Adjacent Doublet Research Articles

P-076 Role of N-DRC complex in male fertility

Abstract Study question There are many causes of human infertility, what’s the role of Nexin-dynein regulatory complex (N-DRC) in male fertility? Summary answer Many gene mutants in N-DRC components have been identified in infertile men, which affect the assembly of N-DRC complex and cause defects of sperm. What is known already human infertility affects 10–15% of couples, asthenozoospermia accounting for 18% of infertile men and is a common male infertility phenotype. Nexin-dynein regulatory complex (N-DRC) is a large protein complex regulating device in the flagellum and connects adjacent doublets of microtubules (DMTs). Some N-DRC components were reported to be associated with human disease, such as Primary ciliary dyskinesia (PCD). In recent years, more and more studies reported mutants in subunits of N-DRC are related to male infertility. For example, DRC1, DRC5 and DRC3 mutant were identified in patients displayed multiple morphological abnormalities of the flagella (MMAF) and obstructive azoospermia. Study design, size, duration We searched articles about N-DRC and male infertility in pumped and select research studies and the tidied up the gene mutants, gene knock out mouse modals and IVF outcomes of different N-DRC components in infertile men. Besides, we summarized the parameters of semen and ultrastructure of sperm flagella in these patients. Participants/materials, setting, methods We searched articles about N-DRC and male infertility in pumped and select research studies and the tidied up the gene mutants, gene knock out mouse modals and IVF outcomes of different N-DRC components in infertile men. Main results and the role of chance N-DRC is a highly conserved structural complex located in the axoneme of the flagellum and cilium, containing at least 11 different subunits (DRC1∼DRC11). Patients who were diagnosed as MMAF and male infertility carrying homozygous DRC1 mutants. TEM analyses of the patients’ sperm revealed the flagellar axoneme structure is disordered. A frameshift mutant of DRC5 was identified in an infertile male with asthenozoospermia, but the patient’s problem of infertility could be successfully solved by vitro Fertilization (IVF). A recent study identified two bi-allelic DRC3 frameshift variants in two unrelated patients, one patient displayed MMAF and the other present obstructive azoospermia. The sperm flagella function of DRC5 deletion mice was abrogated but flagellum ultrastructure remained normal and the mice behaved as asthenozoospermia and sterile. DRC3, DRC7 and DRC9 knockout mice exhibit MMAF and male infertility, and in DRC7 KO mice, the axoneme of sperm is disorganized and it was difficult to detect the ‘9 + 2’ microtubule structure. DRC1 mutant mice on the C57BL/6 background died before puberty, KO DRC6 in mice neither has no obvious effects on sperm motility function nor destroy the “9 + 2” arrangement of microtubules in cilia, and male mice was fertile. Limitations, reasons for caution More samples of MMAF and asthenozoospermia men to sequence N-DRC gene, and track their assisted reproductive outcomes. Wider implications of the findings The conclusions in our study uncover certain mechanism of N-DRC deficient in MMAF and asthenozoospermia phenotype, which could provide a new basis for diagnosis of male infertility. Trial registration number not applicable

Homozygous variant in DRC3 (LRRC48) gene causes asthenozoospermia and male infertility.

Human infertility affects 10-15% of couples. Asthenozoospermia accounts for 18% of men with infertility and is a common male infertility phenotype. The nexin-dynein regulatory complex (N-DRC) is a large protein complex in the sperm flagellum that connects adjacent doublets of microtubules. Defects in the N-DRC can disrupt cilia/flagellum movement, resulting in primary ciliary dyskinesia and male infertility. Using whole-exome sequencing, we identified a pathological homozygous variant of the dynein regulatory complex subunit 3 (DRC3) gene, which expresses leucine-rich repeat-containing protein 48, a component of the N-DRC, in a patient with asthenozoospermia. The variant ENST00000313838.12: c.644dup (p. Glu216GlyfsTer36) causes premature translational arrest of DRC3, resulting in a dysfunctional DRC3 protein. The patient's semen count, color, and pH were normal according to the reference values of the World Health Organization guidelines; however, sperm motility and progressive motility were reduced. DRC3 protein was not detected in the patient's sperm and the ultrastructure of the patient's sperm flagella was destroyed. More importantly, the DRC3 variant reduced its interaction with other components of the N-DRC, including dynein regulatory complex subunits 1, 2, 4, 5, 7, and 8. Our data not only revealed the essential biological functions of DRC3 in sperm flagellum movement and structure but also provided a new basis for the clinical genetic diagnosis of male infertility.

Integrated modeling of the Nexin-dynein regulatory complex reveals its regulatory mechanism

Cilia are hairlike protrusions that project from the surface of eukaryotic cells and play key roles in cell signaling and motility. Ciliary motility is regulated by the conserved nexin-dynein regulatory complex (N-DRC), which links adjacent doublet microtubules and regulates and coordinates the activity of outer doublet complexes. Despite its critical role in cilia motility, the assembly and molecular basis of the regulatory mechanism are poorly understood. Here, using cryo-electron microscopy in conjunction with biochemical cross-linking and integrative modeling, we localize 12 DRC subunits in the N-DRC structure of Tetrahymena thermophila. We also find that the CCDC96/113 complex is in close contact with the DRC9/10 in the linker region. In addition, we reveal that the N-DRC is associated with a network of coiled-coil proteins that most likely mediates N-DRC regulatory activity.

Remodeling and activation mechanisms of outer arm dyneins revealed by cryo-EM.

Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs' conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryo-electron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with coarse-grained molecular dynamic simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.

X-Ray Fiber Diffraction and Numerical Simulation Studies on the Change in the Helical Symmetry of Chlamydomonas Flagellar Axonemes Coupled with the Change in Ca2+ Concentrations

Cilia and flagella are motile organelles found in various types of eukaryotes. In their axonemes, dynein arms generate shear between adjacent doublets, and this shear is converted into complex waveforms. The conversion mechanism has remained a long-standing unresolved issue. Since Chlamydomonas flagella change their waveforms from a flagellar type to a ciliary type, responding to the increase in intracellular Ca2+ concentrations, this response may provide a hint for the mechanism of the flagellar waveform generation.

Direction-Finding With Spread Array of Identical Doublets

In this letter, we present a fully sparse array geometry termed as Spread Array with Identical Doublets (SAID) for high accuracy direction-finding and mutual coupling effect reduction. In SAID configuration, the distances of two adjacent doublets and between two components of a doublet are all set to be larger than a half-wavelength. A direction estimation algorithm is further derived to yield unambiguous source direction estimates to make the SAID array configuration practically feasible. Numerical examples are finally provided to justify the SAID in terms of the estimation accuracy improvement and the mutual coupling reduction, by using a practical mutual coupling model.

Cryo electron tomography with volta phase plate reveals novel structural foundations of the 96-nm axonemal repeat in the pathogen Trypanosoma brucei.

The 96-nm axonemal repeat includes dynein motors and accessory structures as the foundation for motility of eukaryotic flagella and cilia. However, high-resolution 3D axoneme structures are unavailable for organisms among the Excavates, which include pathogens of medical and economic importance. Here we report cryo electron tomography structures of the 96-nm repeat from Trypanosoma brucei, a protozoan parasite in the Excavate lineage that causes African trypanosomiasis. We examined bloodstream and procyclic life cycle stages, and a knockdown lacking DRC11/CMF22 of the nexin dynein regulatory complex (NDRC). Sub-tomogram averaging yields a resolution of 21.8 Å for the 96-nm repeat. We discovered several lineage-specific structures, including novel inter-doublet linkages and microtubule inner proteins (MIPs). We establish that DRC11/CMF22 is required for the NDRC proximal lobe that binds the adjacent doublet microtubule. We propose that lineage-specific elaboration of axoneme structure in T. brucei reflects adaptations to support unique motility needs in diverse host environments.

An evaluation of interferences in heat production from low enthalpy geothermal doublets systems

Required distance between doublet systems in low enthalpy geothermal heat exploitation is often not fully elucidated. The required distance aims to prevent negative interference influencing the utilisation efficiency of doublet systems. Currently production licence areas are often issued based on the expected extent of the reinjected cold water plume on the moment of thermal breakthrough. The production temperature, however, may not immediately drop to non-economic values after this moment. Consequently, heat production could continue increasing the extent of the cold water plume. Furthermore, the area influenced by pressure because of injection and production spreads beyond the cold water plume extent, influencing not only the productivity of adjacent doublet systems but also the shape of cold water plumes. This affects doublet life time, especially if adjacent doublets have different production rates. In this modelling based study a multi parameter analysis is carried out to derive dimensionless relations between basic doublet design parameters and required doublet distance. These parameters include the spacing between injector and producer of the same doublet, different production rates, aquifer thickness and minimal required production temperature. The results of this study can be used to minimize negative interference or optimise positive interference aiming at improving geothermal doublet deployment efficiency.

Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation.

It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements.

The geometric clutch at 20: stripping gears or gaining traction?

It has been 20 years since the geometric clutch (GC) hypothesis was first proposed. The core principle of the GC mechanism is fairly simple. When the axoneme of a eukaryotic flagellum is bent, mechanical stress generates forces transverse to the outer doublets (t-forces). These t-forces can push doublets closer together or pry them apart. The GC hypothesis asserts that changes in the inter-doublet spacing caused by t-forces are responsible for the activation and deactivation of the dynein motors, that creates the beat cycle. A series of computer models utilizing the clutch mechanism has shown that it can simulate ciliary and flagellar beating. The objective of the present review is to assess where things stand with the GC hypothesis in the clarifying light of new information. There is considerable new evidence to support the hypothesis. However, it is also clear that it is necessary to modify some of the original conceptions of the hypothesis so that it can be consistent with the results of recent experimental and ultrastructural studies. In particular, dynein deactivation by t-forces must be able to occur with dyneins that remain attached to the B-subtubule of the adjacent doublet.

The dynein-triggered ciliary motion in embryonic nodes: an exploratory study based on computational models.

The cilia, presenting a rotational movement in the embryonic nodes, play a crucial role in the left-right specification during embryogenesis. The characteristic architecture of these cilia is based on a cylindrical arrangement of 9 doublet microtubules and the motion of the cilia is triggered by the dynein motors located between adjacent doublets by converting the chemical energy into mechanical work. Restricted by the inherent difficulties of experiments, the dynein activation patterns in moving cilia cannot be directly observed. Thus, the mechanism of nodal ciliary movement is still unclear. In this study, we present computational models of the nodal ciliary ultrastructure based on tomographic images of the ciliary body. By employing time accurate three-dimensional solid mechanics analysis, we investigate the dynein-triggered sliding between adjacent doublet microtubules and simulate the induced ciliary bending. As an exploratory study, two dynein activation patterns are proposed and their rationality is discussed. The mathematical model presented by this paper provides a platform to investigate various assumptions of dynein activity, facilitating us to propose the most possible dynein activation pattern and therefore improving our understandings regarding the protein-beating problems of cilia.

Heart of the Beat (the Flagellar Beat, that Is)

Heart of the Beat (the Flagellar Beat, that Is)

Asymmetry of inner dynein arms and inter-doublet links in Chlamydomonas flagella

Although the widely shared “9 + 2” structure of axonemes is thought to be highly symmetrical, axonemes show asymmetrical bending during planar and conical motion. In this study, using electron cryotomography and single particle averaging, we demonstrate an asymmetrical molecular arrangement of proteins binding to the nine microtubule doublets in Chlamydomonas reinhardtii flagella. The eight inner arm dynein heavy chains regulate and determine flagellar waveform. Among these, one heavy chain (dynein c) is missing on one microtubule doublet (this doublet also lacks the outer dynein arm), and another dynein heavy chain (dynein b or g) is missing on the adjacent doublet. Some dynein heavy chains either show an abnormal conformation or were replaced by other proteins, possibly minor dyneins. In addition to nexin, there are two additional linkages between specific pairs of doublets. Interestingly, all these exceptional arrangements take place on doublets on opposite sides of the axoneme, suggesting that the transverse functional asymmetry of the axoneme causes an in-plane bending motion.

Clockwise Translocation of Microtubules by Flagellar Inner-Arm Dyneins In Vitro

Cilia and flagella are equipped with multiple species of dyneins that have diverse motor properties. To assess the properties of various axonemal dyneins of Chlamydomonas, in vitro microtubule translocation by isolated dyneins was examined with and without flow of the medium. With one inner-arm dynein species, dynein c, most microtubules became aligned parallel to the flow and translocated downstream after the onset of flow. When the flow was stopped, the gliding direction was gradually randomized. In contrast, with inner-arm dyneins d and g, microtubules tended to translocate at a shallow right angle to the flow. When the flow was stopped, each microtubule turned to the right, making a curved track. The clockwise translocation was not accompanied by lateral displacement, indicating that these dyneins generate torque that bends the microtubule. The torque generated by these dyneins in the axoneme may modulate the relative orientation between adjacent doublet microtubules and lead to more efficient functioning of total dyneins.

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

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

Two Anti-radial Spoke Monoclonal Antibodies Inhibit Chlamydomonas Axonemal Motility by Different Mechanisms

In the 9 + 2 axoneme, radial spokes are structural components attached to the A-tubules of the nine outer doublet microtubules. They protrude toward the central pair microtubule complex with which they have transient but regular interactions for the normal flagellar motility to occur. Flagella of Chlamydomonas mutants deficient in entire radial spokes or spoke heads are paralyzed. In this study the importance of two radial spoke proteins in the flagellar movement is exemplified by the potent inhibitory action of two monoclonal antibodies on the axonemal motility of demembranated-reactivated Chlamydomonas models. We show that one of these proteins is localized on the stalk of the radial spokes, whereas the other is a component of the head of the same structure and most likely correspond to radial spoke protein 2 and 1, respectively. Fine motility analysis by videomicrography further indicates that these two anti-radial spoke protein antibodies at low concentration affect motility of demembranated-reactivated Chlamydomonas by changing the flagellar waveform without modifying axonemal beat frequency. They also modify wave amplitude differently during motility inhibition. This brings more direct evidence for the involvement of both radial spoke stalk and head in the fine tuning of the waveform during flagellar motility.

Structural-Functional Relationships of the Dynein, Spokes, and Central-Pair Projections Predicted from an Analysis of the Forces Acting within a Flagellum

In the axoneme of eukaryotic flagella the dynein motor proteins form crossbridges between the outer doublet microtubules. These motor proteins generate force that accumulates as linear tension, or compression, on the doublets. When tension or compression is present on a curved microtubule, a force per unit length develops in the plane of bending and is transverse to the long axis of the microtubule. This transverse force (t-force) is evaluated here using available experimental evidence from sea urchin sperm and bull sperm. At or near the switch point for beat reversal, the t-force is in the range of 0.25–1.0 nN/ μm, with 0.5 nN/ μm the most likely value. This is the case in both beating and arrested bull sperm and in beating sea urchin sperm. The total force that can be generated (or resisted) by all the dyneins on one micron of outer doublet is also ∼0.5 nN. The equivalence of the maximum dynein force/ μm and t-force/ μm at the switch point may have important consequences. Firstly, the t-force acting on the doublets near the switch point of the flagellar beat is sufficiently strong that it could terminate the action of the dyneins directly by strongly favoring the detached state and precipitating a cascade of detachment from the adjacent doublet. Secondly, after dynein release occurs, the radial spokes and central-pair apparatus are the structures that must carry the t-force. The spokes attached to the central-pair projections will bear most of the load. The central-pair projections are well-positioned for this role, and they are suitably configured to regulate the amount of axoneme distortion that occurs during switching. However, to fulfill this role without preventing flagellar bend formation, moveable attachments that behave like processive motor proteins must mediate the attachment between the spoke heads and the central-pair structure.

Fine structure of the quantum-dot trion

A theory of the fine structure of the excited state of the quantum-dot trion, which is due to spin–spin interactions, is presented. In particular, it is shown that the fine structure is universal for both positive (two holes, one electron) and negative (two electrons, one hole) trions. It consists of four doublets, three of them forming a compact group of levels separated from the remaining doublet by the energy of the exchange interaction between identical particles (i.e. between two electrons or two holes). The energy separation between the adjacent doublets in the six-level group is of the order of the much smaller energy of the exchange interaction between particles belonging to different bands, i.e. of the exciton exchange energy.

Computer simulation of flagellar movement VIII: coordination of dynein by local curvature control can generate helical bending waves.

Computer simulations have been carried out with a model flagellum that can bend in three dimensions. A pattern of dynein activation in which regions of dynein activity propagate along each doublet, with a phase shift of approximately 1/9 wavelength between adjacent doublets, will produce a helical bending wave. This pattern can be termed "doublet metachronism." The simulations show that doublet metachronism can arise spontaneously in a model axoneme in which activation of dyneins is controlled locally by the curvature of each outer doublet microtubule. In this model, dyneins operate both as sensors of curvature and as motors. Doublet metachronism and the chirality of the resulting helical bending pattern are regulated by the angular difference between the direction of the moment and sliding produced by dyneins on a doublet and the direction of the controlling curvature for that doublet. A flagellum that is generating a helical bending wave experiences twisting moments when it moves against external viscous resistance. At high viscosities, helical bending will be significantly modified by twist unless the twist resistance is greater than previously estimated. Spontaneous doublet metachronism must be modified or overridden in order for a flagellum to generate the planar bending waves that are required for efficient propulsion of spermatozoa. Planar bending can be achieved with the three-dimensional flagellar model by appropriate specification of the direction of the controlling curvature for each doublet. However, experimental observations indicate that this "hard-wired" solution is not appropriate for real flagella.

The molecular motors of cilia and eukaryotic flagella.

Axonemal dyneins occur in two rows (as inner and outer arms) on each of the nine doublets. Axonemal dynein binds reversibly to the B-microtubule and has an ATP-insensitive anchorage to the A-microtubule of the adjacent doublet. The heavy chains have the form of globular heads and are responsible for chemo-mechanical transduction. The B-tubule-binding site is on a tenuous extension of the head. There is only one type of ODA. A 12 nm shift in the globular heads is associated with the hydrolysis cycle. There are three types of IDA. No functional changes have been recognized in their complex conformation. There is plentiful evidence that the axonemal dyneins produce interdoublet displacement. Doubt remains on how much sliding occurs per cycle of ATP hydrolysis. The mechanism for transforming sliding into bending is not yet explained.