Inner-arm Dynein Research Articles

Mutations in ZMYND10, a Gene Essential for Proper Axonemal Assembly of Inner and Outer Dynein Arms in Humans and Flies, Cause Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a ciliopathy characterized by airway disease, infertility, and laterality defects, often caused by dual loss of the inner dynein arms (IDAs) and outer dynein arms (ODAs), which power cilia and flagella beating. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, we found that 6/38 (16%) carried biallelic mutations in the conserved zinc-finger gene BLU (ZMYND10). ZMYND10 mutations conferred dynein-arm loss seen at the ultrastructural and immunofluorescence level and complete cilia immotility, except in hypomorphic p.Val16Gly (c.47T>G) homozygote individuals, whose cilia retained a stiff and slowed beat. In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, Zmynd10 is exclusively expressed in cells with motile cilia: chordotonal sensory neurons and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Drosophila Zmynd10 with an equivalent c.47T>G (p.Val16Gly) missense change rescued mutant male sterility less than the wild-type did. Tagged Drosophila ZMYND10 is localized primarily to the cytoplasm, and human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein altered in PCD. Using a fly model of PCD, we conclude that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly and that its variants cause ciliary dysmotility and PCD with laterality defects.

The Zebrafish Orthologue of the Dyslexia Candidate Gene DYX1C1 Is Essential for Cilia Growth and Function

DYX1C1, a susceptibility gene for dyslexia, encodes a tetratricopeptide repeat domain containing protein that has been implicated in neuronal migration in rodent models. The developmental role of this gene remains unexplored. To understand the biological function(s) of zebrafish dyx1c1 during embryonic development, we cloned the zebrafish dyx1c1 and used morpholino-based knockdown strategy. Quantitative real-time PCR analysis revealed the presence of dyx1c1 transcripts in embryos, early larval stages and in a wide range of adult tissues. Using mRNA in situ hybridization, we show here that dyx1c1 is expressed in many ciliated tissues in zebrafish. Inhibition of dyx1c1 produced pleiotropic phenotypes characteristically associated with cilia defects such as body curvature, hydrocephalus, situs inversus and kidney cysts. We also demonstrate that in dyx1c1 morphants, cilia length is reduced in several organs including Kupffer’s vesicle, pronephros, spinal canal and olfactory placode. Furthermore, electron microscopic analysis of cilia in dyx1c1 morphants revealed loss of both outer (ODA) and inner dynein arms (IDA) that have been shown to be required for cilia motility. Considering all these results, we propose an essential role for dyx1c1 in cilia growth and function.

Identification of the Outer-Inner Dynein Linker as a Hub Controller for Axonemal Dynein Activities

In flagella, the outer dynein arm (ODA) and inner dynein arm (IDA) play distinct roles in generating beating motion. However, functional communications between the two dyneins have not been investigated. Here, we demonstrated by cryo-electron microscopy and chemical crosslinking that intermediate chain 2 (IC2) of ODAs interacts with the dynein regulatory complex in the axoneme and constitutes part of the outer-inner dynein (OID) linker. Furthermore, we identified IC2 as a functional hub between ODAs and IDAs based on the phenotypes of Chlamydomonas mutants expressing biotinylation-tagged IC2. The flagella of the IC2 mutant showed activated microtubule sliding and enhanced ATPase activities of ODAs, as well as an altered waveform, indicating attenuated IDA activity. We concluded that the OID linker controls both ODAs and IDAs and regulates flagellar beating.

Mutations inCCDC39andCCDC40are the Major Cause of Primary Ciliary Dyskinesia with Axonemal Disorganization and Absent Inner Dynein Arms

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed "radial spoke defect." We sequenced CCDC39 and CCDC40 in 54 "radial spoke defect" families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice, and frameshift predicting early protein truncation, which suggests this defect is caused by "null" alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganization and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as "IDA and microtubular disorganisation defect," rather than "radial spoke defect."

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

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

Immunofluorescence microscopy (IFM) analysis of primary ciliary dyskinesia (PCD) patients with suspected inner dynein arm defects (IDA)

Primary ciliary dyskinesia (PCD), characterized by abnormal motility of cilia or flagella, is caused by defects of structural components such as inner dynein arms (IDAs). Recently high-speed videomicroscopy has substituted transmission electron microscopy (TEM) analysis as the “gold standard” for diagnosis. However, TEM is still the most widely used diagnostic tool in many countries. A recent study reported that isolated IDA defects is the most frequent (> 50%) ciliary defect in PCD, as detected by TEM (Theegarten, 2011). IFM analysis has shown in several studies that it can ascertain diagnosis such as in PCD variants caused by DNAH5, DNAI1, DNAI2, KTU, LRRC50, CCDC39 and CCDC40 mutations. IFM can provide a complete view of the ciliary axoneme and identify “partial” axonemal defects which may be misinterpreted by TEM (e.g. present in KTU mutant cilia). Here we performed IFM analysis of known PCD patients to identify the composition of dynein arm defects, including IDAs. Using antibodies specific to the IDA marker DNALI1, we determined that the frequency of isolated IDA defects is unexpected low (thus far, only 12 of ~800 PCD patients, ~1.5%). In contrast, we observe that IDA defects usually accompany DRC defects (absent/abnormal Gas11) or ODA defects (absent/abnormal DNAH5). Because the DNALI1 antibody is targeted against only 3 IDA isoforms out of 7, we are currently screening additional IDA-specific antibodies for IFM analysis to target the other IDA sub-types which escaped our analysis. However, our results suggest the percentage of isolated IDA defects might be lower than previously expected.

Delineation of CCDC39/CCDC40 mutation spectrum and associated phenotypes in primary ciliary dyskinesia

Background CCDC39 and CCDC40 genes have recently been implicated in primary ciliary dyskinesia (PCD) with inner dynein arm (IDA) defects and axonemal disorganisation; their contribution to the disease is, however, unknown. Aiming to delineate the CCDC39/CCDC40 mutation spectrum and associated phenotypes, this study screened a large cohort of patients with IDA defects, in whom clinical and ciliary phenotypes were accurately described. Methods All CCDC39 and CCDC40 exons and intronic boundaries were sequenced in 43 patients from 40 unrelated families. The study recorded and compared clinical features (sex, origin, consanguinity, laterality defects, ages at first symptoms and at phenotype evaluation, neonatal respiratory distress, airway infections, nasal polyposis, otitis media, bronchiectasis, infertility), ciliary beat frequency, and quantitative ultrastructural analyses of cilia and sperm flagella. Results Biallelic CCDC39 or CCDC40 mutations were identified in 30/34 (88.2%) unrelated families with IDA defects associated with axonemal disorganisation (22 and eight families, respectively). Fourteen of the 28 identified mutations are novel. No mutation was found in the six families with isolated IDA defects. Patients with identified mutations shared a similar phenotype, in terms of both clinical features and ciliary structure and function. The sperm flagellar ultrastructure, analysed in 4/7 infertile males, showed evidence of abnormalities similar to the ciliary ones. Conclusions CCDC39 and CCDC40 mutations represent the major cause of PCD with IDA defects and axonemal disorganisation. Patients carrying CCDC39 or CCDC40 mutations are phenotypically indistinguishable. CCDC39 and CCDC40 analyses in selected patients ensure mutations are found with high probability, even if clinical or ciliary phenotypes cannot prioritise one analysis over the other.

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.

Delineation of CCDC39/CCDC40 mutation spectrum and associated phenotypes in primary ciliary dyskinesia

BackgroundCCDC39 and CCDC40 genes have recently been implicated in primary ciliary dyskinesia (PCD) with inner dynein arm (IDA) defects and axonemal disorganisation; their contribution to the disease is, however, unknown....

3PS003 Tubulin polyglutamylation regulates axonemal motility by modulating the function of a specific inner-arm dynein species(The 50th Annual Meeting of the Biophysical Society of Japan)

3PS003 Tubulin polyglutamylation regulates axonemal motility by modulating the function of a specific inner-arm dynein species(The 50th Annual Meeting of the Biophysical Society of Japan)

3PS009 In vitro analyses on two adjacent inner-arm dyneins, subspecies e and c(The 50th Annual Meeting of the Biophysical Society of Japan)

3PS009 In vitro analyses on two adjacent inner-arm dyneins, subspecies e and c(The 50th Annual Meeting of the Biophysical Society of Japan)

Highly Purified Inner-Arm Dynein E from Chlamydomonas Reinhardtii Studied with in vitro Motility Assay

Green alga Chlamydomonas reinhardtii has two flagella at the front of the cell and beats them to swim. A flagellium is a biomolecular machine that consists of various proteins in a highly regulated manner. It is considered that an alignment of the various species of dynein proteins, which generate power using energy released upon ATP hydrolysis, plays important roles in the flagellum beating. Recently, great progress has been made in understanding of construction of a Chlamydomonas flagellum by the use of cryo-electron microscopic tomography. Dyneins are arranged in 96-nm periodic manner for a direction of a longitudinal axis of a flagellum. 8 species of dyneins are called as “inner-arm dyneins”. In 96-nm period, they make specific 4 pairs and are arranged in equally-spaced 4 places. Making use of in vitro motility assay, we can evaluate activities of dyneins as “microtubule sliding”. Dynein “c” slides microtubules relatively fast among the 8 inner-arms, and is well-studied. It has shown that dynein c pairs with dynein “e” in a flagellium. However, properties of dynein e are not known much. One reason for the difficulty is that anion exchange chromatography, a major purification method for inner arm dyneins, cannot separate dynein e and dynein d well. We obtained highly purified dynein e and c using two steps of anion exchange chromatography. We will present properties of dynein e and of a mixture of dynein e and c.

Diagnostic yield of nasal scrape biopsies in primary ciliary dyskinesia: A multicenter experience

Examination of ciliary ultrastructure remains the cornerstone diagnostic test for primary ciliary dyskinesia (PCD), a disease of abnormal ciliary structure and/or function. Obtaining a biopsy with sufficient interpretable cilia and producing quality transmission electron micrographs (TEM) is challenging. Methods for processing tissues for optimal preservation of axonemal structures are not standardized. This study describes our experience using a standard operating procedure (SOP) for collecting nasal scrape biopsies and processing TEMs in a centralized laboratory. We enrolled patients with suspected PCD at research sites of the Genetic Disorders of Mucociliary Clearance Consortium. Biopsies were performed according to a SOP whereby curettes were used to scrape the inferior surface of the inferior turbinate, with samples placed in fixative. Specimens were shipped to a central laboratory where TEMs were prepared and blindly reviewed. Four hundred forty-eight specimens were obtained from 107 young children (0-5 years), 189 older children (5-18 years), and 152 adults (> 18 years), and 88% were adequate for formal interpretation. The proportion of adequate specimens was higher in adults than in children. Fifty percent of the adequate TEMs showed normal ciliary ultrastructure, 39% showed hallmark ultrastructural changes of PCD, and 11% had indeterminate findings. Among specimens without clearly normal ultrastructure, 72% had defects of the outer and/or inner dynein arms (IDA), while 7% had central apparatus defects with or without IDA defects. In summary, nasal scrape biopsies can be performed in the outpatient setting and yield interpretable samples, when performed by individuals with adequate training and experience according to an SOP.

Tubulin Polyglutamylation Regulates Axonemal Motility by Modulating Activities of Inner-Arm Dyneins

Tubulin polyglutamylation is a modification that adds multiple glutamates to the gamma-carboxyl group of a glutamate residue in the C-terminal tails of alpha- and beta-tubulin [1, 2]. This modification has been implicated in the regulation of axonal transport and ciliary motility. However, its molecular function in cilia remains unknown. Here, using a novel Chlamydomonas reinhardtii mutant (tpg1) that lacks a homolog of human TTLL9, a glutamic acid ligase enzyme [3], we found that the lack of a long polyglutamate side chain in alpha-tubulin moderately weakens flagellar motility without noticeably impairing the axonemal structure. Furthermore, the double mutant of tpg1 with oda2, a mutation that leads to loss of outer-arm dynein, completely lacks motility. More surprisingly, when treated with protease and ATP, the axoneme of this paralyzed double mutant displayed faster microtubule sliding than the motile oda2 axoneme. These and other results suggest that polyglutamylation directly regulates microtubule-dynein interaction mainly by modulating the function of inner-arm dyneins.

3P176 Highly purified inner-arm dynein e from Chlamydomonas reinhardtii studied with in vitro motility assay(Molecular motor,The 48th Annual Meeting of the Biophysical Society of Japan)

3P176 Highly purified inner-arm dynein e from Chlamydomonas reinhardtii studied with in vitro motility assay(Molecular motor,The 48th Annual Meeting of the Biophysical Society of Japan)

3P166 Tubulin Polyglutamylation Regulates Flagellar Motility by Affecting Functions of Inner-Arm Dyneins(Molecular motor,The 48th Annual Meeting of the Biophysical Society of Japan)

3P166 Tubulin Polyglutamylation Regulates Flagellar Motility by Affecting Functions of Inner-Arm Dyneins(Molecular motor,The 48th Annual Meeting of the Biophysical Society of Japan)

Ratchetlike Properties of In Vitro Microtubule Translocation by a Chlamydomonas Inner-Arm Dynein Species c in the Presence of Flow

To investigate the force generation properties of Chlamydomonas axonemal inner-arm dyneins in response to external force, we analyzed microtubule gliding on dynein-coated surfaces under shear flow. When inner-arm dynein c was used, microtubule translocation in the downstream direction accelerated with increasing flow speed in a manner that depended on the dynein density and ATP concentration. In contrast, the microtubule translocation velocity in the upstream direction was unaffected by the flow speed. The number of microtubules on the glass surface was almost constant with and without flow, suggesting that gliding acceleration was not simply caused by weakened dynein-microtubule binding. With other inner-arm dynein species, the microtubule gliding velocity was unaffected by the flow regardless of the flow direction or nucleotide concentration. The flow-generated force acting on a single dynein was estimated to be as small as ∼0.03 pN/dynein. These results indicate that dynein c possesses a ratchetlike property that allows acceleration only in one direction by a very small external force. This property should be important for slow- and fast-moving dyneins to function simultaneously within the axoneme.

Identification of dyneins that localize exclusively to the proximal portion of Chlamydomonas flagella

The movements of cilia and flagella are driven by multiple species of dynein heavy chains (DHCs), which constitute inner- and outer-dynein arms. In Chlamydomonas, 11 DHC proteins have been identified in the axoneme, but 14 genes encoding axonemal DHCs are present in the genome. Here, we assigned each previously unassigned DHC gene to a particular DHC protein and found that DHC3, DHC4 and DHC11 encode novel, relatively low abundance DHCs. Immunofluorescence microcopy revealed that DHC11 is localized exclusively to the proximal approximately 2 microm region of the approximately 12 microm long flagellum. Analyses of growing flagella suggested that DHC3 and DHC4 are also localized to the proximal region. By contrast, the DHC of a previously identified inner-arm dynein, dynein b, displayed an inverse distribution pattern. Thus, the proximal portion of the flagellar axoneme apparently differs in dynein composition from the remaining portion; this difference might be relevant to the special function performed by the flagellar base.

Central pair apparatus enhances outer‐arm dynein activities through regulation of inner‐arm dyneins

The beating of eukaryotic cilia and flagella is controlled by multiple species of inner-arm and outer-arm dyneins. To clarify the regulation on axonemal beating by nucleotide conditions and central-pair microtubules, microtubule sliding in disintegrating Chlamydomonas axonemes of various mutants and in vitro microtubule gliding by isolated axonemal dyneins were examined. In the in vitro motility assays with outer-arm dyneins (alphabeta and gamma), microtubule translocation velocity decreased at high concentrations of ATP, while this inhibition was canceled by the simultaneous presence of ADP or ribose-modified analogues, mantATP/ADP. In contrast, motility of inner-arm dyneins was rather insensitive to these nucleotides. The velocity of sliding disintegration in axonemes lacking the central pair was less than that in wild-type axonemes at high ATP concentrations, but was overcome by the presence of ADP or mantATP/ADP. While these nucleotides did not activate the sliding velocity in other mutant axonemes, they increased the extent of sliding, except for axonemes lacking outer-arm dynein. Experiments with axonemes lacking inner-arm dynein f using casein kinase 1 inhibitor suggest that the regulation of outer-arm dynein by the central pair is effected through the activation of inner-arm dynein f, and possibly by other interactions. These results indicate that the central pair activates outer-arm dyneins on specific outer-doublet, resulting in amplification of the axonemal bending force.

Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins.