubule lattice [30], and is only released upon depolymerization. Presumably the lattice constraints that maintain the tubulin.GTP-like conformation in microtubules are also present in the zinc-induced sheets used for the electron crystallographic studies.
Tubulin and microtubule structure Downing and Nogales
lg
Figure 2
Illl
+ end
13
]lJ ii t
t - end i i
(a)
(b)
(c)
(d)
(e)
(f) Current Opinion in Cell Biology
Determination of microtubule polarity. (a, d) Cryo-electron microscopy images of a 14-3 (14 protofilaments, 3 star) and a 14-4 (14 protofilaments, 4 star) microtubule, respectively (see text). Both microtubules are oriented with their plus ends at the top. (b, e) Corresponding computer-generated diffraction patterns. The relative positions of the peaks in the diffraction pattern, marked J3 and 111 for the 14-3 microtubule, and 14 and Jlo for the 14-4 microtubule, indicate that the microtubule in (a) is left-handed and that in (d) is right-handed. (c, f) Filtered images, obtained by Fourier synthesis with only the equatorial components of the diffraction pattern, in which the lengthwise features of the protofilaments are reinforced, thus showing the arrowhead pattern along the microtubule more cleady. This arrowhead pattern results from the superposition of the front and back views of the microtubule wall. The arrowheads point to the minus end in (c) and to the plus end in (f). Figure provided courtesy of Denis Chr~tien.
Tubulin-GTE tubulin.GMPCPP (guanylyl-(~,[~)-methylene-diphosphonate, a nonhydrolyzable analog of GTP) and tubulin.GDP in the presence of BeF 3 or taxoids form microtubules of almost indistinguishable structure [31,32]. This observation supports the hypothesis that the presence of the y phosphate (or a mimic group) or taxol
results in the same conformation of tubulin, corresponding to the 'straight' conformation. This conformation is in turn stabilized within the microtubule lattice. Microtubules made of tubulin.GDP have a slightly shorter distance between monomers along the protofilament (1-1.5/~ shorter) than those formed with nonhydrolyzable analogs [31,32].
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Cytoskeleton
Figure 3
mainly by being pulled apart as the protofilaments curl away from each other. On the other hand, hydrolysis could also have a more direct effect on interprotofilament interactions. The atomic structure suggests ways to investigate the relative importance of the different subunit contacts, perhaps by modeling the interaction energies of contacts within the lattice, or by mutational studies designed to change the balance between the lateral and longitudinal interactions.
13-tl
Figure 4
(a)
(b)
~-tL
C~rren! Opinion in Cell Biology
Ribbon diagram of the tubulin dimer. 13tubulin with bound GDP is at the top; ct tubulin with bound GTP is at the bottom. The orientation corresponds approximately to a lateral view of the dimer in a microtubule with the axis running vertically and the plus end at the top (see text), The left-hand side corresponds to the outside microtubule surface, the right-hand side to the inside. Thus, the structure in this view would fit into the microtubule reconstruction at the position marked with lighter grey in Figure lb. N, amino terminus; C, carboxyl terminus.
This is a very small change compared with that seen upon depolymerization of tubulin.GDP microtubules, where the conformational change in the 'unconstrained' tubulin.GDP results in a dramatic curvature of the protofilaments [28,29]. In spite of the small difference, it has now been shown that kinetochores can tell the difference between GTP- and GDP-bound tubulin within a microtubule [33°], and it has been proposed that this is the mechanism by which they preferentially attach near the microtubule plus end. The structural model of tubulin supports the notion that hydrolysis induces a local conformational change near the dimer interface that results in a kink, as suggested in Figure 4, which accounts for the curvature seen in images of tubulin-GDP rings and of protofilaments peeling away from disassembling microtubules [28,29]. These images suggest that lateral contacts between subunits are lost
Current Opinion in Cell Biology
Schematic diagram of a hypothetical conformational change in the structure of a microtubule protofilament upon loss of the GTP-bound 13-tubulin cap. (a) Capped protofilarnent in the 'straight' conformation as in stable rnicrotubules. (b) Uncapped protofilament (possessing GDP-bound 13-tubulin at the end) in the 'curved' conformation as in rings or depolymerizing microtubules. The proposed conformational change occurs at the longitudinal interface between tubulin dirners, near the nucleotide binding site in 13tubulin, when tubulin.GDP escapes the constraints of the microtubule lattice.
The nucleotide position in the amino-terminal domain of the structure is right at the longitudinal interface between monomers along the protofilament, so that the nucleotide in each subunit interacts with the next monomer along the protofilament, both at the interdimer ~md at the intradimer interfaces. With the definition of the dimer boundary as shown in Figure 3, the N-site in ~ tubulin is shown to be found at the intradimer interface. The N-site will then always be occluded in the dimer, regardless of the state of assembly. The nucleotide at the E-site, on the other hand, will be partially exposed in the dimer,
Tubulin and microtubule structure Downing and Nogales
where it can exchange with the solution, but will become occluded upon polymerization, resulting in its loss of exchangeability. There has been some disagreement in the literature on whether the plus ends of microtubules are crowned by cx or by 13 subunits [34,35], although the latter possibility seems to be the most favored one today. In the dimer model shown in Figure 3, the exchangeable site in 13 tubulin will be exposed at one end of the microtubule. It is most logical to conclude that this end is the plus end on the basis of the observation that nucleotide exchange only occurs at the plus end [36]. Furthermore, antibodies to the amino-terminal region of ot tubulin were found to bind only to the minus end of microtubules [37°°], further supporting the idea that 13 tubulin is on the plus end°
Conclusions Microtubules have intrigued scientists since their discovery because of both their critical importance to the cell and their interesting biophysical properties. Important clues to the processes of assembly and disassembly of microtubules have recently been gained using biochemical [1"], electron microscopy [15,28,29], and X-ray scattering [21"] techniques. We know more about the effects of nucleotides and stabilizing drugs such as taxoids both on the structure of the microtubule [31,32] and on the interaction of microtubules with motor proteins [9•',10",11"]. T h e existence of an atomic model of tubulin [27 •° ] is now bringing together biochemical, genetic and structural information to give us a picture of the properties of tubulin and microtubules at the molecular level. Knowledge of the structure of tubulin should open the possibilities for new and interesting experiments in the field of microtubule study that will lead us to a more complete understanding of microtubule structure and function.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • ••
of special interest of outstanding interest
1. •
Caplow M, Shanks J: Evidence that a single monolayer tubulin.GTP cap is both necessary and sufficient to stabilize microtubules. Mol Biol Cell 1996, 7:663-675. The minus ends of microtubules assembled from tubulin.GTP were transiently stabilized against dilution-induced disassembly by reaction with tubulin.GMPCPP. The minimum size of a tubulin.GMPCPP cap sufficient to prevent disassembly was estimated from the lifetime of the microtubulas following dilution, and from the time required for the loss of a single tubulin.GMPCPP subunit from the miorotubule end. The authors concluded that a microtubule capped with 13-14 tubulin.GMPCPP subunits switches to disassembly after only one dissociation event. 2. •
Tran PT, Walker RA, Salmon ED: A metsstable intermediate state of microtubule dynamic instability that differs significantly between plus and minus ends, J Cell Biol 1997, 138:105-117. Microtubules were cut using a UV microbeam or severed with a glass microneedle while observed by dark field microscopy or video-enhanced differential interference contrast microscopy, respectively. The severed plus ends rapidly shortened, as expected by the lack of a GTP cap, but minus ends immediately resumed elongation. These results suggest the existence of a
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metastable kinetic intermediate in dynamic instability between the elongation and shortening rates. 3. --
Luduefia RF: The multiple forms of tubulin: different gene products and covalent modifications./nt Rev Cytol 1998, 178:207-275. This is an extensive review of the functional significance of tubulin isotypes and the regulation of their expression, from protists to vertebrates. The review also covers all the known post-translational modifications on tubulin. 4.
Wilson PG, Borisy GG: Evolution of the multl-tubulin hypothesis. Bioessays 1997, 19:451-454.
5. ••
Raft EC, Fackenthai JD, Hutchens JA, Hoyle HD, Turner FR: Microtubule architecture specified by a ~-tubulin isoform. Science 1997, 275:70-74. In order to test whether orthologous ~ tubulins from different species are functionally equivalent, the moth He/iothis virescens ~2 homologue was expressed in Drosophila testes. Co-expression of the moth and fly ~ isotypes resulted in the formation of accessory microtubules with the structural characteristics of those from the moth (e.g. higher protofilament number). These experiments demonstrate that the architecture of the microtubule cytoskele o ton can be directed by a component ~ tubulin. 6.
Kikkawa M, Ishikawa T, Wakabayashi T, Hirokawa N: Threedimensional structure of the kinesin head-microtubule complex. Nature 1995, 376:274-277.
7.
Hirose K, Lockhart A, Cross RA, Amos LA: Nucleotide-dependent angular change in kinesin motor domain bound to tubulin. Nature 1995, 376:277-279.
8.
Hirose K, Amos WB, Lockhart A, Cross RA, Amos LA: Threedimensional cryoelectron microscopy of 16-protefilament microtubules: structure, polarity and interaction with motor proteins. J Struct Biol 1997, 118:140-148.
9. .•
Sosa H, Milligan RA: Three-dimensional structure of neddecorated microtubules obtained by a back-projection method. J Mo/Biol 1996, 260:743-755. Using cryoelectron microscopy and a back-projection method the authors obtained reconstructions of microtubules decorated with the motor domain of ned. A view of the reconstructions along the microtubule axis from the minus end shows that the ned motor domain tilts anticlockwise, while the tubulin subunits tilt clockwise. 10. •
Arnal I, Metoz F, DeBonis S, Wade RH: Three-dimensional structure of functional motor proteins on microfubules" Curr Bio/1996, 6:1265-1270. Three-dimensional maps of motor-microtubule complexes obtained by cryoelectron microscopy and helical reconstruction showed that the motors have one attached head and one unattached head per tubulin dimer. The unattached heads of kinesin and ned had distinctly different conformations. 11. •
Hirose K, Lockhart A, Cross RA, Amos I.A: Three-dimensional cryoelectron microscopy of dimeric kinesin and ned motor domains on microtubules" Proc Nat/Acad Sci USA 1996, 93:9539-9544, Naturally occurring 16-protofilament microtubules were decorated with dimeric kinesin and ned motor domains and images were reconstructed by cryoelectron microscopy and helical methods. The reconstructions show that the attached heads of kinesin and ned bind to tubulin in the same way, while the unattached heads point in opposite directions; the second kinesin head points towards the plus microtubule end, the second ned head towards the minus end. 12. •.
Sosa H, Dias DP, Hoenger A, Whittaker M, Wilson-Kubalek E, Sablin E, Fletterick R.I, Vale RD, Milligan RA: A model for the microtubule-Ncd motor protein complex obtained by cryoelectron microscopy and image analysis. Cell 1997, 90:217224. The atomic model of the ncd motor domain was fitted to cryoelectron microscopy reconstructions of microtubules decorated with the same ncd construct. Docking revealed a large contact surface between ncd and the microtubule; the surface includes a loop of conserved residues among kinesins. 13.
Amos L, Hirose K: The structure of microtubule-motor complexes. Curt Bio/1997, 9:4-! 1.
14.
Wade RH, Hyman AA: Microtubule structure and dynamics. Curt Opin Cell Biol 1997, 9:12-17.
15.
Chrbtian D, Fuller SD, Karsenti E: Structure of growing microfubule ends: two-dimensional sheets close into tubes at variable rates, J CellBio11995, 129:1311-1328.
16.
Sosa H, Hoenger A, Milligan RA: Three different approaches for calculating the three dimensional structure of microtubules decorated with kinesin motor proteins. J Struct Biol 1997, 118:149-158.
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17.
Metoz F, Amal I, Wade RH: Tomography without tilt: three dimensional imaging of microtubule/motor complexes. J Struct Bit)/1997, 118:159-168.
18. •
Hoenger A, Milligan RA: Motor domains of kinesin and ncd interact with microtubule protofilaments with the same binding geometry. J Mo/Biol 1997, 265:553-564. The structure and microtubule-binding properties of kinesin and ncd were compared using negative stain electron microscopy and tilt series of tubulin sheets decorated with the motor domains of those proteins. Both motors bind to the crest of the protofilament and make contact with both c¢ and tubulin. The geometry of attachment is very similar. The binding seemed to induce a conformational change in tubulin that the authors suggested could take an active role in motor directionality. 19.
Hoenger A, Milligan RA: Polarity of 2-D and 3-D maps of tubulin sheets and motor-dec•rated sheets. J Mo/Biol 1996, 263:114119.
20. •
Chr6tien D, Kenney JM, Fuller SD, Wade RH: Determination of microtubule polarity by cryo-electron microscopy. Structure 1996, 4:1031-1040. Microtubules nucleated from centrosomes were studied by cry•electron microscopy to relate their arrowhead moir6 pattern (resulting from the superposition of the front and back of the microtubule and related to the skew of the protofilaments) to their polarity. The authors show that the arrowheads point towards the plus end of microtubules with a right-handed skew, and to the minus end of microtubules with a left-handed skew. The handedness can be determined by looking at tilted views of the microtubule or by direct analysis of its diffraction pattern. Diaz JF, Andreu JM, Diakun G, Towns-Andrews E, Bordas J: Structural intermediates in the assembly of taxoid-induced microtubules and GDP-tubulin double rings: time-resolved X-ray scattering. Biophys J 1996, 70:2408-2420. The self-association of tubulin.GDP was studied by synchrotron time-resolved X-ray solution scattering. Tubulin-GDP stays as a dimer at low magnesium concentrations. At higher concentrations of divalent cations and in the presence of taxoid it assembles into either double rings or microtubules. The taxoid induces the formation of microtubular sheets that later close into a cylinder,
26.
27. Nogales E, Wolf SG, Downing KH: Atomic model of the tubulin o= dimer. Nature 1997, in press. This paper provides the first atomic model of tubulin. The model was fitted to a 3.7 A density map obtained by electron crystallography of zinc-induced tubulin sheets, and includes a molecule of GTP bound to o¢ tubulin and molecules of GDP and taxol bound to ~ tubulin. The model is related to previous biochemical and mutagenesis data that dealt with cysteine crosslinking, drug binding, and nucleotide binding, hydrolysis and exchangeability. 98.
Mandelkow E-M, Mandelkow E, Milligan RA: Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study. J Cell Biol 1991, 114:977-991.
99.
Tran PT, Joshi P, Salmon ED: How tubulin subunits are lost from the shortening ends of microtubules. J Struct Bio11997, 118:107-118.
30.
Caplow M, Ruhlen RL, Shanks J: The free energy of hydrolysis of a microtubule-bound nucleotide triphosphate is near zero: all of the free energy for hydrolysis is stored in the microtubule lattice. J Cell Bio/1994, 127:779-788.
31.
Vale RD, Coppin CM, Malik F, Kull FJ, Milligan RA: Tubulin GTP hydrolysis influences the structure, mechanical properties, end kinesin-driven transport of microtubules. J Biol Chem 1994, 269:23769-23775.
32.
HymenAA, Chr~tien D, Arnal I, Wade RH: Structural changes accompanying GTP hydrolysis of microtubules: information from a slowly hydrolyzable analog guanylyl-(¢~)-methylenediphosphonate. J Cell Biol 1995, 128:117-125.
21. •
22.
23.
Dlaz JF, Pantcs E, Bordas J, Andreu JM: Solution structure of GDP-tubulin double rings to 3 nm resolution and comparison with microtubules. J Mol Biol 1994, 238:214-225. Andreu JM, Diaz JF, Gil R, de Pereda JM, Garcia de Lacoba M, Peyrot V, Briand C, Towns-Andrews E, Bordas J: Solution structure of tax•tare-induced microtubules to 3-nm resolution. J Biol Chem 1994, 260:31785-31792.
24. •
De Pereda JM, Andreu JM: Mapping surface sequences of the tubulin dimer and tax•l-induced microtubules with limited proteolysis. Biochemistry 1996, 35:14184-14202. Proteolysis studies of tubulin dimers and microtubules with 12 different proteases revealed 80 different points of nicking by proteolysis that were mapped to (x and ~ tubulin sequences with the aid of site-directed antibodies. Although the majority of the sites remained accessible in microtubules, two sites in ot and one in ~ became protected upon polymerization. 25.
Nogales E, Wolf SG, Khan IA, Ludue~a RF, Downing KH: Structure of tubulin at 6.5 A and location of the tax•l-binding site. Nature 1995, 375:424-427.
Nogales E, Wolf SG, Downing KH: Visualizing the secondary structure of tubulin: Three-dimensional map at 4A. J Struct Bio/ 1997, 118:119-127.
33. •
Severin FF, Sorger PK, Hyman AA: Kinetochores distinguish GTP from GDP forms of the microtubule lattice. Nature 1997, 388:888-891. The authors monitor the attachment of reconstituted kinetochores from yeast to microtubules containing regions made of tubulin.GDP and tubulin.GMPCPP. Kinetochores bound preferentially to GMPCPP regions. They also studied the binding of kinetochores to microtubules of known polarity made with tubulin.GMPCPP, and observed that kinetochores bind preferentially to the plus ends. 34.
Hirose K, Fan J, Amos LA: Re-examination of the polarity of microtubules and sheets decorated with kinesin motor domain. J Mol Bio/1995, 251:329-333.
35.
Wolf SG, Nogales E, Kikkawa M, Gratzinger D, Hirokawa N, Downing KH: Interpreting a medium-resolution model of tubulin: comparison of zinc-sheet and microtubule structure. J Mol Bio11996, 263:485-501.
36.
Mitchison TJ: Localization of an exchangeable GTP binding site at the plus end of microtubules. Science 1993, 261:1044-1047.
37. •o
Fan J, Griffiths AD, Lockhart A, Cross RA, Amos LA: Microtubule minus ends can be labeled with a phage display antibody specific to ~-tubulin. J Mol Biol 1996, 259:325-330. The amino-terminal 100 residues of c( tubulin were bacterially expressed, and a clone was selected from antibody*expressing phagemid particles that reacted with the expressed (x tubulin amino terminus and native tubulin, but not with expressed ~ tubulin amino terminus. Gold beads coated with the antibody were found to bind exclusively to minus ends of axonemes, indicating that c( tubulin crowns the minus end of microtubules.
Tubulin and microtubule structure Kenneth H Downing* and Eva Nogales Our knowledge of microtubule structure and its relationship to microtubule function continues to grow. Cryo-electron microscopy has given us new images of the microtubule polymerization and depolymerization processes and of the interaction of these polymers with motor proteins. We now know more about the effect of nucleotide state on the structure and dynamic instability of microtubules. The atomic model of tubulin, very recently obtained by electron crystallography, is bringing new insight into the properties of this protein and its self-assembly into microtubules, and promises to inspire new experimental efforts that should lead us to an understanding of the microtubule system at the
system is maintained by a cap of tubulin.GTP at the ends, and when this cap is lost the microtubule can come apart. This property is the basis for the dynamic instability of microtubules. It has been shown that a cap of one single tubulin.GTP per protofilament is sufficient to stabilize the microtubule end [1°]. Recent experiments have added a new twist to the dynamic instability problem by showing that microtubules may not immediately begin shortening after being cut, even though there could be no G T P cap at the cut site [2°]. This observation indicates that there is a third kinetic state in addition to the generally recognized growing and shortening states.
molecular level.
Addresses Lawrence Berkeley National Laboratory, Donner Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA *e-maih [email protected]
Current Opinion in Cell Biology 1998, 10:16-22 http :/Ibiomednet.comlelecreflO95506 7401000016 © Current Biology Ltd ISSN 0955-0674
Introduction Microtubules are ubiquitous cytoskeletal structures that are formed by the self-assembly of a13 tubulin heterodimers. Around thirteen parallel protofilaments, each of which is a linear arrangement of dimers, form the microtubule wall to which a variety of microtubule-associated proteins and motor proteins bind. Microtubules are involved in diverse functions that include cell movement, vesicle transport, and chromosome segregation during mitosis. Essential to microtubule functions are both their polarity and their dynamic nature. Numerous ligands also bind to tubulin, affecting its assembly properties, among them several drugs that have proven to have anticancer properties. T h e present review is dedicated to recent progress in the structural and functional characterization of microtubules and their building block, tubulin. (x a n d 13 t u b u l i n o~ and 13 tubulin (proteins o f - 4 5 0 amino acids each) are highly homologous. Each monomer binds a guanine nucleotide, which is nonexchangeable in et tubulin (the nucleotide binding site in ct tubulin is known as the N-site) and exchangeable in 13 tubulin (the binding site in 13 tubulin is known as the E-site). G T P at the E-site is required for microtubule assembly, and its hydrolysis follows addition of a dimer to the microtubule end, upon which it becomes non-exchangeable. T h e stability of the
Both c~ and 13 subunits exist in several isotypic forms and undergo a variety of post-translational modifications. T h e catboxy-terminal residues of both ~t and 13 monomers are highly varied among isotypes, and thus have been proposed to be involved in discrimination among isotypes. T h e significance of the various tubulin isotypes is receiving increasing attention. Luduefia [3 °°] has reviewed isotype diversity and several hypotheses about the evolution and function of isotypes. It has long been recognized that different isotypes are often found with different distributions in different cell types, or in different sets of microtubules in a single cell, and yet they can often co-polymerize. Recent experiments continue to demonstrate that isotypes are partially but not completely interchangeable. An isotype expressed in an unnatural context can usually form microtubules, but these may have a different character (e.g. number of protofilaments) or lack the function of the normal isotype in that context (see [4] for a review). Experiments with transgenic Drosophila, incorporating a moth 13tubulin [5°°], have shown that when expressed alone in spermatids the moth tubulin failed to form microtubules, when expressed as a small fraction of the total [3 tubulin, it caused formation of aberrant axonemes, some with moth-like accessory tubules. Studies of microtubule structure Structural information on microtubules came originally from electron microscopy studies of negatively stained samples. These studies showed the general arrangement of subunits in the microtubule, but were limited in resolution and suffered from artifacts of negative stain and dehydration. T h e development of cryoelectron microscopy of vitrified biological specimens has provided a very reliable image of the undisturbed microtubule structure in the last six or seven years. Furthermore, rapid freezing has allowed the capturing of different structural states, leading to important insights into the structural intermediates in both the the assembly and the disassembly of microtubules. Of particular interest are the changes in the curvature of protofilaments that have been shown to
Tubulin and microtubule structure Downing and Nogales
accompany the assembly and disassembly processes (see below). The study of the interaction between microtubules and motor proteins using cryoelectron microscopy has been an especially hot topic for the past three years [6,7,8,9°',10",11"]. This flurry of activity in the study of microtubule-motor complexes in at least five different labs is a result of the convergence of maturing cryoelectron microscopy methodology and molecular biology that has made it possible to obtain good quantities of well-characterized motor constructs. In a recent article the atomic model of the ncd (non-claret disjunctional) motor domain was ftted into a reconstruction of an ncd-decorated microtubule [12"°]. Reconstructions of vitrified microtubules decorated with motor protein constructs, at a resolution of about 30.~, have revealed interesting facts not only about the interaction of tubulin with kinesin and ncd, but also about the arrangement of tubulin subunits within the microtubule [13,14]. These studies have confirmed that tubulin forms a B-lattice, where the main lateral contacts across protofilaments are between subunits of the same type (i.e. (x-c~ and 13-13). Microtubules with 13 protofilaments, the most common type in vivo, have a 'seam' along their length in which the lateral contacts are reversed (i.e. ~-~ and ~--oc contacts occur), resulting in loss of helical symmetry. Although the relevance of this 'seam' is still being debated, it could be related to the microtubule polymerization process, as indicated by time-resolved cryoelectron microscopy which shows that microtubules grow by elongation of open sheets that later close into a cylinder [15]. The study of microtubules using helical reconstruction methods (a set of techniques used in electron microscopy to obtain 3-D maps of objects with helical symmetry) forces the selection of certain microtubules within a population for which the right combination of protofilament number and star helices results in true helical symmetry (a star helix of 3, the most common in microtubules, indicates that 3 helices are required to cover the entire surface of the microtubule). Reconstructions have been obtained with 10-2 (i.e. 10 protofilament, 2 star) microtubules [6], 15-4 [10°,16], or 16--4 [8,11"] naturally occurring microtubules from crickets. Figure 1 illustrates the features seen in these reconstructions. A different kind of approach, the back-projection method, takes advantage of the supertwist in microtubule protofilaments to do 'tomography without tilt' [9"',17]. While reconstructions by helical and back-projection methods give very similar results [16,17], the latter has the advantage that it can resolve the seam in non-helical structures. The helical approach has the advantage that it is less computationally demanding, but caution has to be taken as seams have been found unexpectedly in microtubules that seemed to obey the 'perfect helix' rules and thus had been assumed to be helical [16]. Finally, studies of open microtubules in negative stain using tilt series reconstructions have
17
Figure 1
(a)
(b)
(¢)
Current Opinion in Cell Biology
Three-dimensional view of a microtubule. The microtubule structure was obtained by cryoelectron microscopy of vitrified samples and helical reconstruction of 15-4 microtubules (15 protofilaments, 4 star). Different sections through the density are shown. (a) Section showing the outside microtubule surface. (b) Inside surface of the microtubule. The cross section corresponding to one dimer (80 A in length) is marked in light grey. The orientation of the microtubule in (a) and (b) is such that the plus end corresponds to the top of the figure. (c) End-on view of the microtubule from the minus end showing the clockwise slew of the protofilaments. This figure was made with a reconstruction provided by Ronald Milligan and Michael Whittaker.
also been used to study the interaction of tubulin with kinesin and ncd. These studies have suggested a possible
18
Cytoskeleton
conformational change in tubulin upon binding of motor proteins [18"]. While caution has to be taken due to possible negative stain artifacts, these reconstructions have the highest resolution of these complexes to date. Determination of microtubule polarity by kinesin decoration of microtubules growing from axonemes resulted originally in opposite conclusions from different research groups, although a consensus now seems to have been found [13,19]. New methods have been developed to determine the polarity of microtubules more directly. Images of vitrified microtubules obtained by cryoelectron microscopy display a lengthwise arrowhead pattern that arises from the superposition of the front and back views of the microtubule wall. It has been shown that these arrowhead moir6 patterns point toward the plus end when the protofilament skew is right-handed, and toward the minus end when it is left-handed [20"] (see Figure 2). The hand of the skew can be obtained by comparing views of the microtubule tilted to different angles, or by examination of the diffraction patterns from microtubule images (see Figure 2b,e). Microtubule polarity has now also been related to three-dimensional reconstructions of both motor-decorated and undecorated microtubules [9"']. In an end-on view of the microtubule from the minus end, there is a clockwise slew of the tubulin monomers (see Figure lc), while there is an anticlockwise slew of the motor.
Further information on microtubules and other tubulin polymers has come from X-ray solution scattering. This technique has the advantage of good sample preservation and continuous time resolution. Interpretation of the data relies on electron microscopy information and/or on computational models that simulate the scattering profiles for different polymer forms. T h e technique has been recently used to characterize the assembly of tubulin.GDP in the presence of taxoids [21"]. T h e scattering profiles have been interpreted by reference to the known scattering from tubulin dimers, double rings [22] and microtubules [23]. Double rings or microtubules were formed from tubulin.GDP depending on the divalent ion concentration, the presence of taxoid and the temperature. Both assemblies require the initial presence of small linear tubulin oligomers, whose formation is magnesium induced. Systematic proteolysis studies on tubulin dimers and microtubules have been conducted in an effort to gain information both on the surface structure of tubulin and on the sequence segments that could be involved in microtubule polymerization [24°]. T h e studies show five major proteolytic regions within each monomer. Two of the nicking points found in (x tubulin and one of the points found in 13tubulin become protected upon polymerization, indicating their possible involvement in subunit contacts within the microtubule.
Visualization of tubulin structure Electron crystallography of zinc-induced tubulin sheets has been used during the past few years to gain higher and higher resolution reconstructions of tubulin [25,26]. An atomic model of tubulin has recently been obtained using this technique [27"']. T h e model shows that the (x and 13tubulin monomers have basically identical structures (Figure 3). Each monomer is formed by two interacting beta sheets surrounded by alpha helices. T h e monomer structure is very compact, but can be divided into three functional domains. T h e amino-terminal domain forms a Rossmann fold (five alpha helices and six parallel beta strands), at the base of which sits the nucleotide. T h e intermediate domain is formed by three sequential alpha helices followed by a mixed beta sheet and two more helices, and contains the taxol-binding site. T h e carboxy-terminal domain is all alpha helical and overlaps the two previous domains, making the 'crest' of the protofilament on the outside surface of the microtubule where microtubule-associated proteins and motor proteins bind. Although the last 10 residues in (x tubulin and the last 18 residues in ~ tubulin are not seen in the map, presumably because of disorder resulting from their high charge density, it is clear that they extend from a point that is near the ridge of the protofilament. These residues are the most highly variable part of the tubulin molecule, and the main determinants of isotype variety. T h e fact that they are exposed on the outside of the microtubule is certain to promote the idea that there are isotype-selective proteins interacting with the microtubule that may provide the functional basis for microtubule isotype variety and which would be crucial to constructing specific types of microtubules and determining their particular function.
In the atomic model, the two monomers have remarkably similar structures, even though they are in different nucleotide-bound states ( G T P is bound to (~ tubulin and GDP and taxol are bound to 13 tubulin). Researchers in the tubulin field like to talk about two tubulin conformations; a 'straight' conformation of tubulin with bound G T P at the E-site in 13 tubulin (tubulin.GTP), and a 'curved' conformation containing tubulin.GDP. Within the microtubule, tubulin.GDP is kept in a 'straight' conformation by the subunit contacts within the lattice. Electron microscopy studies have shown that, upon loss of the G T P cap, protofilaments curl and peel off [28,29], revealing the 'curved' conformation of the tubulin.GDP and often forming closed rings. Such images are in agreement with studies showing that the free energy of hydrolysis following addition of a new dimer to a microtubule end is stored in the microtubule lattice [30], and is only released upon depolymerization. Presumably the lattice constraints that maintain the tubulin.GTP-like conformation in microtubules are also present in the zinc-induced sheets used for the electron crystallographic studies.
Tubulin and microtubule structure Downing and Nogales
lg
Figure 2
Illl
+ end
13
]lJ ii t
t - end i i
(a)
(b)
(c)
(d)
(e)
(f) Current Opinion in Cell Biology
Determination of microtubule polarity. (a, d) Cryo-electron microscopy images of a 14-3 (14 protofilaments, 3 star) and a 14-4 (14 protofilaments, 4 star) microtubule, respectively (see text). Both microtubules are oriented with their plus ends at the top. (b, e) Corresponding computer-generated diffraction patterns. The relative positions of the peaks in the diffraction pattern, marked J3 and 111 for the 14-3 microtubule, and 14 and Jlo for the 14-4 microtubule, indicate that the microtubule in (a) is left-handed and that in (d) is right-handed. (c, f) Filtered images, obtained by Fourier synthesis with only the equatorial components of the diffraction pattern, in which the lengthwise features of the protofilaments are reinforced, thus showing the arrowhead pattern along the microtubule more cleady. This arrowhead pattern results from the superposition of the front and back views of the microtubule wall. The arrowheads point to the minus end in (c) and to the plus end in (f). Figure provided courtesy of Denis Chr~tien.
Tubulin-GTE tubulin.GMPCPP (guanylyl-(~,[~)-methylene-diphosphonate, a nonhydrolyzable analog of GTP) and tubulin.GDP in the presence of BeF 3 or taxoids form microtubules of almost indistinguishable structure [31,32]. This observation supports the hypothesis that the presence of the y phosphate (or a mimic group) or taxol
results in the same conformation of tubulin, corresponding to the 'straight' conformation. This conformation is in turn stabilized within the microtubule lattice. Microtubules made of tubulin.GDP have a slightly shorter distance between monomers along the protofilament (1-1.5/~ shorter) than those formed with nonhydrolyzable analogs [31,32].
20
Cytoskeleton
Figure 3
mainly by being pulled apart as the protofilaments curl away from each other. On the other hand, hydrolysis could also have a more direct effect on interprotofilament interactions. The atomic structure suggests ways to investigate the relative importance of the different subunit contacts, perhaps by modeling the interaction energies of contacts within the lattice, or by mutational studies designed to change the balance between the lateral and longitudinal interactions.
13-tl
Figure 4
(a)
(b)
~-tL
C~rren! Opinion in Cell Biology
Ribbon diagram of the tubulin dimer. 13tubulin with bound GDP is at the top; ct tubulin with bound GTP is at the bottom. The orientation corresponds approximately to a lateral view of the dimer in a microtubule with the axis running vertically and the plus end at the top (see text), The left-hand side corresponds to the outside microtubule surface, the right-hand side to the inside. Thus, the structure in this view would fit into the microtubule reconstruction at the position marked with lighter grey in Figure lb. N, amino terminus; C, carboxyl terminus.
This is a very small change compared with that seen upon depolymerization of tubulin.GDP microtubules, where the conformational change in the 'unconstrained' tubulin.GDP results in a dramatic curvature of the protofilaments [28,29]. In spite of the small difference, it has now been shown that kinetochores can tell the difference between GTP- and GDP-bound tubulin within a microtubule [33°], and it has been proposed that this is the mechanism by which they preferentially attach near the microtubule plus end. The structural model of tubulin supports the notion that hydrolysis induces a local conformational change near the dimer interface that results in a kink, as suggested in Figure 4, which accounts for the curvature seen in images of tubulin-GDP rings and of protofilaments peeling away from disassembling microtubules [28,29]. These images suggest that lateral contacts between subunits are lost
Current Opinion in Cell Biology
Schematic diagram of a hypothetical conformational change in the structure of a microtubule protofilament upon loss of the GTP-bound 13-tubulin cap. (a) Capped protofilarnent in the 'straight' conformation as in stable rnicrotubules. (b) Uncapped protofilament (possessing GDP-bound 13-tubulin at the end) in the 'curved' conformation as in rings or depolymerizing microtubules. The proposed conformational change occurs at the longitudinal interface between tubulin dirners, near the nucleotide binding site in 13tubulin, when tubulin.GDP escapes the constraints of the microtubule lattice.
The nucleotide position in the amino-terminal domain of the structure is right at the longitudinal interface between monomers along the protofilament, so that the nucleotide in each subunit interacts with the next monomer along the protofilament, both at the interdimer ~md at the intradimer interfaces. With the definition of the dimer boundary as shown in Figure 3, the N-site in ~ tubulin is shown to be found at the intradimer interface. The N-site will then always be occluded in the dimer, regardless of the state of assembly. The nucleotide at the E-site, on the other hand, will be partially exposed in the dimer,
Tubulin and microtubule structure Downing and Nogales
where it can exchange with the solution, but will become occluded upon polymerization, resulting in its loss of exchangeability. There has been some disagreement in the literature on whether the plus ends of microtubules are crowned by cx or by 13 subunits [34,35], although the latter possibility seems to be the most favored one today. In the dimer model shown in Figure 3, the exchangeable site in 13 tubulin will be exposed at one end of the microtubule. It is most logical to conclude that this end is the plus end on the basis of the observation that nucleotide exchange only occurs at the plus end [36]. Furthermore, antibodies to the amino-terminal region of ot tubulin were found to bind only to the minus end of microtubules [37°°], further supporting the idea that 13 tubulin is on the plus end°
Conclusions Microtubules have intrigued scientists since their discovery because of both their critical importance to the cell and their interesting biophysical properties. Important clues to the processes of assembly and disassembly of microtubules have recently been gained using biochemical [1"], electron microscopy [15,28,29], and X-ray scattering [21"] techniques. We know more about the effects of nucleotides and stabilizing drugs such as taxoids both on the structure of the microtubule [31,32] and on the interaction of microtubules with motor proteins [9•',10",11"]. T h e existence of an atomic model of tubulin [27 •° ] is now bringing together biochemical, genetic and structural information to give us a picture of the properties of tubulin and microtubules at the molecular level. Knowledge of the structure of tubulin should open the possibilities for new and interesting experiments in the field of microtubule study that will lead us to a more complete understanding of microtubule structure and function.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • ••
of special interest of outstanding interest
1. •
Caplow M, Shanks J: Evidence that a single monolayer tubulin.GTP cap is both necessary and sufficient to stabilize microtubules. Mol Biol Cell 1996, 7:663-675. The minus ends of microtubules assembled from tubulin.GTP were transiently stabilized against dilution-induced disassembly by reaction with tubulin.GMPCPP. The minimum size of a tubulin.GMPCPP cap sufficient to prevent disassembly was estimated from the lifetime of the microtubulas following dilution, and from the time required for the loss of a single tubulin.GMPCPP subunit from the miorotubule end. The authors concluded that a microtubule capped with 13-14 tubulin.GMPCPP subunits switches to disassembly after only one dissociation event. 2. •
Tran PT, Walker RA, Salmon ED: A metsstable intermediate state of microtubule dynamic instability that differs significantly between plus and minus ends, J Cell Biol 1997, 138:105-117. Microtubules were cut using a UV microbeam or severed with a glass microneedle while observed by dark field microscopy or video-enhanced differential interference contrast microscopy, respectively. The severed plus ends rapidly shortened, as expected by the lack of a GTP cap, but minus ends immediately resumed elongation. These results suggest the existence of a
21
metastable kinetic intermediate in dynamic instability between the elongation and shortening rates. 3. --
Luduefia RF: The multiple forms of tubulin: different gene products and covalent modifications./nt Rev Cytol 1998, 178:207-275. This is an extensive review of the functional significance of tubulin isotypes and the regulation of their expression, from protists to vertebrates. The review also covers all the known post-translational modifications on tubulin. 4.
Wilson PG, Borisy GG: Evolution of the multl-tubulin hypothesis. Bioessays 1997, 19:451-454.
5. ••
Raft EC, Fackenthai JD, Hutchens JA, Hoyle HD, Turner FR: Microtubule architecture specified by a ~-tubulin isoform. Science 1997, 275:70-74. In order to test whether orthologous ~ tubulins from different species are functionally equivalent, the moth He/iothis virescens ~2 homologue was expressed in Drosophila testes. Co-expression of the moth and fly ~ isotypes resulted in the formation of accessory microtubules with the structural characteristics of those from the moth (e.g. higher protofilament number). These experiments demonstrate that the architecture of the microtubule cytoskele o ton can be directed by a component ~ tubulin. 6.
Kikkawa M, Ishikawa T, Wakabayashi T, Hirokawa N: Threedimensional structure of the kinesin head-microtubule complex. Nature 1995, 376:274-277.
7.
Hirose K, Lockhart A, Cross RA, Amos LA: Nucleotide-dependent angular change in kinesin motor domain bound to tubulin. Nature 1995, 376:277-279.
8.
Hirose K, Amos WB, Lockhart A, Cross RA, Amos LA: Threedimensional cryoelectron microscopy of 16-protefilament microtubules: structure, polarity and interaction with motor proteins. J Struct Biol 1997, 118:140-148.
9. .•
Sosa H, Milligan RA: Three-dimensional structure of neddecorated microtubules obtained by a back-projection method. J Mo/Biol 1996, 260:743-755. Using cryoelectron microscopy and a back-projection method the authors obtained reconstructions of microtubules decorated with the motor domain of ned. A view of the reconstructions along the microtubule axis from the minus end shows that the ned motor domain tilts anticlockwise, while the tubulin subunits tilt clockwise. 10. •
Arnal I, Metoz F, DeBonis S, Wade RH: Three-dimensional structure of functional motor proteins on microfubules" Curr Bio/1996, 6:1265-1270. Three-dimensional maps of motor-microtubule complexes obtained by cryoelectron microscopy and helical reconstruction showed that the motors have one attached head and one unattached head per tubulin dimer. The unattached heads of kinesin and ned had distinctly different conformations. 11. •
Hirose K, Lockhart A, Cross RA, Amos I.A: Three-dimensional cryoelectron microscopy of dimeric kinesin and ned motor domains on microtubules" Proc Nat/Acad Sci USA 1996, 93:9539-9544, Naturally occurring 16-protofilament microtubules were decorated with dimeric kinesin and ned motor domains and images were reconstructed by cryoelectron microscopy and helical methods. The reconstructions show that the attached heads of kinesin and ned bind to tubulin in the same way, while the unattached heads point in opposite directions; the second kinesin head points towards the plus microtubule end, the second ned head towards the minus end. 12. •.
Sosa H, Dias DP, Hoenger A, Whittaker M, Wilson-Kubalek E, Sablin E, Fletterick R.I, Vale RD, Milligan RA: A model for the microtubule-Ncd motor protein complex obtained by cryoelectron microscopy and image analysis. Cell 1997, 90:217224. The atomic model of the ncd motor domain was fitted to cryoelectron microscopy reconstructions of microtubules decorated with the same ncd construct. Docking revealed a large contact surface between ncd and the microtubule; the surface includes a loop of conserved residues among kinesins. 13.
Amos L, Hirose K: The structure of microtubule-motor complexes. Curt Bio/1997, 9:4-! 1.
14.
Wade RH, Hyman AA: Microtubule structure and dynamics. Curt Opin Cell Biol 1997, 9:12-17.
15.
Chrbtian D, Fuller SD, Karsenti E: Structure of growing microfubule ends: two-dimensional sheets close into tubes at variable rates, J CellBio11995, 129:1311-1328.
16.
Sosa H, Hoenger A, Milligan RA: Three different approaches for calculating the three dimensional structure of microtubules decorated with kinesin motor proteins. J Struct Biol 1997, 118:149-158.
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Cytoskeleton
17.
Metoz F, Amal I, Wade RH: Tomography without tilt: three dimensional imaging of microtubule/motor complexes. J Struct Bit)/1997, 118:159-168.
18. •
Hoenger A, Milligan RA: Motor domains of kinesin and ncd interact with microtubule protofilaments with the same binding geometry. J Mo/Biol 1997, 265:553-564. The structure and microtubule-binding properties of kinesin and ncd were compared using negative stain electron microscopy and tilt series of tubulin sheets decorated with the motor domains of those proteins. Both motors bind to the crest of the protofilament and make contact with both c¢ and tubulin. The geometry of attachment is very similar. The binding seemed to induce a conformational change in tubulin that the authors suggested could take an active role in motor directionality. 19.
Hoenger A, Milligan RA: Polarity of 2-D and 3-D maps of tubulin sheets and motor-dec•rated sheets. J Mo/Biol 1996, 263:114119.
20. •
Chr6tien D, Kenney JM, Fuller SD, Wade RH: Determination of microtubule polarity by cryo-electron microscopy. Structure 1996, 4:1031-1040. Microtubules nucleated from centrosomes were studied by cry•electron microscopy to relate their arrowhead moir6 pattern (resulting from the superposition of the front and back of the microtubule and related to the skew of the protofilaments) to their polarity. The authors show that the arrowheads point towards the plus end of microtubules with a right-handed skew, and to the minus end of microtubules with a left-handed skew. The handedness can be determined by looking at tilted views of the microtubule or by direct analysis of its diffraction pattern. Diaz JF, Andreu JM, Diakun G, Towns-Andrews E, Bordas J: Structural intermediates in the assembly of taxoid-induced microtubules and GDP-tubulin double rings: time-resolved X-ray scattering. Biophys J 1996, 70:2408-2420. The self-association of tubulin.GDP was studied by synchrotron time-resolved X-ray solution scattering. Tubulin-GDP stays as a dimer at low magnesium concentrations. At higher concentrations of divalent cations and in the presence of taxoid it assembles into either double rings or microtubules. The taxoid induces the formation of microtubular sheets that later close into a cylinder,
26.
27. Nogales E, Wolf SG, Downing KH: Atomic model of the tubulin o= dimer. Nature 1997, in press. This paper provides the first atomic model of tubulin. The model was fitted to a 3.7 A density map obtained by electron crystallography of zinc-induced tubulin sheets, and includes a molecule of GTP bound to o¢ tubulin and molecules of GDP and taxol bound to ~ tubulin. The model is related to previous biochemical and mutagenesis data that dealt with cysteine crosslinking, drug binding, and nucleotide binding, hydrolysis and exchangeability. 98.
Mandelkow E-M, Mandelkow E, Milligan RA: Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study. J Cell Biol 1991, 114:977-991.
99.
Tran PT, Joshi P, Salmon ED: How tubulin subunits are lost from the shortening ends of microtubules. J Struct Bio11997, 118:107-118.
30.
Caplow M, Ruhlen RL, Shanks J: The free energy of hydrolysis of a microtubule-bound nucleotide triphosphate is near zero: all of the free energy for hydrolysis is stored in the microtubule lattice. J Cell Bio/1994, 127:779-788.
31.
Vale RD, Coppin CM, Malik F, Kull FJ, Milligan RA: Tubulin GTP hydrolysis influences the structure, mechanical properties, end kinesin-driven transport of microtubules. J Biol Chem 1994, 269:23769-23775.
32.
HymenAA, Chr~tien D, Arnal I, Wade RH: Structural changes accompanying GTP hydrolysis of microtubules: information from a slowly hydrolyzable analog guanylyl-(¢~)-methylenediphosphonate. J Cell Biol 1995, 128:117-125.
21. •
22.
23.
Dlaz JF, Pantcs E, Bordas J, Andreu JM: Solution structure of GDP-tubulin double rings to 3 nm resolution and comparison with microtubules. J Mol Biol 1994, 238:214-225. Andreu JM, Diaz JF, Gil R, de Pereda JM, Garcia de Lacoba M, Peyrot V, Briand C, Towns-Andrews E, Bordas J: Solution structure of tax•tare-induced microtubules to 3-nm resolution. J Biol Chem 1994, 260:31785-31792.
24. •
De Pereda JM, Andreu JM: Mapping surface sequences of the tubulin dimer and tax•l-induced microtubules with limited proteolysis. Biochemistry 1996, 35:14184-14202. Proteolysis studies of tubulin dimers and microtubules with 12 different proteases revealed 80 different points of nicking by proteolysis that were mapped to (x and ~ tubulin sequences with the aid of site-directed antibodies. Although the majority of the sites remained accessible in microtubules, two sites in ot and one in ~ became protected upon polymerization. 25.
Nogales E, Wolf SG, Khan IA, Ludue~a RF, Downing KH: Structure of tubulin at 6.5 A and location of the tax•l-binding site. Nature 1995, 375:424-427.
Nogales E, Wolf SG, Downing KH: Visualizing the secondary structure of tubulin: Three-dimensional map at 4A. J Struct Bio/ 1997, 118:119-127.
33. •
Severin FF, Sorger PK, Hyman AA: Kinetochores distinguish GTP from GDP forms of the microtubule lattice. Nature 1997, 388:888-891. The authors monitor the attachment of reconstituted kinetochores from yeast to microtubules containing regions made of tubulin.GDP and tubulin.GMPCPP. Kinetochores bound preferentially to GMPCPP regions. They also studied the binding of kinetochores to microtubules of known polarity made with tubulin.GMPCPP, and observed that kinetochores bind preferentially to the plus ends. 34.
Hirose K, Fan J, Amos LA: Re-examination of the polarity of microtubules and sheets decorated with kinesin motor domain. J Mol Bio/1995, 251:329-333.
35.
Wolf SG, Nogales E, Kikkawa M, Gratzinger D, Hirokawa N, Downing KH: Interpreting a medium-resolution model of tubulin: comparison of zinc-sheet and microtubule structure. J Mol Bio11996, 263:485-501.
36.
Mitchison TJ: Localization of an exchangeable GTP binding site at the plus end of microtubules. Science 1993, 261:1044-1047.
37. •o
Fan J, Griffiths AD, Lockhart A, Cross RA, Amos LA: Microtubule minus ends can be labeled with a phage display antibody specific to ~-tubulin. J Mol Biol 1996, 259:325-330. The amino-terminal 100 residues of c( tubulin were bacterially expressed, and a clone was selected from antibody*expressing phagemid particles that reacted with the expressed (x tubulin amino terminus and native tubulin, but not with expressed ~ tubulin amino terminus. Gold beads coated with the antibody were found to bind exclusively to minus ends of axonemes, indicating that c( tubulin crowns the minus end of microtubules.