Peripheral Microtubule Doublets Research Articles

CCDC189 depletion leads to oligo-astheno-teratozoospermia and male infertility in mice†.

In male reproductive system, proteins containing the coiled-coil domain (CCDC) are predominantly expressed in specific regions including the testis, epididymis, seminal vesicle, and prostate. They play a vital role in centriole formation, sperm motility and flagellar development in male gametes. Despite being highly expressed in the testis, the exact physiological function of the coiled-coil domain-containing 189 (Ccdc189) gene remain largely unclear. Our research provides a comprehensive and detailed investigation into the localization of CCDC189 protein within the testis seminiferous tubules. CCDC189 specifically expressed in spermatocytes, round spermatids, and elongating spermatids in mouse testis. The deletion of Ccdc189 in mouse leads to male infertility, characterized by significantly reduced sperm counts and motility. Abnormally shaped spermatozoa with irregular tails, exhibiting shortened and twisted morphology, were observed in the seminiferous tubules. Electron microscopy revealed disordered and missing peripheral microtubule doublets (MTD) and outer dense fibers (ODF) in the sperm flagella, accompanied by a consistent absence of central pairs (CP). The knockout of Ccdc189 resulted in oligo-astheno-teratozoospermia, which is characterized by low sperm count and reduced sperm motility and abnormal morphology. Furthermore, we identified poly(A)-binding protein cytoplasmic 1 (PABPC1) and PABPC2 as interacting proteins with CCDC189. These proteins belong to the PABP family and are involved in regulating mRNA translational activity in spermatogenic cells by specifically binding to poly(A) tails at the 3' ends of mRNAs.

P-154 Structural changes of the human sperm tail during the early post-fertilization

Abstract Study question What happens to the sperm tails that are no longer needed after the goal of fertilization has been achieved? Summary answer The sperm tail lost its fibrous sheath and changed to a coiled morphology with a whisker root-like structure at the tip during the early post-fertilization. What is known already The sperm tail has been thought to play only a role in allowing the head and neck to reach the oocyte. The flagellum is found in the mammalian sperm tail and consists of a middle, main, and terminal part. It also contains an axon composed of two central microtubules surrounded by nine peripheral microtubule doublets (9d + 2s microtubule pattern) surrounded by an accessory structure called the fibrous sheath (FS), but few studies have been conducted on the post-fertilization fate of the mammalian sperm tail. Study design, size, duration To investigate sperm tail structure from the pronuclear stage to the second cleavage, we used abnormally fertilized oocytes derived from either conventional in vitro fertilization (cIVF, n = 41) or intracytoplasmic sperm injection (ICSI, n = 11). In this study, abnormally fertilized oocytes were collected between March 2022 and November 2023 and subjected to immunofluorescence analysis and field emission scanning electron microscopy (FE-SEM) analysis. Participants/materials, setting, methods Under ethical approval, abnormally fertilized oocytes were donated by infertile couples undergoing cIVF or ICSI cycles at the Yamashita Shonan Yume Clinic in Japan. Informed consent was obtained from all participants. Immunofluorescence assay was performed to analyze sperm tail morphology using antibodies against H3K27me3, α-tubulin, and AKAP4. FE-SEM analysis was performed to detect the 9d + 2s structure characteristic of the sperm tail axon. Main results and the role of chance Tri-pronuclear zygotes derived from conventional in vitro fertilization (cIVF-derived 3PN) had two male pronuclei, whereas those derived from intracytoplasmic sperm injection (ICSI-derived 3PN) had one. Each zygote had the same number of coiled microtubule structures as the male pronucleus, and the 9d + 2s structure characteristic of the axon was identified, suggesting a sperm tail. No difference in sperm tail shape was observed between ICSI-derived 3PN and cIVF-derived 3PN. AKAP4, which is specifically localized in the FS associated with the principal piece of the flagellum, was observed in sperm tails attached to the zona pellucida but not in those in the oocyte, suggesting that the FS of the flagellum is lost before the pronuclear stage, leaving the microtubules of the axonemes exposed. The sperm tail was attached to one of the spindle poles during the first cleavage of abnormally fertilized oocytes, but no differences in the structure of the spindle pole with or without the sperm tail were observed. The tip of the sperm tail was branched like a “whisker root”, suggesting that the sperm tail was degraded from the end, but the degree of tip branching did not differ from the pronuclear stage to the second cleavage. Limitations, reasons for caution Normal fertilized oocytes were not used in this study because they are used for clinical treatment. The cell biological significance of sperm tail branching is currently unknown and requires further experimentation. Wider implications of the findings These findings shed light on the processes that occur during human fertilization, suggest new roles for the sperm tail, and may have implications for assisted fertilization approaches in the future. Trial registration number not applicable

A splice donor variant of GAS8 induces structural disorganization of the axoneme in sperm flagella and leads to nonsyndromic male infertility.

Motile cilia and flagella are closely related organelles structured around a highly conserved axoneme whose formation and maintenance involve proteins from hundreds of genes. Defects in many of these genes have been described to induce primary ciliary dyskinesia (PCD) mainly characterized by chronic respiratory infections, situs inversus and/or infertility. In men, cilia/flagella-related infertility is usually caused by asthenozoospermia due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we investigated a cohort of 196 infertile men displaying a typical MMAF phenotype without any other PCD symptoms. Analysis of WES data identified a single case carrying a deleterious homozygous GAS8 variant altering a splice donor consensus site. This gene, also known as DRC4, encodes a subunit of the Nexin-Dynein Regulatory Complex (N-DRC), and has been already associated to male infertility and mild PCD. Confirming the deleterious effect of the candidate variant, GAS8 staining by immunofluorescence did not evidence any signal from the patient's spermatozoa whereas a strong signal was present along the whole flagella length in control cells. Concordant with its role in the N-DRC, transmission electron microscopy evidenced peripheral microtubule doublets misalignments. We confirm here the importance of GAS8 in the N-DRC and observed that its absence induces a typical MMAF phenotype not necessarily accompanied by other PCD symptoms.

LRRC23 deficiency causes male infertility with idiopathic asthenozoospermia by disrupting the assembly of radial spokes.

Asthenozoospermia (AZS) is the primary cause of infertility in males. The radial spoke (RS) is an axonemal structure, connecting the peripheral doublet microtubules with the central pair of microtubules. This T-shaped multiprotein complex functions as a mechanochemical sensor to promote sperm motility. LRRC23 is a novel subunit of the RS complex that is necessary for flagellar assembly and movement in mice. However, the importance of LRRC23 in modulating RS formation in humans remains unclear. Here, we identified a homozygous nonsense mutation in LRRC23 (c.376C>T:p. Arg126X) in an infertile AZS patient whose parents were consanguineous. We verified the adversity of this novel mutation because of its ability to disrupt LRRC23 synthesis and impair RSs integrity. Furthermore, we demonstrated an interaction between LRRC23 and RSPH3 in vitro, indicating that LCCR23 is associated with RS in humans. Meanwhile, the LRRC23-mutant patient had a good prognosis following intracytoplasmic sperm injection. This study provides strong preliminary evidence that LRRC23 defects are potential causative factors of AZS in humans, which expands our knowledge for improved genetic counseling and better reproductive recommendations for patients with AZS.

P-720 Biallelic variants in IQCN cause sperm flagella assembly defects and male infertility

Abstract Study question What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? Summary answer Deficiency in IQCN is responsible for sperm flagella assembly defects and abnormal computer-assisted sperm analysis (CASA) parameters, which result in male infertility. What is known already Manchette is a transient structure that is involved in the shaping of the spermatid nucleus and protein transport of flagella. Most recently, our group reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN led to total fertilization failure (TFF) and defective acrosome structure phenotypes. However, the related function of IQCN in sperm flagella assembly is still unknown. Study design, size, duration Fifty infertile males were recruited from the Reproductive and Genetic Hospital of China International Trust Investment Corporation (CITIC)-Xiangya from January 2014 to October 2022. Participants/materials, setting, methods Genomic DNA was extracted from the peripheral blood samples of the affected individuals for whole-exome sequencing. The ultrastructure of the flagella was detected by transmission electron microscopy (TEM). CASA was used to test the parameters of sperm motility. An Iqcn knockout (Iqcn-/-) mouse model was generated by CRISPER-Cas9 technology. Immunoprecipitation (IP) followed by liquid chromatography‒mass spectrometry was used to select the differentially expressed IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. Main results and the role of chance Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our infertile cohort. The sperm from the affected individuals showed irregular “9 + 2” structure of sperm flagella, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn-/- male mice. The CASA parameters in the sperm of Iqcn-/- male mice were significantly lower than those in Iqcn+/+ male mice. The partial of peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or the chaotic arrangement of DMTs were observed in the principal piece and end piece of sperm flagella. The hyperactivation ability of sperm from Iqcn-/- male mice was decreased than sperm from Iqcn+/+ male mice. The CDC42 and IFT protein families are hub proteins that interact with IQCN and regulate flagella assembly during spermiogenesis. Deficiency in IQCN causes the loss of binding with CDC42 and IFT74, which leads to the absence or abnormal localization of CDC42 and IFT74 in mouse and human spermatozoa. Limitations, reasons for caution More cases are needed to demonstrate the relationship between the IQCN variations and the phenotypes. Wider implications of the findings Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, thus providing a genetic marker for sperm motility deficiency and male infertility. Trial registration number N.A.

Biallelic variants in IQCN cause sperm flagellar assembly defects and male infertility.

What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? Deficiency in IQCN causes sperm flagellar assembly defects and male infertility. The manchette is a transient structure that is involved in the shaping of the human spermatid nucleus and protein transport within flagella. Our group recently reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN lead to total fertilization failure and defective acrosome structure phenotypes. However, the function of IQCN in sperm flagellar assembly is still unknown. Fifty men with infertility were recruited from a university-affiliated center from January 2014 to October 2022. Genomic DNA was extracted from the peripheral blood samples of all 50 individuals for whole-exome sequencing. The ultrastructure of the spermatozoa was assessed by transmission electron microscopy. Computer-assisted sperm analysis (CASA) was used to test the parameters of curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). An Iqcn knockout (Iqcn-/-) mouse model was generated by CRISPR-Cas9 technology to evaluate sperm motility and the ultrastructure of the flagellum. Hyperactivation and sperm fertilizing ability were assessed in a mouse model. Immunoprecipitation followed by liquid chromatography-mass spectrometry was used to detect IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our cohort of infertile men. The sperm from the affected individuals showed an irregular '9 + 2' structure of the flagellum, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn-/- male mice. VSL, VCL, and VAP in the sperm of Iqcn-/- male mice were significantly lower than those in Iqcn+/+ male mice. Partial peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or a chaotic arrangement of DMTs was observed in the principal piece and end piece of the sperm flagellum. Hyperactivation and IVF ability were impaired in Iqcn-/- male mice. In addition, we investigated the causes of motility defects and identified IQCN-binding proteins including CDC42 and the intraflagellar transport protein families that regulate flagellar assembly during spermiogenesis. More cases are needed to demonstrate the relation between IQCN variants and phenotypes. Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, providing a genetic marker for sperm motility deficiency and male infertility. This work was supported by the National Natural Science Foundation of China (81974230 and 82202053), the Changsha Municipal Natural Science Foundation (kq2202072), the Hunan Provincial Natural Science Foundation (2022JJ40658), and the Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-Xiangya (YNXM-202114 and YNXM-202201). No conflicts of interest were declared. N/A.

Rsph9 is critical for ciliary radial spoke assembly and central pair microtubule stability.

In the "9+2"-type motile cilia, radial spokes (RSs) protruded from the nine peripheral microtubule doublets surround and interact with the central pair (CP) apparatus to regulate ciliary beat. RSPH9 is the human homologue of the essential protozoan RS head protein Rsp9. Its mutations in human primary ciliary dyskinesia patients, however, cause CP loss in a small portion of airway cilia without affecting the ciliary localization of other head proteins. We characterized mouse Rsph9 and investigated its function in ependymal motile cilia. Rsph9 was specifically expressed in mouse tissues containing motile cilia and upregulated during multiciliation. Its ciliary localization complied with its putative role as an RS subunit. Depletion of Rsph9 by RNAi in mouse ependymal cilia resulted in a near complete CP loss and altered the ciliary beat pattern from planar to rotational. Multiple RS proteins, including those in the head, were also markedly downregulated in the Rsph9-depleted cilia. Rsph9 is essential for both the RS head assembly and the CP maintenance in mammalian ependymal cilia. Our results help to understand the assembly and functions of mammalian RS and pathology of RS-related ciliopathy.

Loss of Calmodulin- and Radial-Spoke-Associated Complex Protein CFAP251 Leads to Immotile Spermatozoa Lacking Mitochondria and Infertility in Men

Flagella and motile cilia share a 9+ 2 microtubule-doublet axoneme structure, and asthenozoospermia (reduced spermatozoa motility) is found in 76% of men with primary ciliary dyskinesia (PCD). Nevertheless, causal genetic variants in a conserved axonemal component have been found in cases of isolated asthenozoospermia: 30% of men with multiple morphological anomalies of sperm flagella (MMAF) carry bi-allelic mutations in DNAH1, encoding one of the seven inner-arm dynein heavy chains of the 9+ 2 axoneme. To further understand the basis for isolated asthenozoospermia, we used whole-exome and Sanger sequencing to study two brothers and two independent men with MMAF. In three men, we found bi-allelic loss-of-function mutations in WDR66, encoding cilia- and flagella-associated protein 251 (CFAP251): the two brothers were homozygous for the frameshift chr12: g.122359334delA (p.Asp42Metfs∗4), and the third individual was compound heterozygous for chr12: g.122359542G>T (p.Glu111∗) and chr12: g.122395032_122395033delCT (p.Leu530Valfs∗4). We show that CFAP251 is normally located along the flagellum but is absent in men carrying WDR66 mutations and reveal a spermatozoa-specific isoform probably generated during spermatozoon maturation. CFAP251 is a component of the calmodulin- and radial-spoke- associated complex, located adjacent to DNAH1, on the inner surface of the peripheral microtubule doublets of the axoneme. In Tetrahymena, the CFAP251 ortholog is necessary for efficient coordinated ciliary beating. Using immunofluorescent and transmission electron microscopy, we provide evidence that loss of CFAP251 affects the formation of the mitochondrial sheath. We propose that CFAP251 plays a structural role during biogenesis of the spermatozoon flagellum in vertebrates.

Budowa rzęsek – od ultrastruktury do funkcji

Rzęski są strukturami zachowanymi w toku ewolucji, występującymi u większości Eukaryota. Ze względu na strukturę i pełnione funkcje wyróżnia się dwa typy rzęsek: nieruchome rzęski pierwotne, tworzone w fazie spoczynkowej cyklu komórkowego oraz rzęski ruchome. Rzęski pierwotne są odpowiedzialne za odbieranie i przekazywanie sygnałów ze środowiska do wnętrza komórki, natomiast rzęski ruchome umożliwiają ruch pojedynczych komórek, a w organizmach wielokomórkowych, w tym u człowieka, przemieszczanie wydzielin lub drobin wzdłuż powierzchni komórek nabłonka wyścielającego m.in. drogi oddechowe, jajowód i komory mózgowia. Szkielet obu typów rzęsek, tzw. aksomena, zbudowany jest z dziewięciu obwodowych par mikrotubul. Rzęski ruchome mają dodatkowo dwie mikrotubule centralne, które wraz z przyłączonymi do nich kompleksami białkowymi tworzą kompleks pary centralnej, oraz makrokompleksy białek przyłączone do mikrotubul obwodowych. Makrokompleksy te są rozmieszczone periodycznie wzdłuż mikrotubul obwodowych, tworząc wzór powtarzający się co 96 nm. W każdym powtórzeniu znajdują się cztery zewnętrzne ramiona dyneinowe, siedem wewnętrznych ramion dyneinowych, trzy promienie łączące, po jednym kompleksie N-DRC i MIA, oraz inne, mniejsze kompleksy. Skoordynowane działanie tych makrokompleksów jest niezbędne do prawidłowego ruchu rzęsek.

Two-Way Traffic Determined by Different Tracks in Cilia

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

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

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

Slow Axonemal Dynein e Facilitates the Motility of Faster Dynein c

We highly purified the Chlamydomonas inner-arm dyneins e and c, considered to be single-headed subspecies. These two dyneins reside side-by-side along the peripheral doublet microtubules of the flagellum. Electron microscopic observations and single particle analysis showed that the head domains of these two dyneins were similar, whereas the tail domain of dynein e was short and bent in contrast to the straight tail of dynein c. The ATPase activities, both basal and microtubule-stimulated, of dynein e (kcat = 0.27 s–1 and kcat,MT = 1.09 s–1, respectively) were lower than those of dynein c (kcat = 1.75 s–1 and kcat,MT = 2.03 s–1, respectively). From in vitro motility assays, the apparent velocity of microtubule translocation by dynein e was found to be slow (Vap = 1.2 ± 0.1 μm/s) and appeared independent of the surface density of the motors, whereas dynein c was very fast (Vmax = 15.8 ± 1.5 μm/s) and highly sensitive to decreases in the surface density (Vmin = 2.2 ± 0.7 μm/s). Dynein e was expected to be a processive motor, since the relationship between the microtubule landing rate and the surface density of dynein e fitted well with first-power dependence. To obtain insight into the in vivo roles of dynein e, we measured the sliding velocity of microtubules driven by a mixture of dynein e and c at various ratios. The microtubule translocation by the fast dynein c became even faster in the presence of the slow dynein e, which could be explained by assuming that dynein e does not retard motility of faster dyneins. In flagella, dynein e likely acts as a facilitator by holding adjacent microtubules to aid dynein c’s power stroke.

The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena

Cilia and flagella are motile organelles that play various roles in eukaryotic cells. Ciliary movement is driven by axonemal dyneins (outer arm and inner arm dyneins) that bind to peripheral microtubule doublets. Elucidating the molecular mechanism of ciliary movement requires the genetic engineering of axonemal dyneins; however, no expression system for axonemal dyneins has been previously established. This study is the first to purify recombinant axonemal dynein with motile activity. In the ciliated protozoan Tetrahymena, recombinant outer arm dynein purified from ciliary extract was able to slide microtubules in a gliding assay. Furthermore, the recombinant dynein moved processively along microtubules in a single-molecule motility assay. This expression system will be useful for investigating the unique properties of diverse axonemal dyneins and will enable future molecular studies on ciliary movement.

Protein tagging reveals new insights into signaling in flagella

In this issue, Oda et al. (2014. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201312014) use mutant analysis, protein tagging, and cryoelectron tomography to determine the detailed location of components in flagellar radial spokes—a complex of proteins that connect the peripheral microtubule doublets to the central pair. Remarkably, this approach revealed an interaction between radial spokes and the central pair based on geometry rather than a specific signaling mechanism, highlighting the importance of studying a system in three dimensions.

Sperm morphology, ultrastructure, and motility in pikeperch Sander lucioperca (Percidae, Teleostei) associated with various activation media

Spermatozoa morphology, ultrastructure, and spermatozoa motility traits were studied in pikeperch (Sander lucioperca) after activation in various media (AM 1 – 45mM NaCl, 5mM KCl, 20mM Tris, pH 8.5; AM 2 – 100mM sucrose, 20mM Tris, pH 8.5; AM 3 – 100mM sucrose, 1mM CaCl<sub>2</sub>, 20mM Tris, pH 8.5) during a 48-hour storage period. The spermatozoon was acrosomeless and differentiated into a spherical nucleus (head), midpiece, and flagellum. The nucleus length and width measured 1.83 ± 0.03 and 1.63 ± 0.02 mm, respectively. The midpiece was located laterally to the nucleus and possessed proximal and distal centrioles and 2–4 mitochondria. Flagellar length was 33.2 ± 0.90 µm, and a pair of lateral fin-like structures projections was observed. The axoneme consisted of nine peripheral doublet microtubules and a single central pair. After a 24 h storage in all activation media at all sampling times post-activation (15, 45, 90, and 120 s), spermatozoa motility was significantly decreased. Spermatozoa were motile after the 48-hour storage at all sampling times post-activation only in AM 3. After the 48-hour storage, no motile spermatozoa were observed in AM 2 and AM 1 at 90 and 120 s post-activation, respectively. Differences in spermatozoa velocity varied with activation medium during storage. After the 48-hour storage in AM 1 and AM 2, decrease of spermatozoa velocity at 15 s post-activation was observed, while in AM 3, velocity was decreased only after the 48-hour storage. Pikeperch spermatozoa morphology and ultrastructure was found similar to that of most freshwater teleosts, with differences in the arrangement of midpiece, number of mitochondria, and position of centrioles. Viable pikeperch sperm was observed after the 48-hour storage. Motility of spermatozoa was improved by addition of Ca<sup>2+</sup> to the activation medium, where higher spermatozoa velocity was observed.

Slow Dynein E with No Retardation Effects: Implication of Indirect Cooperation with Fast Dynein C

We highly purified the Chlamydomonas inner-arm dynein “e” and “c,” which reside in a pairwise fashion along the peripheral doublet microtubules. Electron microscopic observations and single particle analysis showed that the head domains of these two dyneins were similar while the tail domain of dynein e was short and bent in contrast to that of dynein c. The ATPase activities, both basal and microtubule-stimulated, of dynein e (kcat = 0.27 s−1 and kcat,MT = 1.09 s−1, respectively) were lower than those of dynein c (kcat = 1.75 s−1 and kcat,MT = 2.03 s−1, respectively). From in vitro motility assays, microtubule translocation by dynein e was found to be slow (V = 1.2 ± 0.1 µm/s) and appeared independent of the surface density, whereas that by dynein c was very fast (Vmax = 15.8 ± 1.5 µm/s) and highly sensitive to the decrease in the surface density (Vmin = 2.2 ± 0.7 µm/s). Dynein e was expected to be a processive motor, judged from the microtubule landing assay. To obtain insight into the in vivo roles of dynein e, we measured the sliding velocity of microtubules driven by a mixture of dyneins e and c of various ratios. The microtubule translocation by the “fast” dynein c became even faster in the presence of the “slow” dynein e, suggesting that dynein e would not retard the microtubule translocation by a fast dynein. In flagella, dynein e may hold adjacent microtubules for the sake of dynein c's power stroke. Moreover, dynein e may be recruited into the flagellar motion when the local velocity of microtubule sliding is not high.

Fused (Stk36) is a ciliary protein required for central pair assembly and motile cilia orientation in the mammalian oviduct

Motile cilia on the inner lining of the oviductal epithelium play a central role in ovum transport toward the uterus and subsequent fertilization by sperm. While the basic ultrastructure of 9+2 motile cilia (nine peripheral microtubule doublets surrounding a central pair) has been characterized, many important steps of ciliogenesis remain poorly understood. Our previous studies on mammalian Fused (Fu) (Stk36), a putative serine-threonine kinase, reveal a critical function of Fu in central pair construction and cilia orientation of motile cilia that line the tracheal and ependymal epithelia. These findings identify a novel regulatory component for these processes. In this study, we show that Fu is expressed in the multi-ciliated oviductal epithelium in several vertebrates, suggesting a conserved function of Fu in the oviduct. In support of this, analysis of Fu-deficient mouse oviducts uncovers a similar role of Fu in central pair construction and cilia orientation. We also demonstrate that Fu localizes to motile cilia and physically associates with kinesin Kif27 located at the cilium base and known central pair components Spag16 and Pcdp1. Our results delineate a novel pathway for central pair apparatus assembly and add important insight to the biogenesis and function of oviductal motile cilia.

Structural biology of cytoplasmic and axonemal dyneins

Dyneins are microtubule-based, ATP-driven motor proteins with six tandemly linked AAA+ domains, a long N-terminal tail and a coiled-coil stalk. Cytoplasmic dyneins function as individual homodimers and are responsible for minus-end-oriented transport along microtubules. Axonemal dyneins of flagella/cilia are anchored in arrays to peripheral microtubule doublets by their N-terminal tails, and generate sliding motions of adjacent microtubule doublets toward the plus end. The coiled-coil stalk is responsible for communication between the AAA+ domains and the microtubule binding domain. A number of isoforms of axonemal dyneins are integrated to generate bending motion. In this article I will review recent structural studies and address the question as to how dyneins generate force and cause bending in flagella/cilia.

Axonemal radial spokes

The radial spoke (RS) is a complex of at least 23 proteins that works as a mechanochemical transducer between the central-pair apparatus and the peripheral microtubule doublets in eukaryotic flagella and motile cilia. The RS contributes to the regulation of the activity of dynein motors, and thus to flagellar motility. Despite numerous biochemical, physiological and structural studies, the mechanism of the function of the radial spoke remains unclear. Detailed knowledge of the 3D structure of the RS protein complex is needed in order to understand how RS regulates dynein activity. Here we review the most important findings on the structure of the RS, including results of our recent cryo-electron tomographic analysis of the RS protein complex.

Ciliary Abnormalities Due to Defects in the Retrograde Transport Protein DYNC2H1 in Short-Rib Polydactyly Syndrome

The short-rib polydactyly (SRP) syndromes are a heterogeneous group of perinatal lethal skeletal disorders with polydactyly and multisystem organ abnormalities. Homozygosity by descent mapping in a consanguineous SRP family identified a genomic region that contained DYNC2H1, a cytoplasmic dynein involved in retrograde transport in the cilium. Affected individuals in the family were homozygous for an exon 12 missense mutation that predicted the amino acid substitution R587C. Compound heterozygosity for one missense and one null mutation was identified in two additional nonconsanguineous SRP families. Cultured chondrocytes from affected individuals showed morphologically abnormal, shortened cilia. In addition, the chondrocytes showed abnormal cytoskeletal microtubule architecture, implicating an altered microtubule network as part of the disease process. These findings establish SRP as a cilia disorder and demonstrate that DYNC2H1 is essential for skeletogenesis and growth.