Doublet Microtubules Research Articles

The ABCs of Centriole Architecture: The Form and Function of Triplet Microtubules.

The centriole is a defining feature of many eukaryotic cells. It nucleates a cilium, organizes microtubules as part of the centrosome, and is duplicated in coordination with the cell cycle. Centrioles have a remarkable structure, consisting of microtubules arranged in a barrel with ninefold radial symmetry. At their base, or proximal end, centrioles have unique triplet microtubules, formed from three microtubules linked to each other. This microtubule organization is not found anywhere else in the cell, is conserved in all major branches of the eukaryotic tree, and likely was present in the last eukaryotic common ancestor. At their tip, or distal end, centrioles have doublet microtubules, which template the cilium. Here, we consider the structures of the compound microtubules in centrioles and discuss potential mechanisms for their formation and their function. We propose that triplet microtubules are required for the structural integrity of centrioles, allowing the centriole to serve as the essential nucleator of the cilium.

Two-Way Traffic Determined by Different Tracks in Cilia

The cilium and the flagellum are conserved organelles that play fundamental roles in cellular signaling, sensing, and motility. Cilia and flagella have a complex microtubule-based axoneme that includes nine peripheral microtubule doublets, each comprised of a complete A-tubule and an incomplete B-tubule. However, the function of this distinctive geometry has remained unknown until now. In a recent study, Ludek Stepanek and Gaia Pigino use the model organism Chlamydomonas to reveal a new understanding of the functional significance of the A- and B-tubule structure [1]. It is known that ciliary microtubule doublets function as “railways” for intraflagellar transport (IFT), the process required for the assembly and disassembly of cilia. Large protein complexes, known as IFT trains, rapidly traverse up and down the cilium to move ciliary “building blocks” between the cell body and the distal tip of the cilium where the assembly of the cilium occurs. Electron microscopy (EM) has shown that the IFT trains move along the doublets. However, it has not been clear how the railway is organized to avoid collisions between anterograde and retrograde trains.

IFT56 regulates vertebrate developmental patterning by maintaining IFTB complex integrity and ciliary microtubule architecture.

Cilia are key regulators of animal development and depend on intraflagellar transport (IFT) proteins for their formation and function, yet the roles of individual IFT proteins remain unclear. We examined the Ift56hop mouse mutant and reveal novel insight into the function of IFT56, a poorly understood IFTB protein. Ift56hop mice have normal cilia distribution but display defective cilia structure, including abnormal positioning and number of ciliary microtubule doublets. We show that Ift56hop cilia are unable to accumulate Gli proteins efficiently, resulting in developmental patterning defects in Shh signaling-dependent tissues such as the limb and neural tube. Strikingly, core IFTB proteins are unable to accumulate normally within Ift56hop cilia, including IFT88, IFT81 and IFT27, which are crucial for key processes such as tubulin transport and Shh signaling. IFT56 is required specifically for the IFTB complex, as IFTA components and proteins that rely on IFTA function are unaffected in Ift56hop cilia. These studies define a distinct and novel role for IFT56 in IFTB complex integrity that is crucial for cilia structure and function and, ultimately, animal development.

Posttranslational Modifications of Tubulin and Cilia.

Tubulin undergoes several highly conserved posttranslational modifications (PTMs) including acetylation, detyrosination, glutamylation, and glycylation. These PTMs accumulate on a subset of microtubules that are long-lived, including those in the basal bodies and axonemes. Tubulin PTMs are distributed nonuniformly. In the outer doublet microtubules of the axoneme, the B-tubules are highly enriched in the detyrosinated, polyglutamylated, and polyglycylated tubulin, whereas the A-tubules contain mostly unmodified tubulin. The nonuniform patterns of tubulin PTMs may functionalize microtubules in a position-dependent manner. Recent studies indicate that tubulin PTMs contribute to the assembly, disassembly, maintenance, and motility of cilia. In particular, tubulin glutamylation has emerged as a key PTM that affects ciliary motility through regulation of axonemal dynein arms and controls the stability and length of the axoneme.

Progress in ciliary ion channel physiology.

Mammalian cilia are ubiquitous appendages found on the apical surface of cells. Primary and motile cilia are distinct in both morphology and function. Most cells have a solitary primary cilium (9+0), which lacks the central microtubule doublet characteristic of motile cilia (9+2). The immotile primary cilia house unique signaling components and sequester several important transcription factors. In contrast, motile cilia commonly extend into the lumen of respiratory airways, fallopian tubes, and brain ventricles to move their contents and/or produce gradients. In this review, we focus on the composition of putative ion channels found in both types of cilia and in the periciliary membrane and discuss their proposed functions. Our discussion does not cover specialized cilia in photoreceptor or olfactory cells, which express many more ion channels.

Microtubule binding protein PACRG plays a role in regulating specific ciliary dyneins during microtubule sliding.

The complex waveforms characteristic of motile eukaryotic cilia and flagella are produced by the temporally and spatially regulated action of multiple dynein subforms generating sliding between subsets of axonemal microtubules. Multiple protein complexes have been identified that are associated with the doublet microtubules and that mediate regulatory signals between key axonemal structures, such as the radial spokes and central apparatus, and the dynein arm motors; these complexes include the N-DRC, MIA, and CSC complexes. Previous studies have shown that PACRG (parkin co-regulated gene) forms a complex that is anchored to the axonemal doublet microtubules. Loss of PACRG causes defects in ciliary motility and cilia related diseases. Here, we use an in vitro microtubule sliding assay to demonstrate that PACRG and its interactors are part of a signaling pathway that includes the central apparatus, radial spokes and specific inner dynein arm subforms to control dynein-driven microtubule sliding. Using a biochemical approach, our studies also indicate that PACRG interacts with the radial spokes. © 2016 Wiley Periodicals, Inc.

Alterations in oviductal cilia morphology and reduced expression of axonemal dynein in diabetic NOD mice.

In the present study, we examined the morphology of cilia and expression of the dynein intermediate chain 2 (DNAI2) in the oviduct of non-obese diabetic (NOD) mice. Results obtained with immunohistochemistry showed that DNAI2 expression was reduced in oviducts of diabetic NOD (dNOD) mice, as compared to that observed in the normoglycemic NOD (cNOD) group, especially in the acyclic dNOD mice. Oviductal cilia of dNOD mice appeared to be reduced in number. Results obtained with Western blot analysis revealed that the expression of DNAI2 protein was significantly less in oviducts of dNOD mice as compared to that of cNOD mice corroborating the results obtained with immunohistochemistry. Electron microscopic examination and quantitative imaging of thin sections of Epon-embedded oviducts of both dNOD and cNOD mice confirmed the reduction of the number of cilia in the oviduct of the dNOD group which also displayed aberrant axonemal ultrastructure, including disorganization of the axoneme and alteration of microtubule doublets into singlets as well as disruption of the plasma membrane in many cilia. Taken together, the present findings suggest that structural alterations of oviductal cilia in female diabetic NOD mice might be detrimental to the normal function of these particular cell structures in gamete transport.

Curvature regulation of the ciliary beat through axonemal twist.

Cilia and flagella are hairlike organelles that propel cells through fluid. The active motion of the axoneme, the motile structure inside cilia and flagella, is powered by molecular motors of the axonemal dynein family. These motors generate forces and torques that slide and bend the microtubule doublets within the axoneme. To create regular waveforms, the activities of the dyneins must be coordinated. It is thought that coordination is mediated by stresses due to radial, transverse, or sliding deformations, and which build up within the moving axoneme and feed back on dynein activity. However, which particular components of the stress regulate the motors to produce the observed waveforms of the many different types of flagella remains an open question. To address this question, we describe the axoneme as a three-dimensional bundle of filaments and characterize its mechanics. We show that regulation of the motors by radial and transverse stresses can lead to a coordinated flagellar motion only in the presence of twist. We show that twist, which could arise from torque produced by the dyneins, couples curvature to transverse and radial stresses. We calculate emergent beating patterns in twisted axonemes resulting from regulation by transverse stresses. The resulting waveforms are similar to those observed in flagella of Chlamydomonas and sperm. Due to the twist, the waveform has nonplanar components, which result in swimming trajectories such as twisted ribbons and helices, which agree with observations.

The Central Apparatus of Cilia and Eukaryotic Flagella.

The motile cilium is a complex organelle that is typically comprised of a 9+2 microtubule skeleton; nine doublet microtubules surrounding a pair of central singlet microtubules. Like the doublet microtubules, the central microtubules form a scaffold for the assembly of protein complexes forming an intricate network of interconnected projections. The central microtubules and associated structures are collectively referred to as the central apparatus (CA). Studies using a variety of experimental approaches and model organisms have led to the discovery of a number of highly conserved protein complexes, unprecedented high-resolution views of projection structure, and new insights into regulation of dynein-driven microtubule sliding. Here, we review recent progress in defining mechanisms for the assembly and function of the CA and include possible implications for the importance of the CA in human health.

Functional anatomy of the mammalian sperm flagellum.

The eukaryotic flagellum is the organelle responsible for the propulsion of the male gamete in most animals. Without exception, sperm of all mammalian species use a flagellum for swimming. The mammalian sperm has a centrally located 9 + 2 arrangement of microtubule doublets and hundreds of accessory proteins that together constitute an axoneme. However, they also possess several characteristic peri-axonemal structures that make the mammalian sperm tail function differently. These modifications include nine outer dense fibers (ODFs) that are paired with the nine outer microtubule doublets of the axoneme, and are anchored in a structure called the connecting piece located at the base. The presence of the ODFs and connecting piece, and the absence of a basal body, dictate that physical forces generated by the dynein motors are transmitted to the base of the flagellum through the ODFs. Mammalian sperm flagella also possess a mitochondrial and a fibrous sheath that encircle most of the axoneme. These sheaths and the ODFs add mechanical rigidity to the flagellum creating the functional effect of increasing bend wavelength, which requires the entrainment of more dynein motors in the production of a single wave. The sheaths also act as a retinaculum and maintain the integrity of the central axoneme when large bending torques are generated by dynein. Large torque production is crucial to the process of hyperactivation and the unique motility transitions associated with effective fertilizing capacity. Consequently, these specialized anatomical features are essential for the effective interaction of sperm with the female reproductive tract and ovum. © 2016 Wiley Periodicals, Inc.

Emergence of flagellar beating from the collective behavior of individual ATP-powered dyneins.

Flagella are hair-like projections from the surface of eukaryotic cells, and they play an important role in many cellular functions, such as cell-motility. The beating of flagella is enabled by their internal architecture, the axoneme, and is powered by a dense distribution of motor proteins, dyneins. The dyneins deliver the required mechanical work through the hydrolysis of ATP. Although the dynein-ATP cycle, the axoneme microstructure, and the flagellar-beating kinematics are well studied, their integration into a coherent picture of ATP-powered flagellar beating is still lacking. Here we show that a time-delayed negative-work-based switching mechanism is able to convert the individual sliding action of hundreds of dyneins into a regular overall beating pattern leading to propulsion. We developed a computational model based on a minimal representation of the axoneme consisting of two representative doublet microtubules connected by nexin links. The relative sliding of the microtubules is incorporated by modeling two groups of ATP-powered dyneins, each responsible for sliding in opposite directions. A time-delayed switching mechanism is postulated, which is key in converting the local individual sliding action of multiple dyneins into global beating. Our results demonstrate that an overall nonreciprocal beating pattern can emerge with time due to the spatial and temporal coordination of the individual dyneins. These findings provide insights in the fundamental working mechanism of axonemal dyneins and could possibly open new research directions in the field of flagellar motility.

A Forward Genetic Screen and Whole Genome Sequencing Identify Deflagellation Defective Mutants in Chlamydomonas, Including Assignment of ADF1 as a TRP Channel.

With rare exception, ciliated cells entering mitosis lose their cilia, thereby freeing basal bodies to serve as centrosomes in the formation of high-fidelity mitotic spindles. Cilia can be lost by shedding or disassembly, but either way, it appears that the final release may be via a coordinated severing of the nine axonemal outer doublet microtubules linking the basal body to the ciliary transition zone. Little is known about the mechanism or regulation of this important process. The stress-induced deflagellation response of Chlamydomonas provides a basis to identifying key players in axonemal severing. In an earlier screen we uncovered multiple alleles for each of three deflagellation genes, ADF1, FA1, and FA2. Products of the two FA genes localize to the site of axonemal severing and encode a scaffolding protein and a member of the NIMA-related family of ciliary-cell cycle kinases. The identity of the ADF1 gene remained elusive. Here, we report a new screen using a mutagenesis that yields point mutations in Chlamydomonas, an enhanced screening methodology, and whole genome sequencing. We isolated numerous new alleles of the three known genes, and one or two alleles each of at least four new genes. We identify ADF1 as a TRP ion channel, which we suggest may reside at the flagellar transition zone.

Novel roles for the radial spoke head protein 9 in neural and neurosensory cilia.

Cilia are cell surface organelles with key roles in a range of cellular processes, including generation of fluid flow by motile cilia. The axonemes of motile cilia and immotile kinocilia contain 9 peripheral microtubule doublets, a central microtubule pair, and 9 connecting radial spokes. Aberrant radial spoke components RSPH1, 3, 4a and 9 have been linked with primary ciliary dyskinesia (PCD), a disorder characterized by ciliary dysmotility; yet, radial spoke functions remain unclear. Here we show that zebrafish Rsph9 is expressed in cells bearing motile cilia and kinocilia, and localizes to both 9 + 2 and 9 + 0 ciliary axonemes. Using CRISPR mutagenesis, we show that rsph9 is required for motility of presumptive 9 + 2 olfactory cilia and, unexpectedly, 9 + 0 neural cilia. rsph9 is also required for the structural integrity of 9 + 2 and 9 + 0 ciliary axonemes. rsph9 mutant larvae exhibit reduced initiation of the acoustic startle response consistent with hearing impairment, suggesting a novel role for Rsph9 in the kinocilia of the inner ear and/or lateral line neuromasts. These data identify novel roles for Rsph9 in 9 + 0 motile cilia and in sensory kinocilia, and establish a useful zebrafish PCD model.

Paclitaxel suppresses Tau-mediated microtubule bundling in a concentration-dependent manner

BackgroundMicrotubules (MTs) are protein nanotubes comprised of straight protofilaments (PFs), head to tail assemblies of αβ-tubulin heterodimers. Previously, it was shown that Tau, a microtubule-associated protein (MAP) localized to neuronal axons, regulates the average number of PFs in microtubules with increasing inner radius <RinMT> observed for increasing Tau/tubulin-dimer molar ratio ΦTau at paclitaxel/tubulin-dimer molar ratio ΛPtxl=1/1. MethodsWe report a synchrotron SAXS and TEM study of the phase behavior of microtubules as a function of varying concentrations of paclitaxel (1/32≤ΛPtxl≤1/4) and Tau (human isoform 3RS, 0≤Φ3RS≤1/2) at room temperature. ResultsTau and paclitaxel have opposing regulatory effects on microtubule bundling architectures and microtubule diameter. Surprisingly and in contrast to previous results at ΛPtxl=1/1 where microtubule bundles are absent, in the lower paclitaxel concentration regime (ΛPtxl≤1/4), we observe both microtubule doublets and triplets with increasing Tau. Furthermore, increasing paclitaxel concentration (up to ΛPtxl=1/1) slightly decreased the average microtubule diameter (by ~1 PF) while increasing Tau concentration (up to Φ3RS=1/2) significantly increased the diameter (by ~2–3 PFs). ConclusionsThe suppression of Tau-mediated microtubule bundling with increasing paclitaxel is consistent with paclitaxel seeding more, but shorter, microtubules by rapidly exhausting tubulin available for polymerization. Microtubule bundles require the aggregate Tau-Tau attractions along the microtubule length to overcome individual microtubule thermal energies disrupting bundles. General significanceInvestigating MAP-mediated interactions between microtubules (as it relates to in vivo behavior) requires the elimination or minimization of paclitaxel.

Axonemal Dynein Arms.

Axonemal dyneins form the inner and outer rows of arms associated with the doublet microtubules of motile cilia. These enzymes convert the chemical energy released from adenosine triphosphate (ATP) hydrolysis into mechanical work by causing the doublets to slide with respect to each other. Dyneins form two major groups based on the number of heavy-chain motors within each complex. In addition, these enzymes contain other components that are required for assembly of the complete particles and/or for the regulation of motor function in response to phosphorylations status, ligands such as Ca2+, changes in cellular redox state and which also apparently monitor and respond to the mechanical state or curvature in which any given motor finds itself. It is this latter property, which is thought to result in waves of motor function propagating along the axoneme length. Here, I briefly describe our current understanding of axonemal dynein structure, assembly, and organization.

Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease.

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or ‘primary’ cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.

The nexin link and B-tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme.

We developed quantitative assays to test the hypothesis that the N-DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed "reactivated cell models." ATP-induced reactivation of wild-type cells resulted in the forward swimming of ∼90% of cell models. ATP-induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark-field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild-type reactivated cell models remained intact. The sup-pf4 and drc3 mutants, unlike other drc mutants, retain most of the N-DRC linker that interconnects outer doublet microtubules. Reactivated sup-pf4 and drc3 cell models displayed nearly wild-type levels of forward motility. Thus, the N-DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP-induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N-DRC indicate B-tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N-DRC function. © 2016 Wiley Periodicals, Inc.

Analysis of the forces acting on beating cilia

Detailed analysis of the forces acting on a uniform-diameter beating cilium is carried out to determine the moment generated by the inter-doublet forces acting along the length of a cilium and the results are compared with the sliding-control theory according to which the moment is a function of the interdoublet sliding. In the central part of the cilium the inter-doublet forces are found to be proportional to the inter-doublet sliding. However, in spite of the uniformity of the diameter of the cilium, the proportionality constant, known as the dynamic stiffness, is not constant along its entire length. Significant variations are observed in the regions both near the tip of the cilium and proximal to the cell body. In the tip region the magnitude of the dynamic stiffness is found to decrease. This decrease is probably due to decrease in the number density of the molecular motors in that region and in the number of doublet microtubules. The behavior in the proximal region, on the other hand, does not appear to be well described by the sliding control theory. Our analysis therefore suggests that the dynamics of ciliary beating cannot be adequately described by a simple sliding-control theory with constant dynamic stiffness. Our analysis suggests that the cilium is differentiated into a basal region optimized for the creation of a wave and a central region optimized to support a traveling wave that provides the thrust for the cell.

Microtubule doublets are double-track railways for intraflagellar transport trains.

The cilium is a large macromolecular machine that is vital for motility, signaling, and sensing in most eukaryotic cells. Its conserved core structure, the axoneme, contains nine microtubule doublets, each comprising a full A-microtubule and an incomplete B-microtubule. However, thus far, the function of this doublet geometry has not been understood. We developed a time-resolved correlative fluorescence and three-dimensional electron microscopy approach to investigate the dynamics of intraflagellar transport (IFT) trains, which carry ciliary building blocks along microtubules during the assembly and disassembly of the cilium. Using this method, we showed that each microtubule doublet is used as a bidirectional double-track railway: Anterograde IFT trains move along B-microtubules, and retrograde trains move along A-microtubules. Thus, the microtubule doublet geometry provides direction-specific rails to coordinate bidirectional transport of ciliary components.

Stay on the right track

Cell Biology Cilia contain a well-ordered array of microtubule doublets along their length. A longstanding question in cilium structure and function is why the microtubule arrangement in cilia is so complex. Stepanek and Pigino developed a time-resolved correlative fluorescence and three-dimensional electron microscopy method to show that the doublets provide directionality to intraflagellar transport. One microtubule in the pair moves cargoes up to the ciliary tip. Meanwhile, the other microtubule moves cargoes back to the cell body. These results explain why the axoneme is built out of microtubule doublets and suggest a mechanistic picture of how the logistics of bidirectional intraflagellar transport are regulated. Science , this issue p. [721][1] [1]: /lookup/doi/10.1126/science.aaf4594