Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain

Microtubules are cytoskeletal polymers of tubulin involved in many cellular functions. Their dynamic instability is controlled by numerous compounds and proteins, including colchicine and stathmin family proteins. The way in which microtubule instability is regulated at the molecular level has remained elusive, mainly because of the lack of appropriate structural data. Here, we present the structure, at 3.5 A resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Moreover, the tubulin-colchicine complex sheds light on the mechanism of colchicine's activity: we show that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly.     

Elevation of tubulin levels by microinjection suppresses new tubulin synthesis

Most eukaryotic cells rapidly and specifically depress synthesis of alpha- and beta-tubulin polypeptides in response to microtubule inhibitors which cause microtubule depolymerization and presumably increase the intracellular concentration of free subunits. Other drugs which interfere with microtubule function but which lead to a decrease in the subunit pool size have little effect on the rate of new tubulin synthesis. These findings have previously been interpreted to indicate that cultured cells synthesize tubulin constitutively unless the subunit pool rises above a specified level. At this point an autoregulatory control mechanism is triggered which suppresses new tubulin synthesis through specific loss of tubulin mRNAs. That tubulin RNA levels are dramatically lowered by microtubule depolymerizing drugs is unquestionably correct; that fluctuations in the depolymerized tubulin pool size are responsible for altered RNA levels rests, however, entirely on the presumptive effects of different microtubule drugs. This caveat is not trivial, as these drugs induce gross morphological alterations, and the specificities and detailed mechanisms of action of such drugs remain poorly understood. To investigate the effect of altered levels of tubulin subunits on the rate of new tubulin synthesis in mammalian cells, we have microinjected purified tubulin subunits into cells in culture and analysed the synthesized proteins. We report here that tubulin synthesis is rapidly and specifically suppressed by injection of an amount of tubulin roughly equivalent to 25-50% of the amount initially present in the cell, thus indicating the presence of an eukaryotic, autoregulatory control mechanism which specifies tubulin content in a cultured mammalian cell line.     

In vitro assembly of plant tubulin in the absence of microtubule-stabilizing reagents

The assembly of microtubules is essential for physiological functions of microtubules. Addition of microtubule-stabilizing reagents or microtubule "seeds" is usually necessary for plant tubulin assembly in vitro, which hinders the investigation of plant microtubule dynamics. In the present note, highly purified plant tubulins have been obtained from lily pollen, a non-microtubule-stabilizing reagent or microtubule "seed" system for plant tubulin assembly has been established and the analysis of plant tubulin assembly performed. Experiment results showed that purified tubulin polymerized in vitro, and a typical microtubule structure was observed with electron microscopy. The kinetics curve of tubulin assembly exhibited typical "parabola". The presence of taxol significantly altered the character of plant tubulin assembly, including that abnormal microtubules were assembled and the critical concentration for plant tubulin assembly was decreased exceedingly from 3 mg/mL in the absence of taxol to 0.043 mg/mL in the presence of taxol.     

Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly

The atomic structure of tubulin in a polymerized, straight protofilament is clearly distinct from that in a curved conformation bound to a cellular depolymerizer. The nucleotide contents are identical, and in both cases the conformation of the GTP-containing, intra-dimer interface is indistinguishable from the GDP-containing, inter-dimer contact. Here we present two structures corresponding to the start and end points in the microtubule polymerization and hydrolysis cycles that illustrate the consequences of nucleotide state on longitudinal and lateral assembly. In the absence of depolymerizers, GDP-bound tubulin shows distinctive intra-dimer and inter-dimer interactions and thus distinguishes the GTP and GDP interfaces. A cold-stable tubulin polymer with the non-hydrolysable GTP analogue GMPCPP, containing semi-conserved lateral interactions, supports a model in which the straightening of longitudinal interfaces happens sequentially, starting with a conformational change after GTP binding that straightens the dimer enough for the formation of lateral contacts into a non-tubular intermediate. Closure into a microtubule does not require GTP hydrolysis.     

In vitro assembly of plant tubulin in the absence of microtubule-stabilizing reagents

The assembly of microtubules is essential for physiological functions of microtubules. Addition of microtubule-stabilizing reagents or microtubule “seeds” is usually necessary for plant tubulin assemblyin vitro, which hinders the investigation of plant microtubule dynamics. In the present note, highly purified plant tubulins have been obtained from lily pollen, a non-microtubule-stabilizing reagent or microtubule “seed” system for plant tubulin assembly has been established and the analysis of plant tubulin assembly performed. Experiment results showed that purified tubulin polymerizedin vitro, and a typical microtubule structure was observed with electron microscopy. The kinetics curve of tubulin assembly exhibited typical “parabola”. The presence of taxol significantly altered the character of plant tubulin assembly, including that abnormal microtubules were assembled and the critical concentration for plant tubulin assembly was decreased exceedingly from 3 mg/mL in the absence of taxol to 0.043 mg/mL in ihe presence of taxol.     

Assembly of microtubules from nucleotide-depleted tubulin

In vitro assembly of microtubules from tubulin is considered to have an absolute requirement for added GTP (or a non-hydrolysable GTP-analogue) involving binding at the E(exchangeable)-site located on the beta-subunit of the tubulin dimer. By contrast, GDP inhibits assembly. Nucleotide hydrolysis has been implicated in the dynamic properties of microtubules, treadmilling and mechanical coupling. Here we demonstrate that assembly is not necessarily dependent on the presence of GTP at the E-site; microtubules can be formed efficiently in the absence of GTP in the presence of pyrophosphate. These microtubules, which have normal morphology and lability at cold temperatures, contain N(non-exchangeable)-site GTP and a significant proportion of E-site GDP. This demonstrates the possibility of direct incorporation of GDP-containing tubulin dimer during assembly which probably derives from microtubule-associated protein (MAP)-containing oligomers. This finding has important implications for the mechanism of microtubule elongation. The effects of pyrophosphate suggest that charge neutralization by the bidentate ligand is an essential step in promoting microtubule assembly, and that this interaction involves only a minimal conformational change in the protein.     

How kinesin waits between steps

Kinesin-1 (conventional kinesin) is a dimeric motor protein that carries cellular cargoes along microtubules by hydrolysing ATP and moving processively in 8-nm steps. The mechanism of processive motility involves the hand-over-hand motion of the two motor domains ('heads'), a process driven by a conformational change in the neck-linker domain of kinesin. However, the 'waiting conformation' of kinesin between steps remains controversial-some models propose that kinesin adopts a one-head-bound intermediate, whereas others suggest that both the kinesin heads are bound to adjacent tubulin subunits. Addressing this question has proved challenging, in part because of a lack of tools to measure structural states of the kinesin dimer as it moves along a microtubule. Here we develop two different single-molecule fluorescence resonance energy transfer (smFRET) sensors to detect whether kinesin is bound to its microtubule track by one or two heads. Our FRET results indicate that, while moving in the presence of saturating ATP, kinesin spends most of its time bound to the microtubule with both heads. However, when nucleotide binding becomes rate-limiting at low ATP concentrations, kinesin waits for ATP in a one-head-bound state and makes brief transitions to a two-head-bound intermediate as it walks along the microtubule. On the basis of these results, we suggest a model for how transitions in the ATPase cycle position the two kinesin heads and drive their hand-over-hand motion.     

Assembly of microtubules at the tip of growing axons

The growth of axons in the developing nervous system depends on the elongation of the microtubules that form their principal longitudinal structural element. It is not known whether individual microtubules in the axon elongate at their proximal ends, close to the cell body, and then move forward into the lengthening axon, or whether tubulin subunits are transported to the tip of the axon and assembled there onto the free ends of microtubules. The former possibility is supported by studies of slow axonal transport in mature nerves from which it has been deduced that microtubule assembly occurs principally at the neuronal cell body. By contrast, the polarity of microtubules in axons, which have their 'plus' or 'fast-growing' ends distal to the cell body, suggests that assembly occurs at the growing tip, or growth cone, of the axon. We have addressed this question by topically applying Colcemid (N-desacetyl-N-methylcolchicine), and other drugs which alter microtubule stability, to different regions of isolated nerve cells growing in tissue culture. We find that the sensitivity to these drugs is greatest at the growth cone by at least two orders of magnitude, suggesting that this is a major site of microtubule assembly during axonal growth.     

Processivity of the single-headed kinesin KIF1A through biased binding to tubulin

Conventional isoforms of the motor protein kinesin behave functionally not as 'single molecules' but as 'two molecules' paired. This dimeric structure poses a barrier to solving its mechanism. To overcome this problem, we used an unconventional kinesin KIF1A (refs 5, 6) as a model molecule. KIF1A moves processively as an independent monomer, and can also work synergistically as a functional dimer. Here we show, by measuring its movement with an optical trapping system, that a single ATP hydrolysis triggers a single stepping movement of a single KIF1A monomer. The step size is distributed stochastically around multiples of 8 nm with a gaussian-like envelope and a standard deviation of 15 nm. On average, the step is directional to the microtubule's plus-end against a load force of up to 0.15 pN. As the source for this directional movement, we show that KIF1A moves to the microtubule's plus-end by approximately 3 nm on average on binding to the microtubule, presumably by preferential binding to tubulin on the plus-end side. We propose a simple physical formulation to explain the movement of KIF1A.     

Microtubule basis for left-handed helical growth in Arabidopsis

Left-right asymmetry in plants can be found in helices of stalks, stems and tendrils, and in fan-like petal arrangements. The handedness in these asymmetric structures is often fixed in given species, indicating that genetic factors control asymmetric development. Here we show that dominant negative mutations at the tubulin intradimer interface of alpha-tubulins 4 and 6 cause left-handed helical growth and clockwise twisting in elongating organs of Arabidopsis thaliana. We demonstrate that the mutant tubulins incorporate into microtubule polymers, producing right-handed obliquely oriented cortical arrays, in the root epidermal cells. The cortical microtubules in the mutants had increased sensitivity to microtubule-specific drugs. These results suggest that reduced microtubule stability can produce left-handed helical growth in plants.     

Translocation of vesicles from squid axoplasm on flagellar microtubules

Directed intracellular particle movement is a fundamental process characteristic of all cells. During fast axonal transport, membranous organelles move at rapid rates, from 1 to 5 micron s-1, in either the orthograde or retrograde direction along the neurone and can traverse distances as long as 1 m (for reviews, see refs 1-3). Recent studies indicate that this extreme example of intracellular motility can occur along single microtubules, but the molecules generating the motile force have not been identified or localized. It is not known whether the force-transducing 'motor' is associated with the moving particle or with the microtubule lattice. To distinguish between these hypotheses and to characterize the membrane-cytoskeletal interactions that occur during vesicle translocations, we have developed a reconstituted model for microtubule-based motility. We isolated axoplasmic vesicles from the giant axon of the squid Loligo pealei as described previously. The vesicles (35-475 nm in diameter) were then added to axonemes of Arbacia punctulata spermatozoa that served as a source of microtubules. Axonemes were used because the tubulin subunit lattice of the A-subfibre of a given outer doublet is the same as the subunit lattice of neuronal microtubules along which motility occurs. Moreover, all the microtubules of a single axoneme show the same structural polarity, indicating that the axoneme represents an oriented microtubule substrate. Here we demonstrate that vesicle motility is ATP-dependent, that it is not mediated by the flagellar force-transducing molecule dynein and that the direction of movement is not specified by microtubule polarity.     

Molecular architecture of axonemal microtubule doublets revealed by cryo-electron tomography

The axoneme, which forms the core of eukaryotic flagella and cilia, is one of the largest macromolecular machines, with a structure that is largely conserved from protists to mammals. Microtubule doublets are structural components of axonemes that contain a number of proteins besides tubulin, and are usually found in arrays of nine doublets arranged around two singlet microtubules. Coordinated sliding of adjacent doublets, which involves a host of other proteins in the axoneme, produces periodic beating movements of the axoneme. We have obtained a three-dimensional density map of intact microtubule doublets using cryo-electron tomography and image averaging. Our map, with a resolution of about 3 nm, provides insights into locations of particular proteins within the doublets and the structural features of the doublets that define their mechanical properties. We identify likely candidates for several of these non-tubulin components of the doublets. This work offers insight on how tubulin protofilaments and accessory proteins attach together to form the doublets and provides a structural basis for understanding doublet function in axonemes.     

Assembly dynamics of microtubules at molecular resolution

Microtubules are highly dynamic protein polymers that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro and in vivo, to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule end-binding proteins regulate microtubule dynamics in living cells.     

The Ndc80 kinetochore complex forms oligomeric arrays along microtubules

The Ndc80 complex is a key site of regulated kinetochore-microtubule attachment (a process required for cell division), but the molecular mechanism underlying its function remains unknown. Here we present a subnanometre-resolution cryo-electron microscopy reconstruction of the human Ndc80 complex bound to microtubules, sufficient for precise docking of crystal structures of the component proteins. We find that the Ndc80 complex binds the microtubule with a tubulin monomer repeat, recognizing α- and β-tubulin at both intra- and inter-tubulin dimer interfaces in a manner that is sensitive to tubulin conformation. Furthermore, Ndc80 complexes self-associate along protofilaments through interactions mediated by the amino-terminal tail of the NDC80 protein, which is the site of phospho-regulation by Aurora B kinase. The complex's mode of interaction with the microtubule and its oligomerization suggest a mechanism by which Aurora B could regulate the stability of load-bearing kinetochore-microtubule attachments.     

Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin

Spastin, the most common locus for mutations in hereditary spastic paraplegias, and katanin are related microtubule-severing AAA ATPases involved in constructing neuronal and non-centrosomal microtubule arrays and in segregating chromosomes. The mechanism by which spastin and katanin break and destabilize microtubules is unknown, in part owing to the lack of structural information on these enzymes. Here we report the X-ray crystal structure of the Drosophila spastin AAA domain and provide a model for the active spastin hexamer generated using small-angle X-ray scattering combined with atomic docking. The spastin hexamer forms a ring with a prominent central pore and six radiating arms that may dock onto the microtubule. Helices unique to the microtubule-severing AAA ATPases surround the entrances to the pore on either side of the ring, and three highly conserved loops line the pore lumen. Mutagenesis reveals essential roles for these structural elements in the severing reaction. Peptide and antibody inhibition experiments further show that spastin may dismantle microtubules by recognizing specific features in the carboxy-terminal tail of tubulin. Collectively, our data support a model in which spastin pulls the C terminus of tubulin through its central pore, generating a mechanical force that destabilizes tubulin-tubulin interactions within the microtubule lattice. Our work also provides insights into the structural defects in spastin that arise from mutations identified in hereditary spastic paraplegia patients.     

Polewards chromosome movement driven by microtubule depolymerization in vitro

We constructed complexes between isolated chromosomes and microtubules made from purified tubulin to study the movement of chromosomes towards the 'minus' end of microtubules in vitro, a process analogous to the movement of chromosomes towards the pole of the spindle at anaphase of mitosis. Our results show that the energy for this movement is derived solely from microtubule depolymerization, and indicate that anaphase movement of chromosomes is both powered and regulated by microtubule depolymerization at the kinetochore.     

Insights into microtubule nucleation from the crystal structure of human gamma-tubulin

Microtubules are hollow polymers of alphabeta-tubulin that show GTP-dependent assembly dynamics and comprise a critical part of the eukaryotic cytoskeleton. Initiation of new microtubules in vivo requires gamma-tubulin, organized as an oligomer within the 2.2-MDa gamma-tubulin ring complex (gamma-TuRC) of higher eukaryotes. Structural insight is lacking regarding gamma-tubulin, its oligomerization and how it promotes microtubule assembly. Here we report the 2.7-A crystal structure of human gamma-tubulin bound to GTP-gammaS (a non-hydrolysable GTP analogue). We observe a 'curved' conformation for gamma-tubulin-GTPgammaS, similar to that seen for GDP-bound, unpolymerized alphabeta-tubulin. Tubulins are thought to represent a distinct class of GTP-binding proteins, and conformational switching in gamma-tubulin might differ from the nucleotide-dependent switching of signalling GTPases. A crystal packing interaction replicates the lateral contacts between alpha- and beta-tubulins in the microtubule, and this association probably forms the basis for gamma-tubulin oligomerization within the gamma-TuRC. Laterally associated gamma-tubulins in the gamma-TuRC might promote microtubule nucleation by providing a template that enhances the intrinsically weak lateral interaction between alphabeta-tubulin heterodimers. Because they are dimeric, alphabeta-tubulins cannot form microtubule-like lateral associations in the curved conformation. The lateral array of gamma-tubulins we observe in the crystal reveals a unique functional property of a monomeric tubulin.     

配合物与G-四链体DNA相互作用的研究

端粒在维持基因组稳定、癌症和衰老相关的生理过程中发挥着重要作用,鸟嘌呤通过形成G-四分体平面堆积成G-四链体结构,DNA二级结构参与一些重要的生物调控过程。人们已经在生物体内发现G-四分体结构的存在,它们大量存在于基因的启动区域表明G-四链体可能参与调节基因表达。以G-四链体为新靶标设计能与G-四链体DNA相互作用的小分子,为开发抗癌药物提供了新途径。最近的许多研究证明金属配合物与G-四链体DNA具有有效的相互作用,有望开发成新的抗癌药物。