Doublet Microtubules Research Articles

Non-Processive Force Generation by Mammalian Axonemal Dynein In Situ on Doublet Microtubules

We utilize optical tweezers to examine displacements produced by small numbers of dynein motors located in situ on doublet microtubules from disintegrated mammalian sperm axonemes. In contrast with cytoplasmic dynein, we find that axonemal dynein is not processive, and the duration of individual force-generating interactions with a microtubule are longer than predicted from the velocity of movements generated by large ensembles of motors. These findings suggest that tension is required for rapid release of dynein following a power stroke and may explain how axonemal dynein is adapted to work in arrays within an axoneme, where cyclical bending patterns require motors to function over a range of sliding velocities.

Fused (Stk36) is a ciliary protein required for central pair assembly and motile cilia orientation in the mammalian oviduct

Motile cilia on the inner lining of the oviductal epithelium play a central role in ovum transport toward the uterus and subsequent fertilization by sperm. While the basic ultrastructure of 9+2 motile cilia (nine peripheral microtubule doublets surrounding a central pair) has been characterized, many important steps of ciliogenesis remain poorly understood. Our previous studies on mammalian Fused (Fu) (Stk36), a putative serine-threonine kinase, reveal a critical function of Fu in central pair construction and cilia orientation of motile cilia that line the tracheal and ependymal epithelia. These findings identify a novel regulatory component for these processes. In this study, we show that Fu is expressed in the multi-ciliated oviductal epithelium in several vertebrates, suggesting a conserved function of Fu in the oviduct. In support of this, analysis of Fu-deficient mouse oviducts uncovers a similar role of Fu in central pair construction and cilia orientation. We also demonstrate that Fu localizes to motile cilia and physically associates with kinesin Kif27 located at the cilium base and known central pair components Spag16 and Pcdp1. Our results delineate a novel pathway for central pair apparatus assembly and add important insight to the biogenesis and function of oviductal motile cilia.

WD60/FAP163 is a dynein intermediate chain required for retrograde intraflagellar transport in cilia

Retrograde intraflagellar transport (IFT) is required for assembly of cilia. We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement. Biochemical analysis revealed that FAP163 is present in the flagellar matrix and is actively trafficked by IFT. Furthermore, FAP163 copurified with D1bIC(FAP133) and the LC8 dynein light chain, indicating that it is an integral component of the retrograde IFT dynein. To assess the functional role of FAP163, we generated an RNA interference knockdown of the orthologous protein (WD60) in planaria. The Smed-wd60(RNAi) animals had a severe ciliary assembly defect that dramatically compromised whole-organism motility. Most cilia were present as short stubs that had accumulated large quantities of IFT particle-like material between the doublet microtubules and the membrane. The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz. Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly.

Bending, twisting and beating trunk robot bioinspired from the ‘3 + 0’ axoneme

The axoneme is the skeleton and motor axis of flagella and cilia in eukaryotic organisms. Basically it consists of a series of longitudinal fibers (outer doublets of microtubules) that design a cylinder and whose sliding, due to the coordinated activities of dedicated molecular motors (the dynein arms), is converted into a bending because outer doublets pairs are stabilized by elastic links (the nexine molecules). In spite of these interesting mechanical properties, mechanical and robotics engineers have never considered this amazing molecular machinery as a model. The aim of this paper is to propose the robotic design and the kinematic modeling of the ‘3 + 0’ axoneme that makes motile the flagellum of Diplauxis hatti, the simplest that exists. The model that we propose bends and twists and combines the two movements. It is able to propagate wave trains that could be involved in the development of biomimetic actuators of various mechanisms such as (sub)aquatic robotic propellers as well as robotic trunks.

The N-DRC forms a conserved biochemical complex that maintains outer doublet alignment and limits microtubule sliding in motile axonemes

The nexin-dynein regulatory complex (N-DRC) is proposed to coordinate dynein arm activity and interconnect doublet microtubules. Here we identify a conserved region in DRC4 critical for assembly of the N-DRC into the axoneme. At least 10 subunits associate with DRC4 to form a discrete complex distinct from other axonemal substructures. Transformation of drc4 mutants with epitope-tagged DRC4 rescues the motility defects and restores assembly of missing DRC subunits and associated inner-arm dyneins. Four new DRC subunits contain calcium-signaling motifs and/or AAA domains and are nearly ubiquitous in species with motile cilia. However, drc mutants are motile and maintain the 9 + 2 organization of the axoneme. To evaluate the function of the N-DRC, we analyzed ATP-induced reactivation of isolated axonemes. Rather than the reactivated bending observed with wild-type axonemes, ATP addition to drc-mutant axonemes resulted in splaying of doublets in the distal region, followed by oscillatory bending between pairs of doublets. Thus the N-DRC provides some but not all of the resistance to microtubule sliding and helps to maintain optimal alignment of doublets for productive flagellar motility. These findings provide new insights into the mechanisms that regulate motility and further highlight the importance of the proximal region of the axoneme in generating flagellar bending.

Architectural Insights into a Ciliary Partition

Ciliary compartmentalization plays pivotal roles in ciliogenesis and in various signaling pathways. Here we describe a structure at the ciliary base that appears to have all the features required for compartmentalization and which we thus call the "ciliary partitioning system" (CPS). This complex consists of the terminal plate, which serves as a cytosolic "ciliary pore complex" (CPC), and a membrane region well suited to serve as a diffusion barrier. The CPC isa plate-shaped structure containing nine pores through which the microtubule doublets of the basal body pass. Each pore expands from the doublet B-tubule into an opening well suited for the passage of intraflagellar transport particles. The membrane diffusion barrier encompasses an extended region of detergent-resistant periciliary membrane (ciliary pocket) and a ring complex that connects the CPC to the membrane. Proteomics analysis shows involvement of the ciliary pocket in vesicle trafficking, suggesting that this region plays an active role in membrane transport. The CPC and the ring together form a complete partition defining the ciliary boundary.

Morphology and ultrastructure of beluga (Huso huso) spermatozoa and a comparison with related sturgeons

The ultrastructure and morphology of beluga spermatozoa (Huso huso) (Acipenseridae, Chondrostei) were studied by scanning and transmission electron microscopy. Spermatozoa of 51.27±4.71μm total length (mean±SD) were typical of sturgeon and paddlefish spermatozoa. Each consisted of an elongated nucleus (length: 5.84±0.46μm) with distinct acrosome, a cylindrical midpiece (length: 2.10±0.42μm), and a single flagellum (length: 42.21±3.82μm). The acrosome was umbrella shaped (length: 1.12±0.14μm, width: 0.87±0.10μm) with seven to nine posterolateral projections (length: 0.49±0.09μm). Three endonuclear canals, spirally arranged, traversed the nucleus length from the acrosome to the implantation fossa. The flagellum comprised an axoneme with the typical 9+2 organization of microtubules. Two flat fins were present along the flagellum parallel to the plane of the central doublet of microtubules. These fins arose at different positions, 0.57±0.30 and 4.06±1.32μm from the midpiece and terminated 4.18±1.09 and 4.92±1.34μm from the distal end of the flagellum. Principal component analysis revealed spermatozoan ultrastructure and morphology were similar among sturgeon species, and, although assigned to genus Huso, beluga may be closely related to the genus Acipenser.

Microtubule Doublet Curvature and its Role in Cilia Actuation

Motile cilia are hair-like organelles that beat to produce fluid flow. It is not fully understood how these incredibly complex molecular machines function at the protein level to produce complicated beat patterns. The mechanical properties of microtubule doublets, the main structural element of the cilium, have not been well studied. Here we show that doublet microtubules separated from human airway cilia curl up with an average radius of curvature of 1.39 +- 0.01 µm, determined from transmission electron and atomic force imaging. This result is surprising given that cytoplasmic microtubules have a persistence length on the order of millimeters, but is consistent with curvature seen in the literature. Doublet microtubules were isolated by digesting the axoneme with trypsin and inducing sliding by the addition of ATP. After sliding, transmission electron images showed that dynein motors were still attached, which is consistent with prior studies. We prepared samples with a fraction of the outer dynein arms removed using a 0.6M potassium chloride salt extraction. Removal of dynein was confirmed by SDS-PAGE. Samples with removed dynein had an increased radius of curvature, 2.3 +- 0.1 µm. The straightening of the doublet microtubules with the removal of dynein motors implies that the asymmetric distribution of dynein motors causes a stress that bends the doublet microtubule. Our results suggest an alternative source for curvature in the beating cilium. The distribution of dynein heads bound to a microtubule doublet is known to change dynamically during the cilium beat. Changing distribution of dynein heads bound to the microtubules could induce changes in the curvature of the cilium without the need for the motors to apply a shear force.

Regulations of Microtubule Sliding by Ca<sup>2+</sup> and cAMP and Their Roles in Forming Flagellar Waveforms

The function of Ca(2+) and cAMP in extruding doublet microtubules from sea urchin sperm axoneme and generating flagellar waves was investigated in order to clarify the regulatory mechanism of microtubule sliding and the formation mechanism of beating patterns of cilia and flagella. Almost all potentially asymmetric spermatozoa that were demembranated with Triton in the absence of Ca(2+) and reactivated with MgATP(2-) (Gibbons, B.H. and Gibbons, I.R. (1980). J. Cell Biol., 84: 13-27), beat with planar waves closely resembling those of the intact spermatozoa, whereas potentially symmetric spermatozoa, in which axonemal calmodulin was removed by detergent extraction in the presence of millimolar Ca(2+) (Brokaw, C.J. and Nagayama, S.M. (1985). J. Cell Biol., 100: 1875-1883), beat with three-dimensional waves if they were reactivated with low MgATP(2-). At a high MgATP(2-), almost all demembranated spermatozoa beat with planar waves. cAMP enhanced the three-dimensionality of the flagellar waves at a low Ca(2+). These changes in the flagellar waves were caused by different regulations of the microtubule sliding by calcium, cAMP, and MgATP(2-).

Centrosomal protein CEP104/ChlamydomonasFAP256 moves to the ciliary tip during cilia assembly

The ciliary tip has been implicated in ciliary assembly and disassembly, and signaling, yet information on its protein composition is limited. Using comparative, quantitative proteomics based on the fact that tip proteins will be approximately twice as concentrated in half-length compared with full-length flagella, we have identified FAP256 as a tip protein in Chlamydomonas. FAP256 localizes to the tips of both central pair and outer doublet microtubules (MTs) and it remains at the tip during flagellar assembly and disassembly. Similarly, its vertebrate counterpart, CEP104, localizes on the distal ends of both centrioles of nondividing cells until the mother centriole forms a cilium and then localizes at the tip of the elongating cilium. A null mutant of FAP256 in Chlamydomonas and RNAi in vertebrate cells showed that FAP256/CEP104 is required for ciliogenesis in a high percentage of cells. In those cells that could form cilia, there were structural deformities at the ciliary tips.

The conserved ciliary protein Bug22 controls planar beating ofChlamydomonasflagella

Eukaryotic flagella and cilia can exhibit planar and non-planar beating, and the mechanism controlling these beating patterns is not well understood. Chlamydomonas reinhardtii flagella beat in approximately the same plane with either an asymmetric ciliary-type or symmetric flagellar-type waveform. Each B-tubule of the number 1, 5 and 6 doublets of the flagellar axoneme possesses a beak-like structure. The number 5 and 6 beak structures are implicated in conversion of ciliary motion into flagellar motion. Here, we show that in a null mutant of Bug22, the asymmetric ciliary waveform is converted into a three-dimensional (non-planar) symmetric flagellar waveform. Bug22 is localized to approximately the proximal half to two-thirds of the flagellum, similar to localization of beak-like structures. However, as shown by immunogold labeling, Bug22 associates with axonemal microtubules without apparent preference for any particular doublets. Interestingly, bug22 mutants lack all beak-like structures. We propose that one function of Bug22 is to regulate the anchoring of the beak-like structures to the doublet microtubules and confine flagellar beating to a plane.

3D Ultrastructural Organization of Whole Chlamydomonas reinhardtii Cells Studied by Nanoscale Soft X-Ray Tomography

The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology.

Conserved structural motifs in the central pair complex of eukaryotic flagella.

Cilia and flagella are conserved hair-like appendages of eukaryotic cells that function as sensing and motility generating organelles. Motility is driven by thousands of axonemal dyneins that require precise regulation. One essential motility regulator is the central pair complex (CPC) and many CPC defects cause paralysis of cilia/flagella. Several human diseases, such as immotile cilia syndrome, show CPC abnormalities, but little is known about the detailed three-dimensional (3D) structure and function of the CPC. The CPC is located in the center of typical [9+2] cilia/flagella and is composed of two singlet microtubules (MTs), each with a set of associated projections that extend toward the surrounding nine doublet MTs. Using cryo-electron tomography coupled with subtomogram averaging, we visualized and compared the 3D structures of the CPC in both the green alga Chlamydomonas and the sea urchin Strongylocentrotus at the highest resolution published to date. Despite the evolutionary distance between these species, their CPCs exhibit remarkable structural conservation. We identified several new projections, including those that form the elusive sheath, and show that the bridge has a more complex architecture than previously thought. Organism-specific differences include the presence of MT inner proteins in Chlamydomonas, but not Strongylocentrotus, and different overall outlines of the highly connected projection network, which forms a round-shaped cylinder in algae, but is more oval in sea urchin. These differences could be adaptations to the mechanical requirements of the rotating CPC in Chlamydomonas, compared to the Strongylocentrotus CPC which has a fixed orientation.

Ultrastructure and motility of the spermatozoa of Polypedates leucomystax (Amphibia, Anura, Rhacophoridae)

Sperm morphology is thought to be shaped by evolutionary pressure from the fertilization environment. Rhacophoridae (Amphibia, Anura) include both foam-nesting and nonfoam-nesting species and exhibit a variety of sperm morphologies. Here, we examine the sperm morphology and motility of a foam-nesting Rhacophoridae frog, Polypedates leucomystax. Their spermatozoa have a sickle-shaped head and a thick tail containing two axonemes with their doublet microtubule ones (Db1s) facing one another. These two axonemes are surrounded by hundreds of satellite microtubules that form a hexagonal lattice structure. The spermatozoa move spirally by directly converting their tail movements into propulsion force, similar to the movement of the sickle-shaped spermatozoa of Xenopus laevis. By comparing the spermatozoa of P. leucomystax to those of other foam-nesting Rhacophoridae frogs, Rhacophorus and Chiromantis, and to the nonfoam-nesting Rhacophoridae frog, Buergeria buergeri, we found the following: (i) Spermatozoa of foam-nesting Rhacophoridae share common morphological features, a pair of axonemes and crystallized satellite microtubules. (ii) Spermatozoa of nonfoam-nesting Rhacophoridae do not exhibit these features. (iii) Sperm motility in foam-nesting Rhacophoridae is adapted to viscous environments. (iv) A diversity of sperm morphology and motility exists even among foam-nesting Rhacophoridae frogs. (v) The spermatozoa of Rhacophorus are more adapted to the foam nest than the spermatozoa of Polypedates.

The axonemal structure of mouse respiratory cilia

Mucociliary clearance on the surface of the tracheal lumen is an important component of lung defense against dust mites, viruses. Fluid on the surface of the tracheal lumen flows from the lungs to the oropharynx as a result of effective ciliary motion. However, the axonemal structure and the mechanisms by which discretely distributed ciliary cells generate directional flow have been unknown. In this study, we examined the motion of individual cilia with 7-9 nm spatial precision by labeling the tip of each cilium with quantum dots, and detected an asymmetric beating pattern in individual cilia. Then, in order to understand the molecular mechanisms of asymmetric ciliary motion, we analyzed the axonemal structure of respiratory cilia by cryo-electron tomography and image processing. Interestingly, isolated cilia from the mouse tracheal lumen have curvature in the non-active state, and the densities of two of the eight inner dynein arms—dyneins b/g and e, which have been described in Chlamydomonas flagella—were missing from at least two doublet microtubules in respiratory cilia. These results suggest that the asymmetric axonemal structure contributes to asymmetric ciliary motion. Lastly, we investigated the flow field generated by the asymmetric ciliary motion of sparsely distributed ciliated cells in mouse trachea. Although the flow directions generated by individual ciliated cells were unsteady, the velocity field was found to be directional. These results indicate that mouse respiratory cilia with the asymmetric axonemal structure on sparsely distributed ciliated cells can generate overall directional flow from the lungs to the oropharynx.

A picture tells a thousand words: transmission electron microscopy of the ciliary transition zone in C. elegans

Transmission electron microscopy (TEM) is a valuable tool for elucidating cilium ultrastructure and the effects of ciliary and ciliopathy gene mutations on cilium integrity. We perform TEM on amphid channel sensory cilia in C. elegans, enabling us to visualise cilium compartments, namely the axoneme, the transition zone (TZ) and the transition fiber/basal body region. In worms, cilia extend from the dendritic tips of 60 sensory neurons. Here we show how TEM can be used to identify ultrastructural defects observed at the TZ of a number of ciliopathy disease gene mutants. In WT worms, the ~1 micron TZ is the most proximal part of the axoneme that extends from the far distal dendrite membrane, and is defined by a drawing together of the 9 doublet microtubules (MT) and Y-links that connect the doublet MTs with the ciliary membrane. In worms with mutations in Meckel-Gruber Syndrome (MKS) and Nephronophthisis (NPHP) gene homologues, we have observed a range of TZ abnormalities. These include TZs with Y-link defects (missing arms, reduced numbers) and TZs that fail to dock at the tip of the dendritic compartment. In most instances, TZ defects are observed only in worms where both MKS and NPHP functional modules are genetically disrupted and not in worms where only one module is abrogated. Together, our observations indicate redundant roles for multiple TZ-associated ciliopathy proteins in establishing physical connections between the TZ and the ciliary membrane.

Cloning and characterization of Trypanosoma brucei IFT complex B core

The cilium, a protrusion from the cell surface, has recently attracted immense attention due to its importance in cell signaling and its association with a wide range of human disorders. On the structural level, the cilium consists of a ring of nine microtubule doublets along which the bidirectional movement of ciliary building blocks and turnover products takes place. This transport is termed intraflagellar transport (IFT) and is carried out by two distinct multi-subunit protein complexes, IFT complex A and B, which are responsible for retrograde and anterograde transport, respectively. IFT complex B consists of at least 14 different proteins, eight of which are known to form a salt-stable core complex. Our research focuses on the IFT complex B core. The eight IFT genes from Trypanosoma brucei have been cloned into 4 Duet vectors (Novagen) to enable a simultaneous co-expression of the core complex. Upon co-expression in E. coli, the complex has been purified with minor impurities using Zinc-NTA affinity and size-exclusion chromatography. Six of the eight proteins have been confirmed via mass spectrometry and Western blot. On size-exclusion chromatography, different oligomeric states of the complex can be observed, ranging from >8MDa to around 600 kDa. Preliminary data suggests a liposome-binding capability of the complex, but the responsible subunit(s) remains to be elucidated. Since crystallization of the purified complex has not been successful, future studies will focus on electron microscopy analysis of the recombinant IFT complex B core.

In vivo three-dimensional structural analysis of cilia by cryo-electron tomography

Ciliary bending motion is driven by sliding of microtubule doublets with dynein molecular motors. Dyneins form two complexes: inner and outer dynein arms. The inner arm is supposed to determine the waveform, while the outer arm is likely an accelerator. However, the mechanism to integrate linear motion of dynein into well-orchestrated axonemal bending is still to be investigated. We are analyzing 3D structure of cilia from Tetrahymena, Chlamydomonas, sea urchin sperm and mouse trachea, to reveal the novel architecture of cilia. By comparing 3D structure of Chlamydomonas mutant axonemes, we located all the three outer arm dyneins and the eight inner arm dyneins (Bui et al. (2008) J. Cell Biol. 183, 923). Dyneins, which consist of the N-terminal tail, the ATPase ring, the stalk and the microtubule binding domain, undergo conformational change during the phosphate release: the ATPase ring shifts 8nm toward the distal end, which can explain the minus-end driven motor activity of dynein. Interestingly, in vivo, some dyneins change their conformations, while the other stay in the apo form, suggesting negative cooperativity among dyneins and bending mechanism caused by torsion at the boundary between dynein in the apo form and in the nucleotide-binding form (Movassagh et al. (2010) Nat. Struct. Mol. Biol. 17, 761). We also reconstructed 3D structure of the radial spoke, regulatory complex of 23 proteins (Pigino et al. (2011) J. Cell Biol. 195, 673). Each radial spoke has pseudo-two fold symmetry, indicating dimeric nature. Our 3D structure implies the assembly pathway of the radial spoke. http://www.psi.ch/lbr/takashi-ishikawa

The role of G proteins in the control of intraflagellar transport

Intraflagellar transport (IFT) is a mechanism used for formation of cilia and flagella in eukaryotes and little is known about its regulation. We propose that a RAB-like protein termed RABL4 (IFT27), which is associated with IFT complexes could be involved in IFT regulation. Indeed, since small GTPases are known for being key regulators of many trafficking processes. We used different approaches in order to determine the role of RABL4 during flagellum formation in Trypanosoma brucei. First, GFP::RABL4 experiments showed that the protein is localized in the cell body, at the basal body and within the flagellum, like all IFT proteins studied so far in trypanosomes. Bi-directional movement of GFP fusion protein was observed in the flagellum. RABL4 silencing demonstrated an essential role in flagellum assembly and revealed a mixture of phenotypes. Some cells do not assemble a flagellum, whereas others produce short flagella filled with material looking like IFT complexes. The structure of these short flagella is severely affected, with disrupted microtubule doublets, mislocalisation of the central pair and impaired PFR (an extra-axonemal structure) construction. The latter one can be excessively large, with numerous layers that affect the shape of the flagellum membrane. We propose that RABL4 could participate to the loading of flagellar precursors into IFT complexes for transport and we are currently analyzing GDP or GTP locked versions of the protein to challenge this hypothesis.

One of the Nine Doublet Microtubules of Eukaryotic Flagella Exhibits Unique and Partially Conserved Structures

The axonemal core of motile cilia and flagella consists of nine doublet microtubules surrounding two central single microtubules. Attached to the doublets are thousands of dynein motors that produce sliding between neighboring doublets, which in turn causes flagellar bending. Although many structural features of the axoneme have been described, structures that are unique to specific doublets remain largely uncharacterized. These doublet-specific structures introduce asymmetry into the axoneme and are likely important for the spatial control of local microtubule sliding. Here, we used cryo-electron tomography and doublet-specific averaging to determine the 3D structures of individual doublets in the flagella of two evolutionarily distant organisms, the protist Chlamydomonas and the sea urchin Strongylocentrotus. We demonstrate that, in both organisms, one of the nine doublets exhibits unique structural features. Some of these features are highly conserved, such as the inter-doublet link i-SUB5-6, which connects this doublet to its neighbor with a periodicity of 96 nm. We also show that the previously described inter-doublet links attached to this doublet, the o-SUB5-6 in Strongylocentrotus and the proximal 1–2 bridge in Chlamydomonas, are likely not homologous features. The presence of inter-doublet links and reduction of dynein arms indicate that inter-doublet sliding of this unique doublet against its neighbor is limited, providing a rigid plane perpendicular to the flagellar bending plane. These doublet-specific features and the non-sliding nature of these connected doublets suggest a structural basis for the asymmetric distribution of dynein activity and inter-doublet sliding, resulting in quasi-planar waveforms typical of 9+2 cilia and flagella.