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1.
On and Around Microtubules: An Overview   总被引:1,自引:0,他引:1  
Microtubules are hollow tubes some 25 nm in diameter participating in the eukaryotic cytoskeleton. They are built from αβ-tubulin heterodimers that associate to form protofilaments running lengthwise along the microtubule wall with the β-tubulin subunit facing the microtubule plus end conferring a structural polarity. The α- and β-tubulins are highly conserved. A third member of the tubulin family, γ-tubulin, plays a role in microtubule nucleation and assembly. Other members of the tubulin family appear to be involved in microtubule nucleation. Microtubule assembly is accompanied by hydrolysis of GTP associated with β-tubulin so that microtubules consist principally of ‘GDP-tubulin’ stabilized at the plus end by a short ‘cap’. An important property of microtubules is dynamic instability characterized by growth randomly interrupted by pauses and shrinkage. Many proteins interact with microtubules within the cell and are involved in essential functions such as microtubule growth, stabilization, destabilization, and interactions with chromosomes during cell division. The motor proteins kinesin and dynein use microtubules as pathways for transport and are also involved in cell division. Crystallography and electron microscopy are providing a structural basis for understanding the interactions of microtubules with antimitotic drugs, with motor proteins and with plus end tracking proteins.  相似文献   

2.
Johnson V  Ayaz P  Huddleston P  Rice LM 《Biochemistry》2011,50(40):8636-8644
Microtubule dynamics play essential roles in intracellular organization and cell division. They result from structural and biochemical properties of αβ-tubulin heterodimers and how these polymerizing subunits interact with themselves and with regulatory proteins. A broad understanding of the underlying mechanisms has been established, but fundamental questions remain unresolved. The lack of routine access to recombinant αβ-tubulin represents an obstacle to deeper insight into αβ-tubulin structure, biochemistry, and recognition. Indeed, the widespread reliance on animal brain αβ-tubulin means that very few in vitro studies have taken advantage of powerful and ordinarily routine techniques like site-directed mutagenesis. Here we report new methods for purifying wild-type or mutant yeast αβ-tubulin from inducibly overexpressing strains of Saccharomyces cerevisiae. Inducible overexpression is an improvement over existing approaches that rely on constitutive expression: it provides higher yields while also allowing otherwise lethal mutants to be purified. We also designed and purified polymerization-blocked αβ-tubulin mutants. These "blocked" forms of αβ-tubulin give a dominant lethal phenotype when expressed in cells; they cannot form microtubules in vitro and when present in mixtures inhibit the polymerization of wild-type αβ-tubulin. The effects of blocking mutations are very specific, because purified mutants exhibit normal hydrodynamic properties, bind GTP, and interact with a tubulin-binding domain. The ability to overexpress and purify wild-type αβ-tubulin, or mutants like the ones we report here, creates new opportunities for structural studies of αβ-tubulin and its complexes with regulatory proteins, and for biochemical and functional studies of microtubule dynamics and its regulation.  相似文献   

3.
Microtubules are essential cytoskeletal elements assembled from αβ-tubulin dimers. In high eukaryotes, microtubule nucleation, the de novo assembly of a microtubule from its minus end, is initiated by the γ-tubulin ring complex (γ-TuRC). Despite many years of research, the structural and mechanistic principles of the microtubule nucleation machinery remained poorly understood. Only recently, cryoelectron microscopy studies uncovered the molecular organization and potential activation mechanisms of γ-TuRC. In vitro assays further deciphered the spatial and temporal cooperation between γ-TuRC and additional factors, for example, the augmin complex, the phase separation protein TPX2, and the microtubule polymerase XMAP215. These breakthroughs deepen our understanding of microtubule nucleation mechanisms and will link the assembly of individual microtubules to the organization of cellular microtubule networks.  相似文献   

4.
To obtain information on plant microtubule stability to low temperature and Ca2+, the regulatory domain of polymerized tubulin from maize (Zea mays ev. Black Mexican Sweet) was dissected by limited proteolysis with subtilisin. Tubulin in taxol-stabilized microtubules was cleaved in a subtilisin concentration- and time-dependent manner. Immunoblotting of microtubules with antibodies having mapped epitopes on α- and β-tubulins revealed that cleavage initially removed ≤15 residues from the β-tubulin carboxyl terminus to produce αβs-microtubules. Subsequent cleavage occurred at an extreme site and an internal site within the α-tubulin carboxyl terminus. Electron microscopy revealed that αβs-microtubules were ultra structurally indistinguishable from uncleaved control αβ-micro-tubules. Quantitative polymer sedimentation showed that low temperature treatment (0°C) caused significant depolymerization of αβ-microtubules, but little depolymerization of αβs-microtubules. Ca2+ enhanced the cold-induced depolymerization of both αβ- and αβs-microtubules. However, αβs-microtubules were significantly more stable to depolymerization by cold and Ca2+ than were αβ-micro-tubules. The results showed that maize microtubules containing shortened β-tubulin carboxyl termini are relatively resistant to the combined depolymerizing effects of cold and Ca2+. Thus, the extreme carboxyl terminus of β-tubulin is a crucial element of the plant tubulin regulatory domain and may be involved in the modulation of microtubule stability during the chilling response in plants.  相似文献   

5.
Microtubules are composed of αβ-tubulin heterodimers and have been treated as highly attractive targets for antitumor drugs. A broad range of agents bind to tubulin and interfere with microtubule assembly, including colchicine binding site inhibitors (CBSIs). Tubulin Polymerization Inhibitor I (TPI1), a benzylidene derivative of 9(10H)-anthracenone, is a CBSI that inhibits the assembly of microtubules. However, for a long time, the design and development of anthracenone family drugs have been hindered by the lack of structural information of the tubulin-agent complex. Here we report a 2.3 Å crystal structure of tubulin complexed with TPI1, the first structure of anthracenone family agents. This complex structure reveals the interactions between TPI1 and tubulin, and thus provides insights into the development of new anthracenone derivatives targeting the colchicine binding site.  相似文献   

6.
Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryo-electron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor–microtubule interface and the susceptibility of the complex to drug binding. Despite their non-canonical architecture and mechanochemistry, Schizosaccharomyces pombe microtubules were stabilized by epothilone at the taxane binding pocket. The overall Cut7 footprint on the S. pombe microtubule surface is altered compared to mammalian tubulin microtubules because of their different polymer architectures. However, the core motor–microtubule interaction is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation including cover neck bundle formation. However, the Cut7 ATPase is not blocked by a mammalian-specific kinesin-5 inhibitor, consistent with the non-conserved sequence and structure of its loop5 insertion.  相似文献   

7.
The four-subunit Ndc80 complex, comprised of Ndc80/Nuf2 and Spc24/Spc25 dimers, directly connects kinetochores to spindle microtubules. The complex is anchored to the kinetochore at the Spc24/25 end, and the Ndc80/Nuf2 dimer projects outward to bind to microtubules. Here, we use cryoelectron microscopy and helical image analysis to visualize the interaction of the Ndc80/Nuf2 dimer with microtubules. Our results, when combined with crystallography data, suggest that the globular domain of the Ndc80 subunit binds strongly at the interface between tubulin dimers and weakly at the adjacent intradimer interface along the protofilament axis. Such a binding mode, in which the Ndc80 complex interacts with sequential α/β-tubulin heterodimers, may be important for stabilizing kinetochore-bound microtubules. Additionally, we define the binding of the Ndc80 complex relative to microtubule polarity, which reveals that the microtubule interaction surface is at a considerable distance from the opposite kinetochore-anchored end; this binding geometry may facilitate polymerization and depolymerization at kinetochore-attached microtubule ends.  相似文献   

8.
Abstract

Tubulin isotypes are known to regulate microtubule dynamic instability and contribute to the development of drug resistance in certain types of cancers. Combretastatin-A4 (CA-4) has a potent anti-mitotic, vascular disrupting and anti-angiogenic activity. It binds at the interface of αβ tubulin heterodimers and inhibits microtubules assembly. Interestingly, the CA-4 resistant human lung carcinoma shows alteration of βI and βIII isotype levels, a higher expression of βI tubulin isotype and a decreased expression of βIII tubulin isotypes has been reported in drug resistant cell lines. However, the origin of CA-4 resistance in lung carcinoma is not well understood. Here, we investigate the interaction and binding affinities of αβI, αβIIb, αβIII and αβIVa tubulin isotypes with CA-4, employing molecular modeling approaches. Sequence analysis shows that variations in residue composition at the CA-4 binding pocket of βI, βIII and βIVa tubulin isotypes when compared to template βIIb isotype. Molecular docking result shows that the CA-4 prefers ‘cis’ conformation in all αβ-tubulin isotypes. Molecular dynamics simulation reveal role of H7 helix, T7 loop and H8 helix of β-tubulin in lower binding affinity of αβI and αβIII isotypes for CA-4. The order of binding energy for CA-4 is αβIIb?>?αβIVa?>?αβI?>?αβIII. This suggest that drug resistance is induced in human lung carcinoma cells by altering the expression of β-tubulin isotypes namely βI and βIII which show lowest binding affinities. Our present study can help in designing potential CA-4 analogs against drug-resistant cancer cells showing altered expression of tubulin isotypes. Abbreviations: CA-4 combretastatin-A4

MD molecular dynamics

RMSD root mean square deviation

DSSP dictionary of secondary structure of proteins

VMD visual molecular dynamics

Communicated by Ramaswamy H. Sarma  相似文献   

9.
Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.  相似文献   

10.
Microtubules are long filamentous hollow cylinders whose surfaces form lattice structures of αβ-tubulin heterodimers. They perform multiple physiological roles in eukaryotic cells and are targets for therapeutic interventions. In our study, we carried out all-atom molecular dynamics simulations for arbitrarily long microtubules that have either GDP or GTP molecules in the E-site of β-tubulin. A detailed energy balance of the MM/GBSA inter-dimer interaction energy per residue contributing to the overall lateral and longitudinal structural stability was performed. The obtained results identified the key residues and tubulin domains according to their energetic contributions. They also identified the molecular forces that drive microtubule disassembly. At the tip of the plus end of the microtubule, the uneven distribution of longitudinal interaction energies within a protofilament generates a torque that bends tubulin outwardly with respect to the cylinder''s axis causing disassembly. In the presence of GTP, this torque is opposed by lateral interactions that prevent outward curling, thus stabilizing the whole microtubule. Once GTP hydrolysis reaches the tip of the microtubule (lateral cap), lateral interactions become much weaker, allowing tubulin dimers to bend outwards, causing disassembly. The role of magnesium in the process of outward curling has also been demonstrated. This study also showed that the microtubule seam is the most energetically labile inter-dimer interface and could serve as a trigger point for disassembly. Based on a detailed balance of the energetic contributions per amino acid residue in the microtubule, numerous other analyses could be performed to give additional insights into the properties of microtubule dynamic instability.  相似文献   

11.
Rings of guanosine diphosphate (GDP)-tubulin formed in the presence of divalent cations have been studied using conventional negative stain and cryo-electron microscopy. The structure of such rings resembles that of depolymerizing microtubule ends and corresponds to an “unconstrained” conformation of tubulin in its GDP state. The use of cryo-techniques has allowed us to image the ring polymers free from dehydration and flattening artifacts. Preparations of frozenhydrated GDP-tubulin rings are generally heterogeneous and contain a mixture of double, triple, and incomplete rings, as well as spirals and some rare single rings. Images of different polymer types can be identified and classified into groups that are then amenable for averaging and single particle reconstruction methods. Identifying the differences in tubulin structure, between straight and curve protofilaments, will be important to understand the molecular bases of dynamic instability in microtubules.  相似文献   

12.
NimA-related kinase 6 (NEK6) has been implicated in microtubule regulation to suppress the ectopic outgrowth of epidermal cells; however, its molecular functions remain to be elucidated. Here, we analyze the function of NEK6 and other members of the NEK family with regard to epidermal cell expansion and cortical microtubule organization. The functional NEK6-green fluorescent protein fusion localizes to cortical microtubules, predominantly in particles that exhibit dynamic movement along microtubules. The kinase-dead mutant of NEK6 (ibo1-1) exhibits a disturbance of the cortical microtubule array at the site of ectopic protrusions in epidermal cells. Pharmacological studies with microtubule inhibitors and quantitative analysis of microtubule dynamics indicate excessive stabilization of cortical microtubules in ibo1/nek6 mutants. In addition, NEK6 directly binds to microtubules in vitro and phosphorylates β-tubulin. NEK6 interacts and co-localizes with NEK4 and NEK5 in a transient expression assay. The ibo1-3 mutation markedly reduces the interaction between NEK6 and NEK4 and increases the interaction between NEK6 and NEK5. NEK4 and NEK5 are required for the ibo1/nek6 ectopic outgrowth phenotype in epidermal cells. These results demonstrate that NEK6 homodimerizes and forms heterodimers with NEK4 and NEK5 to regulate cortical microtubule organization possibly through the phosphorylation of β-tubulins.  相似文献   

13.
We have used cryo-electron microscopy of kinesin-decorated microtubules to resolve the structure of the motor protein kinesin's crucial nucleotide response elements, switch I and the switch II helix, in kinesin's poorly understood nucleotide-free state. Both of the switch elements undergo conformational change relative to the microtubule-free state. The changes in switch I suggest a role for it in "ejecting" adenosine diphosphate when kinesin initially binds to the microtubule. The switch II helix has an N-terminal extension, apparently stabilized by conserved microtubule contacts, implying a microtubule activation mechanism that could convey the state of the bound nucleotide to kinesin's putative force-delivering element (the "neck linker"). In deriving this structure, we have adapted an image-processing technique, single-particle reconstruction, for analyzing decorated microtubules. The resulting reconstruction visualizes the asymmetric seam present in native, 13-protofilament microtubules, and this method will provide an avenue to higher-resolution characterization of a variety of microtubule- binding proteins, as well as the microtubule itself.  相似文献   

14.
The yeast Saccharomyces cerevisiae has two genes for α-tubulin, TUB1 and TUB3, and one β-tubulin gene, TUB2. The gene product of TUB3, Tub3, represents ~10% of α-tubulin in the cell. We determined the effects of the two α-tubulin isotypes on microtubule dynamics in vitro. Tubulin was purified from wild-type and deletion strains lacking either Tub1 or Tub3, and parameters of microtubule dynamics were examined. Microtubules containing Tub3 as the only α-tubulin isotype were less dynamic than wild-type microtubules, as shown by a shrinkage rate and catastrophe frequency that were about one-third of that for wild-type microtubules. Conversely, microtubules containing Tub1 as the only α-tubulin isotype were more dynamic than wild-type microtubules, as shown by a shrinkage rate that was 50% higher and a catastrophe frequency that was 30% higher than those of wild-type microtubules. The results suggest that a role of Tub3 in budding yeast is to control microtubule dynamics.  相似文献   

15.
《The Journal of cell biology》1994,127(6):1965-1971
Microtubules are constructed from alpha- and beta-tubulin heterodimers that are arranged into protofilaments. Most commonly there are 13 or 14 protofilaments. A series of structural investigations using both electron microscopy and x-ray diffraction have indicated that there are two potential lattices (A and B) in which the tubulin subunits can be arranged. Electron microscopy has shown that kinesin heads, which bind only to beta-tubulin, follow a helical path with a 12-nm pitch in which subunits repeat every 8-nm axially, implying a primarily B-type lattice. However, these helical symmetry parameters are not consistent with a closed lattice and imply that there must be a discontinuity or "seam" along the microtubule. We have used quick-freeze deep-etch electron microscopy to obtain the first direct evidence for the presence of this seam in microtubules formed either in vivo or in vitro. In addition to a conventional single seam, we have also rarely found microtubules in which there is more than one seam. Overall our data indicates that microtubules have a predominantly B lattice, but that A lattice bonds between tubulin subunits are found at the seam. The cytoplasmic microtubules in mouse nerve cells also have predominantly B lattice structure and A lattice bonds at the seam. These observations have important implications for the interaction of microtubules with MAPs and with motor proteins, and for example, suggest that kinesin motors may follow a single protofilament track.  相似文献   

16.
Maurer SP  Fourniol FJ  Bohner G  Moores CA  Surrey T 《Cell》2012,149(2):371-382
Growing microtubule ends serve as transient binding platforms for essential proteins that regulate microtubule dynamics and their interactions with cellular substructures. End-binding proteins (EBs) autonomously recognize an extended region at growing microtubule ends with unknown structural characteristics and then recruit other factors to the dynamic end structure. Using cryo-electron microscopy, subnanometer single-particle reconstruction, and fluorescence imaging, we present a pseudoatomic model of how the calponin homology (CH) domain of the fission yeast EB Mal3 binds to the end regions?of growing microtubules. The Mal3 CH domain bridges protofilaments except at the microtubule seam. By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state. The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization. This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.  相似文献   

17.
Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gain-of-function mutation in the C. elegans α-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress.  相似文献   

18.
PACSINs are intracellular adapter proteins involved in vesicle transport, membrane dynamics and actin reorganisation. In this study, we report a novel role for PACSIN proteins as components of the centrosome involved in microtubule dynamics. Glutathione S-transferase (GST)-tagged PACSIN proteins interacted with protein complexes containing α- and γ-tubulin in brain homogenate. Analysis of cell lysates showed that all three endogenous PACSINs co-immunoprecipitated dynamin, α-tubulin and γ-tubulin. Furthermore, PACSINs bound only to unpolymerised tubulin, not to microtubules purified from brain. In agreement, the cellular localisation of endogenous PACSIN 2 was not affected by the microtubule depolymerising reagent nocodazole. By light microscopy, endogenous PACSIN 2 localised next to γ-tubulin at purified centrosomes from NIH 3T3 cells. Finally, reduction of PACSIN 2 protein levels with small-interfering RNA (siRNA) resulted in impaired microtubule nucleation from centrosomes, whereas microtubule centrosome splitting was not affected, suggesting a role for PACSIN 2 in the regulation of tubulin polymerisation. These findings suggest a novel function for PACSIN proteins in dynamic microtubuli nucleation.  相似文献   

19.
NIMA-related kinase 6 (NEK6) regulates cellular expansion and morphogenesis through microtubule organizaiton in Arabidopsis thaliana. Loss-of-function mutations in NEK6 (nek6/ibo1) cause ectopic outgrowth and microtubule disorganization in epidermal cells. We recently found that NEK6 forms homodimers and heterodimers with NEK4 and NEK5 to destabilize cortical microtubules possibly by direct binding to microtubules and the β-tubulin phosphorylation. Here, we identified a new allele of NEK6 and further analyzed the morphological phenotypes of nek6/ibo1 mutants, along with alleles of nek4 and nek5 mutants. Phenotypic analysis demonstrated that NEK6 is required for the directional growth of roots and hypocotyls, petiole elongation, cell file formation, and trichome morphogenesis. In addition, nek4, nek5, and nek6/ibo1 mutants were hypersensitive to microtubule inhibitors such as propyzamide and taxol. These results suggest that plant NEKs function in directional cell growth and organ development through the regulation of microtubule organization.  相似文献   

20.
Neurons have highly polarized arrangements of microtubules, but it is incompletely understood how microtubule polarity is controlled in either axons or dendrites. To explore whether microtubule nucleation by γ-tubulin might contribute to polarity, we analyzed neuronal microtubules in Drosophila containing gain- or loss-of-function alleles of γ-tubulin. Both increased and decreased activity of γ-tubulin, the core microtubule nucleation protein, altered microtubule polarity in axons and dendrites, suggesting a close link between regulation of nucleation and polarity. To test whether nucleation might locally regulate polarity in axons and dendrites, we examined the distribution of γ-tubulin. Consistent with local nucleation, tagged and endogenous γ-tubulins were found in specific positions in dendrites and axons. Because the Golgi complex can house nucleation sites, we explored whether microtubule nucleation might occur at dendritic Golgi outposts. However, distinct Golgi outposts were not present in all dendrites that required regulated nucleation for polarity. Moreover, when we dragged the Golgi out of dendrites with an activated kinesin, γ-tubulin remained in dendrites. We conclude that regulated microtubule nucleation controls neuronal microtubule polarity but that the Golgi complex is not directly involved in housing nucleation sites.  相似文献   

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