Nexin Links Research Articles

Functional genomics inTrypanosoma bruceiidentifies evolutionarily conserved components of motile flagella

Cilia and flagella are highly conserved, complex organelles involved in a variety of important functions. Flagella are required for motility of several human pathogens and ciliary defects lead to a variety of fatal and debilitating human diseases. Many of the major structural components of cilia and flagella are known, but little is known about regulation of flagellar beat. Trypanosoma brucei, the causative agent of African sleeping sickness, provides an excellent model for studying flagellar motility. We have used comparative genomics to identify a core group of 50 genes unique to organisms with motile flagella. These genes, referred to as T. brucei components of motile flagella (TbCMF) include 30 novel genes, and human homologues of many of the TbCMF genes map to loci associated with human ciliary diseases. To characterize TbCMF protein function we used RNA interference to target 41 TbCMF genes. Sedimentation assays and direct observation demonstrated clear motility defects in a majority of these knockdown mutants. Epitope tagging, fluorescence localization and biochemical fractionation demonstrated flagellar localization for several TbCMF proteins. Finally, ultrastructural analysis identified a family of novel TbCMF proteins that function to maintain connections between outer doublet microtubules, suggesting that they are the first identified components of nexin links. Overall, our results provide insights into the workings of the eukaryotic flagellum, identify several novel human disease gene candidates, reveal unique aspects of the trypanosome flagellum and underscore the value of T. brucei as an experimental system for studying flagellar biology.

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.

Absence of Nexin Links as a Possible Causeof Primary Ciliary Dyskinesia

Transmission electron microscopy of nasal cilia was performed in three patients, two of them siblings, with repeated respiratory infections. Number of microtubuli and dynein arms were within normal limits and they had an ordered arrangement except for a disarray of the microtubuli in some areas of the biopsies from two of the patients. In the normal areas radial spokes and sheaths were easily found but nexin links could not be discerned in any of the patients. The orientation of the cilia was partly random. As all patients repeatedly and constantly had very low nasal NO (range 9-15 ppb; normal findings for persons <10 years old are > 50 ppb), the diagnoses were very likely primary ciliary dyskinesia (PCD). Absence of nexin links may be an ultrastructural variant of PCD. Deficiency of these structures might be the cause of the microtubular disarray observed in some areas of the biopsies.

Elastic extension and jump of the flagellar nexin links: a theoretical mechanical cycle.

The functions of the nexin links of a flagellar axoneme have not been clearly demonstrated. Taking into account both the elastic (Hookean) characteristics and the possible jump of the nexin links, we calculated the sliding to bending conversion of a theoretical model in a tip-ward direction step by step, according to the essential principles proposed by the geometric clutch hypothesis [Lindemann, 1994: J Theoret Biol 168:175-189]: the activity of the dynein arms depends on the transverse forces induced by the axonemal curvature. In our calculations, however, the transverse forces that are involved in the regulation of the activities of the dynein arms were due to the extension of the nexin links located upstream of a given abscissa. This allowed us to define a bent segment as the axonemal portion at whose proximal and distal ends the nexin links were relaxed, and as fully extended as possible, respectively. The model creates an apparent disorder in the orientation of the nexin links as already observed [Bozkurt and Wooley, 1993: Cell Motil Cytoskeleton 24:109-118; Wooley, 1997: J Cell Sci 110:85-94]. We propose that the nexin links are involved in a mechanical cycle, whose 3 stages are (1) rapid extension, (2) slow relaxation, and (3) stand-by. The rapid extension is compatible with the mechanical interactions between the nexin links and the inner dynein arms with which they form the dynein regulatory complex. This was correlated with the oscillating properties of the nexin links along the axoneme that allow them to be one of the regulatory elements of the local ATPase activity of the dynein arms.

Disorders of Ciliary Motility

Clearance of mucus and other debris from the airways is achieved by 3 main mechanisms: mucociliary activity, coughing, and alveolar clearance. Disorders of ciliary structure or function results in impaired clearance, and result in chronic sinopulmonary disease manifested as chronic sinusitis, otitis media, nasal polyposis, and ultimately bronchiectasis. In addition, situs inversus, dextrocardia, and infertility can be associated with dysfunctional ciliary activity. The term primary ciliary dyskinesia has been proposed for the spectrum of these diseases. The term Kartagener syndrome applies to this syndrome when accompanied by infertility and dextrocardia or situs inversus. The more common types of ciliary dysmotility syndromes are characterized by missing dynein arms, central microtubule pairs, inner sheath, radial spokes, or nexin links. In addition to structural defects within the cilia, disordered ciliary beating and disordered ciliary arrays on epithelial cell surfaces have been described in this syndrome. Treatment includes rigorous lung physiotherapy, prophylactic and organism-specific antibiotics, and immunization against common pulmonary pathogens. Late stages of the disease may require surgical intervention for bronchiectasis or lung transplant for end-stage lung disease.

An Integrative Model of Internal Axoneme Mechanics and External Fluid Dynamics in Ciliary Beating

We present a fluid-mechanical model of an individual cilium which incorporates discrete representations of the dynein arms, the passive elastic structure of the axoneme including the microtubules and nexin links. This model, based upon the immersed boundary method (Peskin, 1977), couples the internal force generation of the molecular motors through the passive elastic structure with the external fluid mechanics governed by the Navier–Stokes equations. Detailed geometric information is available, such as the spacing and shear between the microtubules, the local curvature of individual microtubules and the stretching of the nexin links. In addition, the explicit representation of the dynein motors allows us the flexibility to incorporate a variety of activation theories. In this article, we choose a simple activation theory so that the ciliary beat is not present, but is an emergent property of the interacting components of the coupled fluid–axoneme system. We present numerical results from computer simulations of sliding disintegration and ciliary beating with several different viscosities.

Direct measurement of inter-doublet elasticity in flagellar axonemes.

The outer doublet microtubules in ciliary and flagellar axonemes are presumed to be connected with each other by elastic links called the inter-doublet links or the nexin links, but it is not known whether there actually are such elastic links. In this study, to detect the elasticity of the putative inter-doublet links, shear force was applied to Chlamydomonas axonemes with a fine glass needle and the longitudinal elasticity was determined from the deflection of the needle. Wild-type axonemes underwent a high-frequency, nanometer-scale vibration in the presence of ATP. When longitudinal shear force was applied, the average position of the needle tip attached to the axoneme moved linearly with the force applied, yielding an estimate of spring constant of 2.0 (S.D.: 0.8) pN/nm for 1 microm of axoneme. This value did not change in the presence of vanadate, i.e., when dynein does not form strong cross bridges. In contrast, it was at least five times larger when ATP was absent, i.e., when dynein forms strong cross bridges. The measured elasticity did not significantly differ in various mutant axonemes lacking the central-pair microtubules, a subset of inner-arm dynein, outer-arm dynein, or the radial spokes, although it was somewhat smaller in the latter two mutants. It was also observed that the shear displacement in an axoneme in the presence of ATP often took place in a stepwise manner. This suggests that the inter-doublet links can reversibly detach from and reattach to the outer doublets in a cooperative manner. This study thus provides the first direct measure of the elasticity of inter-doublet links and also demonstrates its dynamic nature.

Rotation of the central pair microtubules in eukaryotic flagella.

SUMMARY It has been 20 years since the phenomenon of centralpair rotation in eukaryotic flagella was reported(Omoto, and Kung, 1979). At that time, a model wasproposed in which the central pair functions as adistributor to regulate dynein activity among the outerdoublet microtubules. The video evidence obtainedsince then and gathered together in this essay is con-sistent with this model. Geometric arguments indicatethat there must be circumferential and longitudinalregulation of shear forces to produce effective bendingmotion of an axoneme. Central pair microtubules areideally situated to perform this regulatory function.The regulation of outer doublet sliding by the centralpair, together with the rotation of the latter, where thisoccurs, may explain the wide diversity of two- andthree-dimensional flagellar and ciliary waveforms thatis found in organisms using the same basic 9 1 2structure. ACKNOWLEDGMENTS We thank Mike McLaughlin of Material Media Services, Washing-ton State University for converting 16-mm film to video format, andDenise A. Palmen (Technical Services, Washington State University)for editing the videos, assembling the QuickTime movies, and pro-ducing the jpeg images to use as figures. The research based onvideo images shown in this essay was first published by Kamiya(1982), Omoto and Witman (1981), Gibbons

Cyclical bending movements induced locally by successive iontophoretic application of ATP to an elastase-treated flagellar axoneme.

To elucidate the mechanism of oscillatory bending in cilia and flagella, we studied the effect of protease digestion on the response of axonemes to localized application of ATP. When the axonemes were treated with elastase and then reactivated locally by ATP iontophoresis, a pair of local bends were formed due to localized unidirectional sliding in the vicinity of the ATP pipette. Upon repeated application of ATP, the direction of bending with respect to the sperm head axis changed cyclically from side to side over several cycles. The bends were planar and similar to those observed in axonemes that had not been treated with elastase. In trypsin-treated axonemes, in contrast, repetitive local reactivation did not induce such cyclical bending; instead, it induced a bend that grew only in one direction upon repeated application of ATP. Moreover, the bends were not planar. Electron microscopy of these protease-digested axonemes showed that both the interdoublet (nexin) links and the radial spokes were disrupted, but the effects of these proteases were different; trypsin disrupted 60-70% of these structures whereas elastase disrupted 20-30% of them. In both cases, spokes no. 3 and no. 8 (and no. 7) were more resistant to digestion than the others, although they tended to be more resistant to elastase than to trypsin. The importance of radial spokes and interdoublet links in the generation of cyclical bending and the determination of the bending plane is discussed.

At least one of the protofilaments in flagellar microtubules is not composed of tubulin.

The core of the eukaryotic flagellum is the axoneme, a complex motile organelle composed of approximately 200 different polypeptides. The most prominent components of the axoneme are the central pair and nine outer doublet microtubules. Each doublet microtubule contains an A and a B tubule; these are composed, respectively, of 13 and 10-11 protofilaments, all of which are thought to be made of tubulin. The mechanisms that control the assembly of the doublet microtubules and establish the periodic spacings of associated proteins, such as dynein arms and radial spokes, are unknown. Tektins, a set of microtubule-associated proteins, are present in the axoneme as stable filaments that remain after the extraction of doublet microtubules; they are localized near to where the B tubule attaches to the A tubule and near to the binding sites for radial spokes, inner dynein arms and nexin links. Tektin filaments may contribute in an interesting way to the structural properties of axonemes. We have fractionated doublet microtubules from sea urchin sperm flagella into ribbons of stable protofilaments, which can be shown to originate from the A tubule. Using cryo-electron microscopy, conventional electron microscopy, scanning transmission electron microscopy, three-dimensional reconstruction and kinesin decoration, we have found that one protofilament in the ribbon is not composed of tubulin. This protofilament is an integral protofilament of the A tubule wall, has less mass per unit length than tubulin and does not bind kinesin. Contrary to what is generally assumed, at least one protofilament in the wall of the A tubule is not composed of tubulin. Our data suggest that this nontubulin protofilament is primarily composed of tektins, proteins that show some structural similarity to intermediate filament proteins. A 480 A axial periodicity within these ribbons, revealed by scanning transmission electron microscopy, can be related to the structure of tektin, and may determine the large-scale structure of the axoneme in terms of the binding of dynein, nexin and radial spokes to the doublet microtubule.

Morphology of nexin links in relation to interdoublet sliding in the sperm flagellum

In this work, we examine whether the "nexin" linkages of the flagellum can extend in length to accommodate interdoublet sliding. Flagellar bends of large angle were induced in bull spermatozoa by hypotonic treatment. It is argued that this produces large interdoublet displacements that are, nevertheless, still within physiological limits. Such flagella were examined by the rapid-freeze, deep-etch technique and the nexin linkages identified by their position in relation to the inner dynein arms and by their straplike, bipartite, morphology. They were found to bridge perpendicularly (or occasionally at an angle) between the A- and B-tubules of adjacent doublets. The nexin linkages were no more than approximately nm in length, even in regions in which approximately 200 nm of sliding could be inferred. Variable registration between adjacent nexin rows gave some further support to the assumption that sliding had indeed taken place. From this, it is concluded that elastic deformation of the links, such as would accommodate interdoublet sliding, does not occur; some form of displacement must occur between nexin and the adjacent B-tubule.

Ultrastructural Findings in an Autopsy Eye From a Patient With Usher's Syndrome Type II

We studied an eye taken at autopsy from a 55-year-old woman with retinitis pigmentosa and partial deafness (Usher's syndrome type II). Two years before her death, her visual acuity was R.E.: 20/80 and L.E.: 20/50, and visual fields were constricted to a 30- to 40-degree diameter with a V4e white test light. Both rods and cones were present across the retina except for areas of the midperiphery; remaining photoreceptors had shortened or disrupted outer segments, or both, and some cones had inclusion bodies in inner segments. In the macula, most of the connecting cilia of photoreceptors appeared abnormal, with either extra microtubules or an apparent disruption of the nexin link between doublet microtubules. The pigment epithelium was abnormal across the eye, with diminished apical microvilli and basal infoldings of the plasma membrane. Possible mechanisms by which abnormal cilia could lead to photoreceptor cell death are considered.

The inner dynein arm complex: Compatible images from freeze-etch and thin section methods of microscopy

A study has been made of the inner dynein arm complex in the sperm flagellum of Gallus domesticus. It has been found that images of the complex made from highly contrasted, approx. 20 nm, sections are very largely compatible with images made from replicas of rapidly cryofixed material. This suggests that neither technique seriously distorts the native state. From both types of image, we conclude that the most proximal group of inner dynein arm heads (IDA 1) is related to spoke S1 and consists of 3 heads with fine connections to the B-tubule. IDA 2 consists of 2 such heads and is related to spoke S2. IDA 3 is apparently single headed and lies close to the A-attachment of spoke S3. This arrangement of IDAs repeats every 96 nm. Between the IDAs and outer dynein arms (ODAs), lying close to IDAs 2 and 3, is a strap-like linkage to the next B-tubule; it is argued that this represents the circumferential link, nexin. In sections, but not consistently in replicas, IDA2 lies closer to the plane of the nexin link than does IDA 1. In the presence of ATP and vanadate, little change is seen in the IDA complex except for an ill-defined alteration to IDA 1. It is speculated that the apparently smaller size of the inner arm heads (compared with the ODA major subunit) may have functional implications in relation to maximal sliding velocity.

Excitable dynein model with multiple active sites for large-amplitude oscillations and bend propagation in flagella

The formal excitable dynein model proposed by Murase et al. (1989, J. theor. Biol. 139, 413-430) is modified to produce large-amplitude oscillations and excitability. The present model assumes that (i) each dynein arm has multiple active sites, which are distributed along most of the 24-nm distance between adjacent B-subtubule attachment sites; and (ii) any given dynein molecule tends to produce force continuously during interdoublet sliding in one direction and to produce little force during sliding in the opposite direction. Since no sliding motion occurs without superthreshold perturbations in the form of the sliding displacement, this new model also possesses an excitable nature. Once passive elastic components (e.g. nexin links and radial spokes) are incorporated into this model, oscillations with large amplitudes result. To test the ability of the model for bend propagation without a curvature-control mechanism, forced oscillations are applied to the basal end of the flagellum by the sliding displacement. It is found that bend propagation can occur even in the absence of a curvature-control mechanism.

B‐band protein in sea urchin sperm flagella

AbstractA high‐molecular‐weight polypeptide, named B‐band, was partially purified from sea urchin sperm flagella using selective extraction, hydroxylapatite chromatography, and sucrose density gradient centrifugation. The molecular weight of the B‐band was 440,000 by continuous system of sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Sedimentation coefficient of the B‐band protein was 10.5 S, and its Stokes radius was 10 nm. When examined by low‐angle rotary shadowing electron microscopy, this molecule appeared to be composed of four globular heads and two curved linkers (“double headphone shape”), which was quite different from the shape of 21 S dynein, the outer arm dynein. Flagellar axonemes were also subjected to several chemical dissections. The B‐band was not extracted with treatments that remove both arm structures but was solubilized with treatments that extract other components such as radial spokes and nexin links. The B‐band protein in the axoneme was also more susceptible to trypsin digestion than the arm structures. These results suggest that the B‐band protein is a “double headphone‐shaped” component of the axonemal structures and makes up the elastic structure that might regulate the active sliding between adjacent doublet microtubules.

Ultrastructural diagnosis in the immotile cilia syndrome.

This article reports on the experience with 38 biopsies from nasal mucosa, submitted with the question of immotile cilia syndrome. Fixation in glutaraldehyde with MgSO4 is preferable. At least 50 cilia should be scrutinized in the electron microscope, and dynein arms, radial spokes, sheaths, nexin links and orientation should be tabulated. Six cases displayed virtual absence of inner and outer dynein arms. The orientation of these cilia was random. Two of the patients had situs inversus. In biopsies considered not to represent the immotile cilia syndrome, about four inner and seven outer dynein arms were found per cilium.

Electron microscopic observation of the primary cilium in the pancreatic islets.

A primary cilium often projected from the Golgi region of pancreatic A, B and D cells. The proximal portion of the cilium was found surrounded by a tubular invagination of the plasmalemma, and then the cilium extended into the intercellular canaliculus. The most proximal portion of the ciliary membrane exhibited periodical densities which might correspond to the ciliary necklace. The axoneme of the cilium was basically of the 9 + 0 pattern, i.e., nine peripheral doublets and no central singlet, though it was modified along the length of the cilium. Although a few appendages were projected from each doublet, it was difficult to identify dynein arms and nexin links. At the most proximal portion of the cilium, a "champagne-glass" structure connected each doublet with the ciliary membrane. The distal and proximal centrioles of the diplosome were connected to each other by a striated band. The proximal centriole, which served as a basal body, had accessory structures, such as alar sheets, basal feet and rootlets. Frequent projections of the primary cilium and its elaborate structure suggest that the cilium is not an aberrant structure but rather one which plays a certain role in the islet cell function.

CILIARY DISEASES

The immotile-cilia syndrome has been characterized as an inborn disease in which cilia (and sperm tails) are unable to move in a normal and effective manner (Afzelius, Science 193, 317, 1976). When studied with the electron microscope the ciliary axoneme was found to lack the dynein arms, i.e. the components that are responsible for converting the chemically bound energy into microtubule sliding and hence ciliary movements. Later other types of defects in the ciliary axoneme have been found, such as deficiencies in the so called spokes or in the nexin links. By now our clinical material includes 40 children (aged 0 - 18 years), 18 of which have situs inversus. (There are also 35 adult cases, 20 of which have situs inversus.) Of the forty children about half the cases have a markedly lowered number of dynein arms and in most cases the orientation also is poor. In others the number of dynein arms was only slightly decreased or the dynein arms were shorter than normally. Some had defective spokes or a disorganized axoneme which probably was caused by defective nexin links. In three cases no ciliated cells were seen. Two children had cilia with a normal ultrastructure and a third case had cilia that appeared normal except for the length that was twice the normal one. Evidently the immotile-cilia syndrome is a genetically heterogeneous disease although it is clinically homogeneous. These data can be compared with data from three cases suffering from cystic fibrosis and having cilia with a normal ultra-structure except for the ciliary orientation that in one case was poor.

Immotile cilia syndrome in two Old English Sheepdog litter‐mates

ABSTRACTTwo Old English Sheepdog litter‐mates with chronic respiratory disease since birth had ultrastructural abnormalities of their respiratory tract cilia. The ciliary defects included lack of inner dynein arms, abnormal radial spokes and nexin links, and microtubule doublet displacement. Similar ciliary defects in man result in immotile cilia with the subsequent development of sinusitis, bronchitis or bronchiectasis, male sterility, and, in some cases, situs inversus. Both dogs had sinusitis and bronchiectasis, and one of the dogs also had situs inversus.

Heterogeneity of ciliary morphology in the immotile-cilia syndrome in man

Detailed ultrastructural analysis of the respiratory cilia of three male patients with the immotile-cilia syndrome is presented. They all had similar clinical manifestations, namely, situs inversus totalis, repeated respiratory infections, and sinusitis. The respiratory cilia, however, of these patients showed a variety of structural abnormalities some of which are described for the first time. In the cilia of the first patient both the dynein arms were lacking, and, almost 40% of the cilia had one to nine supernumerary microtubules peripheral to the nine outer doublets. The cilia of the second patient showed an absence of inner dynein arms, unusually prominent outer dynein arms, a spoke defect in 46% of cilia examined, and no morphologically detectable nexin links. The cilia of the last patient lacked both dynein arms but, in contrast to the first two patients, showed virtually no microtubular abnormalities. These patients demonstrate that a variety of structural abnormalities in respiratory cilia can result in virtually identical clinical manifestations.