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1.
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. 相似文献
2.
Xuecheng Ye Tae Kim Elisabeth A. Geyer Luke M. Rice 《Protein science : a publication of the Protein Society》2020,29(6):1429-1439
αβ‐tubulin subunits cycle through a series of different conformations in the polymer lattice during microtubule growing and shrinking. How these allosteric responses to different tubulin:tubulin contacts contribute to microtubule dynamics, and whether the contributions are evolutionarily conserved, remains poorly understood. Here, we sought to determine whether the microtubule‐stabilizing effects (slower shrinking) of the β:T238A mutation we previously observed using yeast αβ‐tubulin would generalize to mammalian microtubules. Using recombinant human microtubules as a model, we found that the mutation caused slow microtubule shrinking, indicating that this effect of the mutation is indeed conserved. However, unlike in yeast, β:T238A human microtubules grew faster than wild‐type and the mutation did not appear to attenuate the conformational change associated with guanosine 5′‐triphosphate (GTP) hydrolysis in the lattice. We conclude that the assembly‐dependent conformational change in αβ‐tubulin can contribute to determine the rates of microtubule growing as well as shrinking. Our results also suggest that an allosteric perturbation like the β:T238A mutation can alter the behavior of terminal subunits without accompanying changes in the conformation of fully surrounded subunits in the body of the microtubule. 相似文献
3.
Plus-end-tracking proteins (+TIPs) are localized at the fast-growing, or plus end, of microtubules, and link microtubule ends to cellular structures. One of the best studied +TIPs is EB1, which forms comet-like structures at the tips of growing microtubules. The molecular mechanisms by which EB1 recognizes and tracks growing microtubule ends are largely unknown. However, one clue is that EB1 can bind directly to a microtubule end in the absence of other proteins. Here we use an in vitro assay for dynamic microtubule growth with two-color total-internal-reflection-fluorescence imaging to investigate binding of mammalian EB1 to both stabilized and dynamic microtubules. We find that under conditions of microtubule growth, EB1 not only tip tracks, as previously shown, but also preferentially recognizes the GMPCPP microtubule lattice as opposed to the GDP lattice. The interaction of EB1 with the GMPCPP microtubule lattice depends on the E-hook of tubulin, as well as the amount of salt in solution. The ability to distinguish different nucleotide states of tubulin in microtubule lattice may contribute to the end-tracking mechanism of EB1. 相似文献
4.
Elie-Caille C Severin F Helenius J Howard J Muller DJ Hyman AA 《Current biology : CB》2007,17(20):1765-1770
Microtubules exist in dynamic equilibrium, growing and shrinking by the addition or loss of tubulin dimers from the ends of protofilaments. The hydrolysis of GTP in beta-tubulin destabilizes the microtubule lattice by increasing the curvature of protofilaments in the microtubule and putting strain on the lattice. The observation that protofilament curvature depends on GTP hydrolysis suggests that microtubule destabilizers and stabilizers work by modulating the curvature of the microtubule lattice itself. Indeed, the microtubule destabilizer MCAK has been shown to increase the curvature of protofilaments during depolymerization. Here, we show that the atomic force microscopy (AFM) of individual tubulin protofilaments provides sufficient resolution to allow the imaging of single protofilaments in their native environment. By using this assay, we confirm previous results for the effects of GTP hydrolysis and MCAK on the conformation of protofilaments. We go on to show that taxol stabilizes microtubules by straightening the GDP protofilament and slowing down the transition of protofilaments from straight to a curved configuration. 相似文献
5.
The three-dimensional structure of zinc-induced tubulin sheets freed of microtubule associated proteins has been determined to 20 Å resolution by electron microscopy and image reconstruction. The determination was carried out with porcine brain tubulin separated from microtubule associated proteins by phosphocellulose chromatography. Negatively stained samples were tilted using the goniometer stage of the electron microscope to provide images of the tubulin sheets ranging in tilt from ?60 ° to +60 °. The micrographs were digitized and subjected to a cross-correlation analysis to compensate for smooth curvature of the lattice in the sheets. For each angle of tilt, an average unit cell was obtained from the cross-correlation analysis and subsequently a Fourier transform was computed for inclusion in the three-dimensional Fourier data set. The transforms of 47 tilted images plus the average of five untilted sheets were combined and an inverse Fourier transform was applied to give a threedimensional reconstruction of the microtubule associated protein-free tubulin sheets. Comparison of the protofilament structure in these sheets with the previously published protofilament structure of zinc-induced tubulin sheets containing microtubule associated proteins reveals a number of consequences of the removal of microtubule associated proteins. (1) The extensive internal contact along the protofilament observed in microtubule associated protein-containing tubulin sheets is maintained in microtubule associated protein-free tubulin sheets. (2) In projection, the protofilaments in microtubule associated protein-free tubulin sheets are 2.2 Å closer together than in microtubule associated protein-tubulin sheets. (3) The deviations of adjacent protofilaments from the plane of the sheets when viewed end-on are more pronounced in the absence of microtubule associated proteins. Differences are also observed at the level of individual tubulin subunits. In particular, the distinct cleft which was found in one class of subunits in tubulin sheets with microtubule associated proteins is absent in the microtubule associated protein-free tubulin sheets. The loss of this cleft and some changes in the shape of the tubulin subunits upon removal of microtubule associated proteins suggest a possible site for the interaction of tubulin with microtubule associated proteins. 相似文献
6.
D J Odde 《Biophysical journal》1997,73(1):88-96
Microtubule assembly is a complex process with individual microtubules alternating stochastically between extended periods of assembly and disassembly, a phenomenon known as dynamic instability. Since the discovery of dynamic instability, molecular models of assembly have generally assumed that tubulin incorporation into the microtubule lattice is primarily reaction-limited. Recently this assumption has been challenged and the importance of diffusion in microtubule assembly dynamics asserted on the basis of scaling arguments, with tubulin gradients predicted to extend over length scales exceeding a cell diameter, approximately 50 microns. To assess whether individual microtubules in vivo assemble at diffusion-limited rates and to predict the theoretical upper limit on the assembly rate, a steady-state mean-field model for the concentration of tubulin about a growing microtubule tip was developed. Using published parameter values for microtubule assembly in vivo (growth rate = 7 microns/min, diffusivity = 6 x 10(-12) m2/s, tubulin concentration = 10 microM), the model predicted that the tubulin concentration at the microtubule tip was approximately 89% of the concentration far from the tip, indicating that microtubule self-assembly is not diffusion-limited. Furthermore, the gradients extended less than approximately 50 nm (the equivalent of about two microtubule diameters) from the microtubule tip, a distance much less than a cell diameter. In addition, a general relation was developed to predict the diffusion-limited assembly rate from the diffusivity and bulk tubulin concentration. Using this relation, it was estimated that the maximum theoretical assembly rate is approximately 65 microns/min, above which tubulin can no longer diffuse rapidly enough to support faster growth. 相似文献
7.
The evolution of the structure of tubulin and its potential consequences for the role and function of microtubules in cells and embryos 总被引:4,自引:0,他引:4
Tuszynski JA Carpenter EJ Huzil JT Malinski W Luchko T Luduena RF 《The International journal of developmental biology》2006,50(2-3):341-358
This paper discusses the results of homology modeling and resulting calculation of key structural and physical properties for close to 300 tubulin sequences, including alpha, beta, gamma, delta and epsilon -tubulins. The basis for our calculations was the structure of the tubulin dimer published several years ago by Nogales et al. (1998), later refined to 3.5 resolution by Lowe et al. (2001). While, it appears that the alpha, beta and gamma-tubulins segregate into distinct structural families, we have found several differences in the physical properties within each group. Each of the alpha, beta and gamma- tubulin groups exhibit major differences in their net electric charge, dipole moments and dipole vector orientations. These properties could influence functional characteristics such as microtubule stability and assembly kinetics, due to their effects on the strength of protein-protein interactions. In addition to the general structural trends between tubulin isoforms, we have observed that the carboxy-termini of alpha and beta-tubulin exists in at least two stable configurations, either projecting away from the tubulin (or microtubule) surface, or collapsed onto the surface. In the latter case, the carboxy-termini form a lattice distinctly different from that of the well-known A and B lattices formed by the tubulin subunits. However, this C-terminal lattice is indistinguishable from the lattice formed when the microtubule-associated protein tau binds to the microtubule surface. Finally, we have discussed how tubulin sequence diversity arose in evolution giving rise to its particular phylogeny and how it may be used in cell- and tissue-specific expression including embryonal development. 相似文献
8.
XMAP215 is a processive microtubule polymerase 总被引:6,自引:0,他引:6
Brouhard GJ Stear JH Noetzel TL Al-Bassam J Kinoshita K Harrison SC Howard J Hyman AA 《Cell》2008,132(1):79-88
Fast growth of microtubules is essential for rapid assembly of the microtubule cytoskeleton during cell proliferation and differentiation. XMAP215 belongs to a conserved family of proteins that promote microtubule growth. To determine how XMAP215 accelerates growth, we developed a single-molecule assay to visualize directly XMAP215-GFP interacting with dynamic microtubules. XMAP215 binds free tubulin in a 1:1 complex that interacts with the microtubule lattice and targets the ends by a diffusion-facilitated mechanism. XMAP215 persists at the plus end for many rounds of tubulin subunit addition in a form of "tip tracking." These results show that XMAP215 is a processive polymerase that directly catalyzes the addition of up to 25 tubulin dimers to the growing plus end. Under some circumstances XMAP215 can also catalyze the reverse reaction, namely microtubule shrinkage. The similarities between XMAP215 and formins, actin polymerases, suggest that processive tip tracking is a common mechanism for stimulating the growth of cytoskeletal polymers. 相似文献
9.
J M Andreu J Bordas J F Diaz J García de Ancos R Gil F J Medrano E Nogales E Pantos E Towns-Andrews 《Journal of molecular biology》1992,226(1):169-184
The structure of microtubules has been characterized to 3 nm resolution employing time-resolved X-ray scattering. This has revealed detailed structural features of microtubules not observed before in solution. The polymerization of highly purified tubulin, induced by the antitumour drug taxol, has been employed as a microtubule model system. This assembly reaction requires Mg2+, is optimal at a 1:1 taxol to tubulin heterodimer molar ratio, proceeds with GTP or GDP and is intrinsically reversible. The X-ray scattering profiles are consistent with identical non-globular alpha and beta-tubulin monomers ordered within the known helical surface lattice of microtubules. Purified tubulin-taxol microtubules have a smaller mean diameter (approx. 22 nm) than those induced by microtubule associated proteins or glycerol (approx. 24 nm), but nearly identical wall substructure to the resolution of the measurements. This is because the majority of the former consist of only 12 protofilaments instead of the typical 13 protofilaments, as confirmed by electron microscopy of thin-sectioned, negatively stained and ice-embedded taxol microtubules. It may be concluded that taxol induces a slight reduction of the lateral contact curvature between tubulin monomers. The main fringe pattern observed in cryo-electron micrographs is consistent with a simple 12 protofilament 3-start skewed lattice model. Cylindrical closure of this lattice can be achieved by tilting the lattice 0.8 degrees with respect to the microtubule axis. The closure implies a discontinuity in the type of lateral contacts between the tubulin monomers (regardless of whether these are of the -alpha-beta- or the -alpha-alpha-/-beta-beta- type), which indicates that lateral contacts and the subunit specificity of taxol binding are, to a large degree, equivalent. 相似文献
10.
Electron microscopy has recently revealed striking structural orderliness in kinetochore proteins and protein complexes that associate with microtubules. In addition to their astonishing appearance and intrinsic beauty, the structures are functionally informative. The Dam1 and Ndc80 complexes bind to the microtubule lattice as rings and chevrons, respectively. These structures give insight into how the kinetochore couples to dynamic microtubules, a process crucial to the accurate segregation of chromosomes. HURP and kinesin-13 arrange tubulin into sleeves and bracelets surrounding the microtubule lattice. These structures might reflect the ability of these proteins to modulate microtubule dynamics by interacting with specialized tubulin configurations. In this review, we compare and contrast the structure of these proteins and their interactions with microtubules to illustrate how they attach to and modulate the dynamics of microtubules. 相似文献
11.
Microtubules are polymers of tubulin subunits (dimers) arranged on a hexagonal lattice. Each tubulin dimer comprises two monomers, the alpha-tubulin and beta-tubulin, and can be found in two states. In the first state a mobile negative charge is located into the alpha-tubulin monomer and in the second into the beta-tubulin monomer. Each tubulin dimer is modeled as an electrical dipole coupled to its neighbors by electrostatic forces. The location of the mobile charge in each dimer depends on the location of the charges in the dimer's neighborhood. Mechanical forces that act on the microtubule affect the distances between the dimers and alter the electrostatic potential. Changes in this potential affect the mobile negative charge location in each dimer and the charge distribution in the microtubule. The net effect is that mechanical forces affect the charge distribution in microtubules. We propose to exploit this effect and use microtubules as mechanical force sensors. We model each dimer as a two-state quantum system and, following the quantum computation paradigm, we use discrete quantum random walk on the hexagonal microtubule lattice to determine the charge distribution. Different forces applied on the microtubule are modeled as different coin biases leading to different probability distributions of the quantum walker location, which are directly connected to different charge distributions. Simulation results show that there is a strong indication that microtubules can be used as mechanical force sensors and that they can also detect the force directions and magnitudes. 相似文献
12.
The dynamics of microtubules in living cells can be seen by fluorescence microscopy when fluorescently labeled tubulin is microinjected into cells, mixing with the cellular tubulin pool and incorporating into microtubules. The subsequent fluorescence distribution along microtubules can appear "speckled" in high-resolution images obtained with a cooled CCD camera (Waterman-Storer and Salmon, 1997. J. Cell Biol. 139:417-434). In this paper we investigate the origins of these fluorescent speckles. In vivo microtubules exhibited a random pattern of speckles for different microtubules and different regions of an individual microtubule. The speckle pattern changed only after microtubule shortening and regrowth. Microtubules assembled from mixtures of labeled and unlabeled pure tubulin in vitro also exhibited fluorescent speckles, demonstrating that cellular factors or organelles do not contribute to the speckle pattern. Speckle contrast (measured as the standard deviation of fluorescence intensity along the microtubule divided by the mean fluorescence intensity) decreased as the fraction of labeled tubulin increased, and it was not altered by the binding of purified brain microtubule-associated proteins. Computer simulation of microtubule assembly with labeled and unlabeled tubulin showed that the speckle patterns can be explained solely by the stochastic nature of tubulin dimer association with a growing end. Speckle patterns can provide fiduciary marks in the microtubule lattice for motility studies or can be used to determine the fraction of labeled tubulin microinjected into living cells. 相似文献
13.
Dysfunction of the microtubule system is emerging as a contributing factor in a number of neurodegenerative diseases. Looking for the potential role played by the microtubule cytoskeleton in neuron degeneration underlying Parkinson's disease (PD), we investigate the influence of the parkinsonism producing neurotoxin 1-methyl-4-phenylpyridinium (MPP+) on microtubule dynamics. We find that it acts as a strong catastrophe promoter causing a decrease of the average length of microtubules assembled from purified tubulin. We also find that it reduces the number of microtubules nucleated from purified centrosomes. Finally, binding assays demonstrate that the neurotoxin binds specifically to tubulin in the microtubule lattice in a close to stoichiometric manner. This paper provides the first evidence that dynamic instability of microtubules is specifically affected by MPP+ and suggests that it could play a role in neuronal cell death underlying PD. 相似文献
14.
The kinesin-related protein MCAK is a microtubule depolymerase that forms an ATP-hydrolyzing complex at microtubule ends 总被引:12,自引:0,他引:12
MCAK belongs to the Kin I subfamily of kinesin-related proteins, a unique group of motor proteins that are not motile but instead destabilize microtubules. We show that MCAK is an ATPase that catalytically depolymerizes microtubules by accelerating, 100-fold, the rate of dissociation of tubulin from microtubule ends. MCAK has one high-affinity binding site per protofilament end, which, when occupied, has both the depolymerase and ATPase activities. MCAK targets protofilament ends very rapidly (on-rate 54 micro M(-1).s(-1)), perhaps by diffusion along the microtubule lattice, and, once there, removes approximately 20 tubulin dimers at a rate of 1 s(-1). We propose that up to 14 MCAK dimers assemble at the end of a microtubule to form an ATP-hydrolyzing complex that processively depolymerizes the microtubule. 相似文献
15.
Molecular interactions between tubulin tails and glutamylases reveal determinants of glutamylation patterns
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Kathiresan Natarajan Sudarshan Gadadhar Judith Souphron Maria M Magiera Carsten Janke 《EMBO reports》2017,18(6):1013-1026
Posttranslational modifications of tubulin currently emerge as key regulators of microtubule functions. Polyglutamylation generates a variety of modification patterns that are essential for controlling microtubule functions in different cell types and organelles, and deregulation of these patterns has been linked to ciliopathies, cancer and neurodegeneration. How the different glutamylating enzymes determine precise modification patterns has so far remained elusive. Using computational modelling, molecular dynamics simulations and mutational analyses we now show how the carboxy‐terminal tails of tubulin bind into the active sites of glutamylases. Our models suggest that the glutamylation sites on α‐ and β‐tubulins are determined by the positioning of the tails within the catalytic pocket. Moreover, we found that the binding modes of α‐ and β‐tubulin tails are highly similar, implying that most enzymes could potentially modify both, α‐ and β‐tubulin. This supports a model in which the binding of the enzymes to the entire microtubule lattice, but not the specificity of the C‐terminal tubulin tails to their active sites, determines the catalytic specificities of glutamylases. 相似文献
16.
The importance of lattice defects in katanin-mediated microtubule severing in vitro 总被引:4,自引:0,他引:4
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The microtubule-severing enzyme katanin uses ATP hydrolysis to disrupt noncovalent bonds between tubulin dimers within the microtubule lattice. Although its microtubule severing activity is likely important for fundamental processes including mitosis and axonal outgrowth, its mechanism of action is poorly understood. To better understand this activity, an in vitro assay was developed to enable the real-time observation of katanin-mediated severing of individual, mechanically unconstrained microtubules. To interpret the experimental observations, a number of theoretical models were developed and compared quantitatively to the experimental data via Monte Carlo simulation. Models that assumed that katanin acts on a uniform microtubule lattice were incompatible with the in vitro data, whereas a model that assumed that katanin acts preferentially on spatially infrequent microtubule lattice defects was found to correctly predict the experimentally observed breaking rates, number and spatial frequency of severing events, final levels of severing, and sensitivity to katanin concentration over the range 6-300 nM. As a result of our analysis, we propose that defects in the microtubule lattice, which are known to exist but previously not known to have any biological function, serve as sites for katanin activity. 相似文献
17.
Vitre B Coquelle FM Heichette C Garnier C Chrétien D Arnal I 《Nature cell biology》2008,10(4):415-421
End binding 1 (EB1) is a plus-end-tracking protein (+TIP) that localizes to microtubule plus ends where it modulates their dynamics and interactions with intracellular organelles. Although the regulating activity of EB1 on microtubule dynamics has been studied in cells and purified systems, the molecular mechanisms involved in its specific activity are still unclear. Here, we describe how EB1 regulates the dynamics and structure of microtubules assembled from pure tubulin. We found that EB1 stimulates spontaneous nucleation and growth of microtubules, and promotes both catastrophes (transitions from growth to shrinkage) and rescues (reverse events). Electron cryomicroscopy showed that EB1 induces the initial formation of tubulin sheets, which rapidly close into the common 13-protofilament-microtubule architecture. Our results suggest that EB1 favours the lateral association of free tubulin at microtubule-sheet edges, thereby stimulating nucleation, sheet growth and closure. The reduction of sheet length at microtubule growing-ends together with the elimination of stressed microtubule lattices may account for catastrophes. Conversely, occasional binding of EB1 to the microtubule lattice may induce rescues. 相似文献
18.
Microtubules polymerize from GTP-liganded tubulin dimers, but are essentially made of GDP-liganded tubulin. We investigate the tug-of-war resulting from the fact that GDP-liganded tubulin favors a curved configuration, but is forced to remain in a straight one when part of a microtubule. We point out that near the end of a microtubule, the proximity of the end shifts the balance in this tug-of-war, with some protofilament bending as result. This somewhat relaxes the microtubule lattice near its end, resulting in a structural cap. This structural cap thus is a simple mechanical consequence of two well-established facts: protofilaments made of GDP-liganded tubulin have intrinsic curvature, and microtubules are elastic, made from material that can yield to forces, in casu its own intrinsic forces. We explore possible properties of this structural cap, and demonstrate 1) how it allows both polymerization from GTP-liganded tubulin and rapid depolymerization in its absence; 2) how rescue can occur; 3) how a third, meta-stable intermediate state is possible and can explain some experimental results; and 4) how the tapered tips observed at polymerizing microtubule ends are stabilized during growth, though unable to accommodate a lateral cap. This scenario thus supports the widely accepted GTP-cap model by suggesting a stabilizing mechanism that explains the many aspects of dynamic instability. 相似文献
19.
Tubulin is known to exist in at least two main conformations: straight when bound to GTP or buried within the microtubule lattice, and curved when bound to GDP. The latter is most obvious during microtubule depolymerization, when protofilaments bend and peel off from microtubule ends. The curved, low-energy subunits form tantalizing ring structures in the presence of stabilizing divalent cations. Interestingly, cellular factors and antimitotic agents that act by depolymerizing microtubules can induce the formation of rings. In these rings, tubulin dimers generally appear kinked at the monomer-monomer interface, either to the same or to a lesser extent than at the dimer-dimer interface, with each agent giving rise to particular subtleties in the structures of the rings and the tubulin dimer itself that may reflect their distinctive mechanisms of action. How these kinks relate to what happens when the stored energy of GTP hydrolysis is released, freeing GDP*tubulin into an unconstrained state, remains an open question. 相似文献
20.
Drummond DR 《Seminars in cell & developmental biology》2011,(9):927-934
The simple mechanistic and functional division of the kinesin family into either active translocators or non-motile microtubule depolymerases was initially appropriate but is now proving increasingly unhelpful, given evidence that several translocase kinesins can affect microtubule dynamics, whilst non-translocase kinesins can promote microtubule assembly and depolymerisation. Such multi-role kinesins act either directly on microtubule dynamics, by interaction with microtubules and tubulin, or indirectly, through the transport of other factors along the lattice to the microtubule tip. Here I review recent progress on the mechanisms and roles of these translocase kinesins. 相似文献