Motile flagellar axonemes with a 9 + 1 microtubule configuration

Electron microscope (EM) studies of the eukaryotic flagellum reveal that the organelle contains a 9 + 2 arrangement of microtubules, the axoneme, with nine doublets surrounding two singlets enveloped by a membrane which is continuous with that of the cell; various linkages and projections are associated with the microtubules. Strong experimental evidence supports the idea that the forces required for bend formation on eukaryotic flagella are derived from active relative sliding of the peripheral doublets. Dynein arms, which project from each peripheral microtubule and possess ATPase activity, interact with a neighbouring doublet and undergo conformational changes which induce sliding. To form and propagate coordinated bends along a flagellum the sliding must be resisted in a controlled manner by structures within the axoneme. The regulatory mechanism responsible for the control of inter-doublet sliding is not known in detail, but ultrastructural studies suggest that interactions between the radial spokes attached to each doublet and the central complex of the axoneme may be involved. We report here the treatment of flagella with a 9 + 2 microtubular structure from the trypanosomid flagellate Crithidia oncopelti to produce motile axonemes with only one central microtubule. We conclude that the complete central complex is not involved in the conversion of microtubule sliding into axonemal bending, but may be both associated with the control of wave propagation and essential for bend initiation.     

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.     

Spontaneous recovery after experimental manipulation of the plane of beat in sperm flagella

It is generally accepted that the oscillatory beating characteristic of sperm flagella is the result of an ATP-induced sliding between the doublet microtubules of the flagellar axoneme, with these longitudinal forces being converted into a lateral bending moment by resistive components of the structure that limit the displacement. However, little is known about the mechanisms that regulate this sliding among the nine doublets of the cylindrical axoneme to produce the coordinated planar bending waves required for efficient sperm propulsion. We have investigated these mechanisms with a new procedure in which the sperm head is held in the tip of a vibrating micropipette. Data obtained by gradually rotating the plane of imposed vibration around the sperm axis indicate that the pattern of active sliding between the outer doublet tubules can rotate relative to the sperm head, and suggest that this active sliding is regulated in part by the central tubule complex.     

Flagellar motility is required for the viability of the bloodstream trypanosome

The 9 + 2 microtubule axoneme of flagella and cilia represents one of the most iconic structures built by eukaryotic cells and organisms. Both unity and diversity are present among cilia and flagella on the evolutionary as well as the developmental scale. Some cilia are motile, whereas others function as sensory organelles and can variously possess 9 + 2 and 9 + 0 axonemes and other associated structures. How such unity and diversity are reflected in molecular repertoires is unclear. The flagellated protozoan parasite Trypanosoma brucei is endemic in sub-Saharan Africa, causing devastating disease in humans and other animals. There is little hope of a vaccine for African sleeping sickness and a desperate need for modern drug therapies. Here we present a detailed proteomic analysis of the trypanosome flagellum. RNA interference (RNAi)-based interrogation of this proteome provides functional insights into human ciliary diseases and establishes that flagellar function is essential to the bloodstream-form trypanosome. We show that RNAi-mediated ablation of various proteins identified in the trypanosome flagellar proteome leads to a rapid and marked failure of cytokinesis in bloodstream-form (but not procyclic insect-form) trypanosomes, suggesting that impairment of flagellar function may provide a method of disease control. A postgenomic meta-analysis, comparing the evolutionarily ancient trypanosome with other eukaryotes including humans, identifies numerous trypanosome-specific flagellar proteins, suggesting new avenues for selective intervention.     

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.     

鳜精巢的组织学和超微结构观察

对鳜精巢的组织学结构和超微结构进行观察．鳜精巢呈叶型结构,由精原细胞、初级精母细胞、次级精母细胞、精细胞、成熟精子等生殖细胞和支持细胞、间质细胞、边界细胞、类肌细胞、成纤维细胞、内皮细胞等非生殖细胞组成．鳜精子形成过程,精细胞大致经历了鞭毛发生,核质凝缩及核位置的改变,线粒体迁移等过程．鳜成熟精子无顶体结构,头短而圆,主要为核占据,核凹窝发达,尾细长,具侧鳍,尾部轴丝为“9 2”结构．     

Synapsin I is a microtubule-bundling protein

Synapsin I, a synaptic vesicle protein, is thought to be involved in the regulation of neurotransmission through its phosphorylation by the cyclic AMP-dependent and Ca2+/calmodulin-dependent protein kinases which become activated upon depolarization of nerve endings. However, despite its recent characterization as a spectrin-binding protein immunologically related to erythrocyte protein 4.1, other interactions of synapsin I with structural proteins remain unknown. We report here that synapsin I can co-cycle with microtubules through three cycles of warm polymerization and cold depolymerization. Synapsin I binds saturably to microtubules stabilized by taxol, with an estimated dissociation constant (Kd) of 4.5 microM and a stoichiometry of 1.2 mol of synapsin binding sites per mol tubulin dimer. Synapsin I also increases the turbidity of tubulin solutions at 37 degrees C, but without causing detectable alterations in the critical concentration required for polymerization. Mixtures of synapsin I and tubulin observed by negative stain electron microscopy contain bundles of microtubules, accounting for the effect of synapsin I on tubulin turbidity. Synapsin I is thus a candidate to mediate or regulate the interaction of synaptic vesicles with microtubules.     

Turnover of fluorescently labelled tubulin and actin in the axon.

The cytoskeleton has an important role in the generation and maintenance of the structure of the axon. Microtubules, neurofilaments and actin, together with various kinds of associated proteins, form highly organized dynamic cytoskeletal structures. Because tubulin and actin molecules are essential cytoskeletal components and are transported down the axon, it is important to understand their dynamic behaviour within the axon. Although previous pulse-labelling studies have indicated that the axonal cytoskeleton is a static complex travelling down the axon, this view has been challenged by the results of several recent experiments. We have now addressed this question by analysing the recovery of fluorescence after photobleaching fluorescent analogues of tubulin and actin in the axons of cultured neurons. We did not observe movement or spreading of bleached zones along the axon, both in neurons injected with fluorescein-labelled tubulin and actin. All bleached zones recovered their fluorescence gradually, however, indicating that microtubules and actin filaments are not static polymers moving forward within the axon, but are dynamic structures that continue to assemble along the length of the axon.     

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.     

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.     

TCP1 complex is a molecular chaperone in tubulin biogenesis.

A role in folding of newly translated proteins in the cytosol of eukaryotes has been proposed for t-complex polypeptide-1 (TCP1), although its molecular targets have not yet been identified. Tubulin is a major cytosolic protein whose assembly into microtubules is critical to many cellular processes. Although numerous studies have focused on the expression of tubulin, little is known about the processes whereby newly translated tubulin subunits acquire conformations that enable them to form alpha-beta-heterodimers. We examined the biogenesis of alpha- and beta-tubulin in rabbit reticulocyte lysate, and report here that newly translated tubulin subunits entered a 900K complex in a protease-sensitive conformation. Addition of Mg-ATP, but not nonhydrolysable analogues, released the tubulin subunits as assembly-competent protein with a conformation that was relatively protease-resistant. The 900K complex purified from reticulocyte lysate contained as its major constituent a 58K protein that cross-reacted with a monoclonal antiserum against mouse TCP1. We conclude that TCP1 functions as a cytosolic chaperone in the biogenesis of tubulin.     

The structure of ribosomal protein S5 reveals sites of interaction with 16S rRNA.

Understanding the process whereby the ribosome translates the genetic code into protein molecules will ultimately require high-resolution structural information, and we report here the first crystal structure of a protein from the small ribosomal subunit. This protein, S5, has a molecular mass of 17,500 and is highly conserved in all lifeforms. The molecule contains two distinct alpha/beta domains that have structural similarities to several other proteins that are components of ribonucleoprotein complexes. Mutations in S5 result in several phenotypes which suggest that S5 may have a role in translational fidelity and translocation. These include ribosome ambiguity or ram, reversion from streptomycin dependence and resistance to spectinomycin. Also, a cold-sensitive, spectinomycin-resistant mutant of S5 has been identified which is defective in initiation. Here we show that these mutations map to two distinct regions of the molecule which seem to be sites of interaction with ribosomal RNA. A structure/function analysis of the molecule reveals discrepancies with current models of the 30S subunit.     

The mechanism of membrane-associated steps in tail-anchored protein insertion

Tail-anchored (TA) membrane proteins destined for the endoplasmic reticulum are chaperoned by cytosolic targeting factors that deliver them to a membrane receptor for insertion. Although a basic framework for TA protein recognition is now emerging, the decisive targeting and membrane insertion steps are not understood. Here we reconstitute the TA protein insertion cycle with purified components, present crystal structures of key complexes between these components and perform mutational analyses based on the structures. We show that a committed targeting complex, formed by a TA protein bound to the chaperone ATPase Get3, is initially recruited to the membrane through an interaction with Get2. Once the targeting complex has been recruited, Get1 interacts with Get3 to drive TA protein release in an ATPase-dependent reaction. After releasing its TA protein cargo, the now-vacant Get3 recycles back to the cytosol concomitant with ATP binding. This work provides a detailed structural and mechanistic framework for the minimal TA protein insertion cycle.     

Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression

Although proteins fulfil most of the requirements that biology has for structural and functional components such as enzymes and receptors, RNA can also serve in these capacities. For example, RNA has sufficient structural plasticity to form ribozyme and receptor elements that exhibit considerable enzymatic power and binding specificity. Moreover, these activities can be combined to create allosteric ribozymes that are modulated by effector molecules. It has also been proposed that certain messenger RNAs might use allosteric mechanisms to mediate regulatory responses depending on specific metabolites. We report here that mRNAs encoding enzymes involved in thiamine (vitamin B(1)) biosynthesis in Escherichia coli can bind thiamine or its pyrophosphate derivative without the need for protein cofactors. The mRNA-effector complex adopts a distinct structure that sequesters the ribosome-binding site and leads to a reduction in gene expression. This metabolite-sensing regulatory system provides an example of a 'riboswitch' whose evolutionary origin might pre-date the emergence of proteins.     

Structure and conserved RNA binding of the PAZ domain

The discovery of RNA-mediated gene-silencing pathways, including RNA interference, highlights a fundamental role of short RNAs in eukaryotic gene regulation and antiviral defence. Members of the Dicer and Argonaute protein families are essential components of these RNA-silencing pathways. Notably, these two families possess an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain whose biochemical function is unknown. Here we report the nuclear magnetic resonance solution structure of the PAZ domain from Drosophila melanogaster Argonaute 1 (Ago1). The structure consists of a left-handed, six-stranded beta-barrel capped at one end by two alpha-helices and wrapped on one side by a distinctive appendage, which comprises a long beta-hairpin and a short alpha-helix. Using structural and biochemical analyses, we demonstrate that the PAZ domain binds a 5-nucleotide RNA with 1:1 stoichiometry. We map the RNA-binding surface to the open face of the beta-barrel, which contains amino acids conserved within the PAZ domain family, and we define the 5'-to-3' orientation of single-stranded RNA bound within that site. Furthermore, we show that PAZ domains from different human Argonaute proteins also bind RNA, establishing a conserved function for this domain.     

Stabilization of sea urchin flagellar microtubules by histone H1.

Complex microtubule assemblies are essential components of eukaryotic cilia and flagella. They are extremely stable and are not affected by agents that normally induce polymer disassembly. The molecular basis of this microtubular stability is unknown, and it is not related to any feature of the constitutive tubulin. In sea urchin sperm flagella, axonemal microtubules are found to be stabilized by a protein identical to histone H1, a result that defines a new role for this histone and provides evidence for a concerted evolution of chromatin and microtubular structures.     

The architecture of active zone material at the frog's neuromuscular junction

Active zone material at the nervous system's synapses is situated next to synaptic vesicles that are docked at the presynaptic plasma membrane, and calcium channels that are anchored in the membrane. Here we use electron microscope tomography to show the arrangement and associations of structural components of this compact organelle at a model synapse, the frog's neuromuscular junction. Our findings indicate that the active zone material helps to dock the vesicles and anchor the channels, and that its architecture provides both a particular spatial relationship and a structural linkage between them. The structural linkage may include proteins that mediate the calcium-triggered exocytosis of neurotransmitter by the synaptic vesicles during synaptic transmission.     

Mapping and sequencing of structural variation from eight human genomes

Genetic variation among individual humans occurs on many different scales, ranging from gross alterations in the human karyotype to single nucleotide changes. Here we explore variation on an intermediate scale--particularly insertions, deletions and inversions affecting from a few thousand to a few million base pairs. We employed a clone-based method to interrogate this intermediate structural variation in eight individuals of diverse geographic ancestry. Our analysis provides a comprehensive overview of the normal pattern of structural variation present in these genomes, refining the location of 1,695 structural variants. We find that 50% were seen in more than one individual and that nearly half lay outside regions of the genome previously described as structurally variant. We discover 525 new insertion sequences that are not present in the human reference genome and show that many of these are variable in copy number between individuals. Complete sequencing of 261 structural variants reveals considerable locus complexity and provides insights into the different mutational processes that have shaped the human genome. These data provide the first high-resolution sequence map of human structural variation--a standard for genotyping platforms and a prelude to future individual genome sequencing projects.     

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.