Nexin Links Research Articles

DRC1 deficiency caused primary ciliary dyskinesia and MMAF in a Chinese patient.

Primary ciliary dyskinesia (PCD) is a heterogeneous disease characterized by the failure of mucociliary clearance. Dynein regulatory complex subunit 1 (DRC1) variants can cause PCD by disrupting the nexin link connecting the outer doublets. In this study, we aimed to investigate the clinical and functional impacts of DRC1 variants on respiratory cilia and sperm. We identified and validated the DRC1 variant by using whole-exome and Sanger sequencing. High-speed video microscopy analysis (HSVA) was used to measure the nasal ciliary beating frequency and pattern in a patient and a healthy control. Hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) were applied to analyze the morphological and ultrastructural sperm defects resulting from the DRC1 variant. NM_145038.5:c.1296 G>A, p.(Trp432*), a novel homozygous DRC1 nonsense variant, was identified in a patient from a consanguineous Chinese family. The patient exhibited bronchiectasis, chronic sinusitis, situs solitus, and male infertility. The markedly reduced nasal nitric oxide production rate (3.0 nL/min) was consistent with PCD diagnosis. HSVA showed reduced bending capacity and higher beating frequency of nasal cilia in the patient compared with those in healthy control. The diagnosis of multiple morphological abnormalities of the sperm flagella (MMAF) was confirmed through sperm HE staining and TEM analysis. Following the intracytoplasmic sperm injection treatment, the patient fathered a healthy daughter. This report is the first to describe a novel DRC1 variant in a patient with PCD and MMAF, and in vitro fertilization was effective for treating infertility in this male patient. Our findings expand the genetic spectrum of PCD and MMAF, and provide a detailed clinical summary and functional analysis of patients with DRC1 variants.

Ccdc113/Ccdc96 complex, a novel regulator of ciliary beating that connects radial spoke 3 to dynein g and the nexin link.

Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein composition and role of radial spokes (RS), nexin links (N-DRC) and dyneins (ODAs and IDAs) is well established. However, how information is transmitted from the central apparatus to the RS and across other ciliary structures remains unclear. Here, we identify a complex comprising the evolutionarily conserved proteins Ccdc96 and Ccdc113, positioned parallel to N-DRC and forming a connection between RS3, dynein g, and N-DRC. Although Ccdc96 and Ccdc113 can be transported to cilia independently, their stable docking and function requires the presence of both proteins. Deletion of either CCDC113 or CCDC96 alters cilia beating frequency, amplitude and waveform. We propose that the Ccdc113/Ccdc96 complex transmits signals from RS3 and N-DRC to dynein g and thus regulates its activity and the ciliary beat pattern.

Effects of Inter-Doublet Coupling on Flagellar Beating

Cilia and flagella are slender organelles that exhibit wavelike oscillations to propel cells or move fluid. The cytoskeletal structure (the axoneme) of the cilium is well known, comprising arrays of the motor protein dynein between 9 pairs of microtubule doublets connected by nexin links and surrounding a central pair of microtubules. However, the mechanism by which dynein creates the oscillatory, propulsive motion remains uncertain. Are dynein forces dynamically regulated, or do oscillatory motions occur spontaneously due to dynamic instabilities under steady dynein activity? How do changes in physical parameters, such as stiffness and nonlinearity of radial spokes (RS) and nexin-dynein regulatory complex (N-DRC), affect the emergence and character of these oscillations? In the current work, we have created a custom finite-element code that allows us to simulate active, unstable, and nonlinear behavior on the nanoscale, unconstrained by the limits of commercial FEM packages. The complex eigenvalues of the system allow us to determine critical values that cause the system to oscillate. Time-domain simulations allow us to compare model behavior and waveforms with those of cilia and allow us to observe the effects of system nonlinearities. We first modeled a single filament under distributed axial follower load to find critical values for dynamic instability and study the effects of mechanical parameters in individual doublets. We then moved to a two-filament model to study the effect of filament-coupling parameters on oscillatory behavior of multiple filaments coupled by passive elements (RS and N-DRC) and active elements (dyneins). Finally, we created a four-doublet, three-dimensional model to show the complex behavior of multiple filaments interacting through internal forces and moments as well as passive elastic and viscous coupling. Using biologically-plausible parameter values, all systems exhibited dynamic instability, leading to oscillations like those observed in real cilia and flagella.

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.

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.

Loss-of-Function GAS8 Mutations Cause Primary Ciliary Dyskinesia and Disrupt the Nexin-Dynein Regulatory Complex

Multiciliated epithelial cells protect the upper and lower airways from chronic bacterial infections by moving mucus and debris outward. Congenital disorders of ciliary beating, referred to as primary ciliary dyskinesia (PCD), are characterized by deficient mucociliary clearance and severe, recurrent respiratory infections. Numerous genetic defects, most of which can be detected by transmission electron microscopy (TEM), are so far known to cause different abnormalities of the ciliary axoneme. However, some defects are not regularly discernable by TEM because the ciliary architecture of the axoneme remains preserved. This applies in particular to isolated defects of the nexin links, also known as the nexin-dynein regulatory complex (N-DRC), connecting the peripheral outer microtubular doublets. Immunofluorescence analyses of respiratory cells from PCD-affected individuals detected a N-DRC defect. Genome-wide exome sequence analyses identified recessive loss-of-function mutations in GAS8 encoding DRC4 in three independent PCD-affected families.

Number of nexin links detectable at standard electron microscopy of normal human nasal cilia and at nexin link deficiency

Background: Eleven years ago we had described three patients with missing nexin links as a possible cause of primary ciliary dyskinesia (PCD). The assumption was substantiated last year by finding a mutation in these patients. Materials and Methods: We counted the nexin links, inner (IDA) and outer (ODA) dynein arms and microtubuli in each of, if possible, 50 cilia in 41 patients with normal cilia, 4 patients with deficiency of nexin links only and 4 with deficiency of nexin links and IDA. Results: In the control group the median number of nexin links was 4.5 per cilium, range 3.4–5.3. In the second group the mean numbers of nexin links per cilium were 1.1–1.4, in the third group 0.8–1.2, per patient. The median number of IDA was in the control group 4.2, range 3.3–5.2. In groups 2 and 3 the numbers were 3.0–3.5 and 0.2–1.0, respectively. Numbers of ODA were normal in all groups. Conclusions: It is possible to reliable count the number of nexin links in nasal human cilia and to distinguish cases with missing nexin links from normal controls.

S88 Electron Tomography Detects Ultrastructural Abnormalities In Patients With Pcd Due To A Dnah11 Defect

Primary Ciliary Dyskinesia (PCD) is a genetically heterogeneous condition. characterised by impaired mucociliary clearance and chronic sino-pulmonary disease. Over 30 causative gene mutations resulting in dysfunctional ciliary motility and structure have been identified. Ciliary ultrastructure examined by Transmission Electron Microscopy (TEM) is currently the diagnostic gold standard. Patients with mutations in the gene DNAH11 have a hyperfrequent ciliary beat and clinical symptoms of PCD. Diagnosis of a DNAH11 defect by TEM is difficult due to apparently normal ciliary ultrastructure. The electron tomography technique, an extension of TEM, produces 3D models of cilia with superior resolution. The aim of this study was to determine if electron tomography can detect ultrastructural abnormalities in patients with DNAH11 defects. Immunofluorescence by specific antibodies for DNAH11 showed localisation to the proximal portion of cilia. Proximal cilia cross sections from araldite embedded nasal brush biopsies were examined. Dual axis tomograms were collected on a Philips CM200 electron microscope. The data were analysed using IMOD software and averaged using PEET. Electron tomography and averaging of cilia cross sections indicated a deficiency in the outer dynein arm, consistent across all patients with a DNAH11 defect (n = 5) compared to healthy controls (n = 3) and patients with PCD due to a defect of the central pair or nexin link (n = 3) (Figure 1). A reduction in outer dynein arm volume of 30% was identified compared to healthy and PCD controls. In conclusion, a mutation in DNAH11 results in a subtle abnormality in ultrastructure. The defects are specific to the ‘forearm’ of the outer dynein arm and only detectable at the base of the cilium where DNAH11 is located. Electron tomography is highly effective in visualising this defect.

Number of Nexin Links Detectable at Standard Electron Microscopy of Normal Human Nasal Cilia and at Nexin Link Deficiency

Background: Eleven years ago we had described three patients with missing nexin links as a possible cause of primary ciliary dyskinesia (PCD). The assumption was substantiated last year by finding a mutation in these patients.Materials and Methods: We counted the nexin links, inner (IDA) and outer (ODA) dynein arms and microtubuli in each of, if possible, 50 cilia in 41 patients with normal cilia, 4 patients with deficiency of nexin links only and 4 with deficiency of nexin links and IDA.Results: In the control group the median number of nexin links was 4.5 per cilium, range 3.4–5.3. In the second group the mean numbers of nexin links per cilium were 1.1–1.4, in the third group 0.8–1.2, per patient. The median number of IDA was in the control group 4.2, range 3.3–5.2. In groups 2 and 3 the numbers were 3.0–3.5 and 0.2–1.0, respectively. Numbers of ODA were normal in all groups.Conclusions: It is possible to reliable count the number of nexin links in nasal human cilia and to distinguish cases with missing nexin links from normal controls.

Tubulin polyglutamylation regulates flagellar motility by controlling a specific inner‐arm dynein that interacts with the dynein regulatory complex

The tpg1 mutant of Chlamydomonas lacks the tubulin polyglutamylase TTLL9 and is deficient in flagellar tubulin polyglutamylation. It exhibits slow swimming, whereas the double mutant with oda2 (a slow-swimming mutant that lacks outer-arm dynein) is completely nonmotile. Thus, tubulin polyglutamylation must be important for the functioning of inner-arm dynein(s). In this study, we show that the tpg1 mutation only slightly affects the motility of mutants that lack dynein "e," one of the seven species of major inner-arm dyneins, whereas it greatly reduces the motility of mutants lacking other inner-arm dynein species. This suggests that dynein e is the main target of motility regulation by tubulin polyglutamylation. Furthermore, the motility of various mutants in the background of the tpg1 mutation raises the possibility that tubulin polyglutamylation also affects the dynein regulatory complex, a dynein e-associated key regulator of flagellar motility, which possibly constitutes the interdoublet (nexin) link. Tubulin polyglutamylation thus may play a central role in the regulation of ciliary and flagellar motility. © 2012 Wiley Periodicals, Inc.

Flexural Rigidity and Shear Stiffness of Flagella

Cilia are thin subcellular organelles that line airways and other passages and bend actively to propel fluid and foreign materials. The ciliary cytoskeleton (the axoneme) consists of nine outer microtubule doublets surrounding a central pair of singlet microtubules. Large bending deformations of the axoneme involve relative sliding of the outer doublets driven by the motor protein dyneins. The genetics and cell biology of the ciliary structure and function have been studied extensively, but the mechanics of the axoneme remain unclear. In this study, we used the unicellular alga Chlamydomonas reinhardtii as the model system for their flagellum replicates the highly conserved molecular structure of the ciliary axoneme. Piconewton forces were applied perpendicularly on the tip of a single flagellum (length L) through a microsphere trapped in optical tweezers. Dividing the force (P) by the corresponding deflection of the flagellar tip (δ) yields the flexural stiffness of the flagellum (K = P/δ), which was then used to calculate the apparent flexural rigidity (EI = KL3/3). The contributions of major structural components to passive mechanical properties were quantified by testing on flagella of specific mutations. The average apparent flexural rigidity of wild-type, pf-3 (without nexin links), and pf-13 flagella (without outer dynein arms) was about 2700 ± 1100, 1300 ± 550, and 650 ± 140 pN·μm2, respectively. In addition, the ratio of elastic shear stiffness (resistance to interdoublet sliding) to true flexural rigidity was estimated by the counterbend response in bent flagella manipulated with a glass microneedle. The quantitative understanding of axonemal mechanics will help illuminate the roles of certain genes and molecular structures in the normal and abnormal axoneme.

Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella

The directional flow generated by motile cilia and flagella is critical for many processes, including human development and organ function. Normal beating requires the control and coordination of thousands of dynein motors, and the nexin-dynein regulatory complex (N-DRC) has been identified as an important regulatory node for orchestrating dynein activity. The nexin link appears to be critical for the transformation of dynein-driven, linear microtubule sliding to flagellar bending, yet the molecular composition and mechanism of the N-DRC remain largely unknown. Here, we used proteomics with special attention to protein phosphorylation to analyze the composition of the N-DRC and to determine which subunits may be important for signal transduction. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry of WT and mutant flagellar axonemes from Chlamydomonas identified 12 N-DRC-associated proteins, including all seven previously observed N-DRC components. Sequence and PCR analyses identified the mutation responsible for the phenotype of the sup-pf-4 strain, and biochemical comparison with a radial spoke mutant revealed two components that may link the N-DRC and the radial spokes. Phosphoproteomics revealed eight proteins with phosphorylated isoforms for which the isoform patterns changed with the genotype as well as two components that may play pivotal roles in N-DRC function through their phosphorylation status. These data were assembled into a model of the N-DRC that explains aspects of its regulatory function.

Bending of the “9+2” axoneme analyzed by the finite element method

Many data demonstrate that the regulation of the bending polarity of the “9+2” axoneme is supported by the curvature itself, making the internal constraints central in this process, adjusting either the physical characteristics of the machinery or the activity of the enzymes involved in different pathways. Among them, the very integrated Geometric Clutch model founds this regulation on the convenient adjustments of the probability of interaction between the dynein arms and the β-tubulin monomers of the outer doublet pairs on which they walk. Taking into consideration (i) the deviated bending of the outer doublets pairs (Cibert, C., Heck, J.-V., 2004. Cell Motil. Cytoskeleton 59, 153–168), (ii) the internal tensions of the radial spokes and the tangential links (nexin links, dynein arms), (iii) a theoretical 5 μm long proximal segment of the axoneme and (iv) the short proximal segment of the axoneme, we have reevaluated the adjustments of these intervals using a finite element approach. The movements we have calculated within the axonemal cylinder are consistent with the basic hypothesis that found the Geometric Clutch model, except that the axonemal side where the dynein arms are active increases the intervals between the two neighbor outer doublet pairs. This result allows us to propose a mechanism of bending reversion of the axoneme, involving the concerted ignition of the molecular engines along the two opposite sides of the axoneme delineated by the bending plane.

Sculpting the dynein regulatory complex

Cryoelectron tomography materializes the (missing) nexin link.

The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella

Cilia and flagella are highly conserved microtubule (MT)-based organelles with motile and sensory functions, and ciliary defects have been linked to several human diseases. The 9 + 2 structure of motile axonemes contains nine MT doublets interconnected by nexin links, which surround a central pair of singlet MTs. Motility is generated by the orchestrated activity of thousands of dynein motors, which drive interdoublet sliding. A key regulator of motor activity is the dynein regulatory complex (DRC), but detailed structural information is lacking. Using cryoelectron tomography of wild-type and mutant axonemes from Chlamydomonas reinhardtii, we visualized the DRC in situ at molecular resolution. We present the three-dimensional structure of the DRC, including a model for its subunit organization and intermolecular connections that establish the DRC as a major regulatory node. We further demonstrate that the DRC is the nexin link, which is thought to be critical for the generation of axonemal bending.

The primary cilia of secretory cells in the human oviduct mucosa

The human oviduct is lined with a simple columnar epithelium composed of ciliated cells and secretory cells. Primary cilia or solitary cilia usually extend from the apical surface of the secretory cells. The axoneme of the primary cilia is composed of nine peripheral microtubule doublets (9 + 0 pattern) that lack dynein arms and nexin links. Displacement of peripheral doublets to the central region, which is suggested to be attributable to the lack of nexin links, is one of the distinctive features of oviductal primary cilia. The basal body that extends the primary cilium connects to its paired centriole by the striated connector. The basal body is associated with the accessory structures, such as alar sheets, basal feet, and striated rootlets. Several basal feet project laterally from the basal body. The cap of the basal foot serves as the microtubule organizing center. Several striated rootlets radiate from the basal body toward the nucleus. The basal body, the paired centriole, and the basal body-associated structures are considered to play important roles in the stabilization and fixing of the cilium in the proper position on the apical cell surface.

Are the local adjustments of the relative spatial frequencies of the dynein arms and the β-tubulin monomers involved in the regulation of the “9+2” axoneme?

The “9+2” axoneme is a highly specific cylindrical machine whose periodic bending is due to the cumulative shear of its 9 outer doublets of microtubules. Because of the discrete architecture of the tubulin monomers and the active appendices that the outer doublets carry (dynein arms, nexin links and radial spokes), this movement corresponds to the relative shear of these topological verniers, whose characteristics depend on the geometry of the wave train. When an axonemal segment bends, this induces the compressed and dilated conformations of the tubulin monomers and, consequently, the modification of the spatial frequencies of the appendages that the outer doublets carry. From a dynamic point of view, the adjustments of the spatial frequencies of the elements of the two facing verniers that must interact create different longitudinal periodic patterns of distribution of the joint probability of the molecular interaction as a function of the location of the doublet pairs around the axonemal cylinder and their spatial orientation within the axonemal cylinder. During the shear, these patterns move along the outer doublet intervals at a speed that ranges from one to more than a thousand times that of sliding, in two opposite directions along the two opposite halves of the axoneme separated by the bending plane, respecting the polarity of the dynein arms within the axoneme. Consequently, these waves might be involved in the regulation of the alternating activity of the dynein arms along the flagellum, because they induce the necessary intermolecular dialog along the axoneme since they could be an element of the local dynamic stability/instability equilibrium of the axoneme. This complements the geometric clutch model [Lindemann, C., 1994. A “geometric clutch” hypothesis to explain oscillations of the axoneme of cilia and flagella. J. Theor. Biol. 168, 175–189].

The Geometric Clutch as a Working Hypothesis for Future Research on Cilia and Flagella

The Geometric Clutch hypothesis contends that the forces transverse to the flagellar axis (t-forces) act on the axonemal scaffold to regulate flagellar beating. T-forces develop as the product of the curvature and the accumulated tension or compression on the doublet microtubules. In this respect, t-force is a mediator of self-organizing behavior. It arises from the collective action of the assemblage of dynein motors on the structural components of the axoneme and, in turn, imparts order to the sequence of activation and deactivation of the dynein. At the switch point of the flagellar beat, the magnitude of the t-force per micron of flagellum is approximately equal to the sum total of dynein force that can be generated per micron of flagellum. This suggests that the t-force could directly overcome the force-producing dynein bridges and terminate their action. However, many questions remain to be answered concerning the behavior of the axonemal scaffold under stress. Little is known of the force-bearing capacity of the radial spokes and the central pair (cp) projections. The properties of these structures will determine how t-force is distributed within the axoneme. The mechanical and elastic properties of the dynein arms and nexin links need to be better understood to determine how they respond to the application of t-force. In the framework of the Geometric Clutch hypothesis these are the issues that are most important to explore if we are to understand how the flagellum works.

Fluid Dynamic Models of Flagellar and Ciliary Beating

We have developed a fluid-mechanical model of a eucaryotic axoneme that couples the internal force generation of dynein molecular motors, the passive elastic mechanics of microtubules, and forces due to nexin links with a surrounding incompressible fluid. This model has been used to examine both ciliary beating and flagellar motility. In this article, we show preliminary simulation results for sperm motility in both viscous and viscoelastic fluids, as well as multiciliary interaction with a mucus layer.

Metachronal wave formation in a model of pulmonary cilia

A three-dimensional simulation of the formation of metachronal waves in rows of pulmonary cilia is presented. The cilia move in a two-layer fluid model. The fluid layer adjacent to the cilia bases is purely viscous while the tips of the cilia move through a viscoelastic fluid. An overlapping fixed-moving grid formulation is employed to capture the effect of the cilia on the surrounding fluid. In contrast with immersed boundary methods, this technique allows a natural enforcement of boundary conditions without the need for smoothing of singular force distributions. The fluid domains are discretized using a finite volume method. The 9 + 2 internal microtubule structure of an individual cilium is modeled using large-deflection, curved, finite-element beams. The microtubule skeleton is cross-linked to itself and to the cilium membrane through spring elements which model nexin links. The cilium membrane itself is considered to be elastic and subject to fluid stresses computed from the moving grid formulation as well as internal forces transmitted from the microtubule skeleton. A cilium is set into motion by the action of dynein molecules exerting forces between adjacent microtubules. Realistic models of the forces exerted by dynein molecules are extracted from measurements of observed cilia shapes.