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At least 19 recordsLinked to original sources
PubMed1997

Isolation of several human axonemal dynein heavy chain genes: genomic structure of the catalytic site, phylogenetic analysis and chromosomal assignment.

Dynein heavy chains (DHCs) are the main components of multisubunit motor ATPase complexes called dyneins. Axonemal dyneins provide the driving force for ciliary and flagellar motility. Recent molecular studies demonstrated that multiple DHC isoforms are produced by separate genes. We describe the isolation of five human axonemal DHC genes. Analysis of the human genomic clones revealed the existence of intronic sequences that were used to demonstrate that human axonemal DHC genes are located on different chromosomes. The cloned human DHC sequences were integrated into an evolutionary approach based on phylogenetic analysis. Tissue expression studies showed that these human axonemal DHCs are expressed in testis and/or trachea, two tissues with axonemal structures that can be altered in primary ciliary dyskinesia, making DHC genes strong candidates in the genesis of these human diseases.

Amino Acid Sequence↗
PubMed1991

Axonemal dynein from Tetrahymena.

Axonemal dynein from Tetrahymena cilia can be separated on a sucrose gradient into two fractions, at least one of which appears to be polymorphic. We have been using immuno-electron microscopy in order to try and locate the different types of dynein molecules within the axonemal structure.

Animals↗
PubMed1997

Identification of dynein heavy chain genes expressed in human and mouse testis: chromosomal localization of an axonemal dynein gene.

Dynein heavy chains are involved in microtubule-dependent transport processes. While cytoplasmic dyneins are involved in chromosome or vesicle movement, axonemal dyneins are essential for motility of cilia and flagella. Here we report the isolation of dynein heavy chain (DHC)-like sequences in man and mouse. Using polymerase chain reaction and reverse-transcribed human and mouse testis RNA cDNA fragments encoding the conserved ATP binding region of dynein heavy chains were amplified. We identified 11 different mouse and eight human dynein-like sequences in testis which show high similarity to known dyneins of different species such as rat, sea urchin or green algae. Sequence similarities suggest that two of the mouse clones and one human clone encode putative cytoplasmic dynein heavy chains, whereas the other sequences show higher similarity to axonemal dyneins. Two of nine axonemal dynein isoforms identified in the mouse testis are more closely related to known outer arm dyneins, while seven clones seem to belong to the inner arm dynein group. Of the isolated human isoforms three clones were classified as outer arm and four clones as inner arm dynein heavy chains. Each of the DHC cDNAs corresponds to an individual gene as determined by Southern blot experiments. The alignment of the deduced protein sequences between human (HDHC) and mouse (MDHC) dynein fragments reveals higher similarity between single human and mouse sequences than between two sequences of the same species. Human and mouse cDNA fragments were used to isolate genomic clones. Two of these clones, gHDHC7 and gMDHC7, are homologous genes encoding axonemal inner arm dyneins. While the human clone is assigned to 3p21, the mouse gene maps to chromosome 14.

Amino Acid Sequence↗
PubMed1992

Distribution of cytoplasmic and axonemal dyneins in rat tissues.

Microtubule-associated protein 1C (MAP 1C) is now defined as brain cytoplasmic dynein. Recent studies have suggested that cytoplasmic dynein is a motor protein responsible for the intracellular microtubule-based motility in neuronal and non-neuronal cells. We have prepared an antibody against bovine brain MAP 1C and have examined the localizations of cytoplasmic dynein in rat tissues. Immunoblots of extracts from the tissues showed that the dynein was present in brain, testis, liver, kidney and lung. Immunohistochemical experiments have demonstrated that dynein is localized in Purkinje cells of cerebellum and axons of central and peripheral nervous systems. In non-neuronal tissues, the antibody staining was intense in many types of cells, such as hepatocytes, epithelia of renal convoluted tubules, secretory cells of adrenal medulla and spermatids. Glomeruli of kidney, bronchial epithelia and type II pneumocytes of lung, pancreatic islets and acini, adrenal cortex and Sertoli cells were moderately positive upon exposure to the cytoplasmic dynein antibody. On the other hand, the localization of axonemal dynein was examined using antibodies against flagellar dynein of sea urchin spermatozoa. Anti-axonemal dynein labeled cilia and flagella in rat tissues whereas anti-MAP 1C did not stain axonemes. We also tested for immunological cross-reactivity between cytoplasmic and axonemal dyneins to probe for molecular similarities. Anti-axonemal dynein reacted with MAP 1C weakly. These results have confirmed that cytoplasmic dyneins are distributed widely among rat organs, not only in neuronal but also in non-neuronal tissues. There is no similarity in the localization of cytoplasmic and axonemal dyneins but there is some similarity in molecular antigenicity.

Animals↗
PubMed1996

Identification of seven rat axonemal dynein heavy chain genes: expression during ciliated cell differentiation.

Axonemal dyneins are molecular motors that drive the beating of cilia and flagella. We report here the identification and partial cloning of seven unique axonemal dynein heavy chains from rat tracheal epithelial (RTE) cells. Combinations of axonemal-specific and degenerate primers to conserved regions around the catalytic site of dynein heavy chains were used to obtain cDNA fragments of rat dynein heavy chains. Southern analysis indicates that these are single copy genes, with one possible exception, and Northern analysis of RNA from RTE cells shows a transcript of approximately 15 kb for each gene. Expression of these genes was restricted to tissues containing axonemes (trachea, testis, and brain). A time course analysis during ciliated cell differentiation of RTE cells in culture demonstrated that the expression of axonemal dynein heavy chains correlated with the development of ciliated cells, while cytoplasmic dynein heavy chain expression remained constant. In addition, factors that regulate the development of ciliated cells in culture regulated the expression of axonemal dynein heavy chains in a parallel fashion. These are the first mammalian dynein heavy chain genes shown to be expressed specifically in axonemal tissues. Identification of the mechanisms that regulate the cell-specific expression of these axonemal dynein heavy chains will further our understanding of the process of ciliated cell differentiation.

Amino Acid Sequence↗
PubMed2002

Axonemal dynein expression in human fetal tracheal epithelium.

Ciliogenesis in human fetal airway epithelium occurs from 11 to 24 gestational weeks. Using genetic and antigenic markers specific for human axonemal dynein heavy chain 9, we characterized temporal aspects of axonemal dynein expression associated with large airway epithelial ciliogenesis during human fetal development. Late in the first trimester, an undifferentiated columnar epithelium is characteristic of the large airways, and immunocytochemical studies exhibited focal localization of axonemal dynein antigen on luminal epithelial cell borders at sites consistent with emergent ciliary beds. From 12 to 22 wk, immunocytochemical labeling of new ciliary beds was prominent, and localization within the cytoplasm of epithelial cells suggested avid synthesis of axonemal dynein in advance of ciliogenic events. Quantitative RT-PCR of tracheal RNA and in situ hybridization studies compared favorably with immunocytochemical findings with the earliest expression of axonemal dynein at 9-10 wk gestation. These studies have documented that axonemal dynein is expressed early in human fetal life during airway epithelial maturation and well before histological or ultrastructural evidence of ciliogenesis is apparent.

Axonemal Dyneins↗
PubMed1998

The 29 kDa light chain that regulates axonemal dynein activity binds to cytoplasmic dyneins.

In earlier studies, Hamasaki et al. (Proc. Natl. Acad. Sci. USA. 88:7918-7922, 1991) and Barkalow et al. (J. Cell Biol. 126:727-735, 1994) found that cAMP- and Ca2+-sensitive phosphorylation of a 29 kDa dynein light chain (p29) extracted from 22S axonemal dynein of Paramecium, regulates the velocity of in vitro microtubule translocation and ciliate swimming speed. In this study we report evidence of recombination of p29 to cytoplasmic dyneins from both rat liver and Paramecium, as well as to a 22S dynein precursor molecule, based on immunoprecipitation and force filtration data. Immunoprecipitation also provides additional evidence for the binding of p29 to 22S axonemal dynein. The results suggest that p29 might regulate cytoplasmic dynein, as well as axonemal dynein function in Paramecium, and that a homologue of p29 may exist in rat liver and other mammalian cells.

Animals↗
PubMed1985

Binding of axonemal dynein to microtubules comprising the cytoplasmic transport system in insect ovarioles.

Dynein isolated from ciliary axonemes of Tetrahymena is shown to bind in a characteristic fashion as arms to microtubules dissected from the nutritive tubes of insect ovarioles. The microtubules in nutritive tubes are associated with the transport of cytoplasmic components along their length, and the significance of their ability to bind axonemal dynein, to the possibility that microtubule/dynein interactions are involved in microtubule-associated movements, generally, is discussed.

Adenosine Triphosphatases↗
PubMed1992

Axonemal dyneins.

Recent advances in our understanding of the axonemal dyneins reveal them to be much more complex than previously believed. A combination of genetic, molecular genetic, ultrastructural and biochemical approaches is now aiding the elucidation of the organization and function of these important mechanochemical transducers.

Amino Acid Sequence↗
PubMed1994

22S axonemal dynein is preassembled and functional prior to being transported to and attached on the axonemes.

In an earlier study we reported the isolation of a cytoplasmic dynein from the cytosol of Paramecium multimicronucleatum. In this study we report the isolation and characterization of two cytosolic axonemal dyneins (22S and 12S) as well as a 19S cytoplasmic dynein from the cytosol of whole or deciliated cells using preformed bovine brain microtubules. These three dynein species were characterized according to mass, morphology, vanadate photocleavage patterns, CTPase/ATPase ratios, Km and Vmax values, temperature optima and reactivity with a mAb. For comparison, 22S and 12S axonemal dyneins (ADs) were also isolated and purified from the demembranated axonemes. The 22S and 12S soluble dyneins appear to be related to ciliary ADs in that the 22S soluble dynein is three-headed while the 12S is a one-headed dynein, as determined by negative staining. Ciliary ADs and their corresponding 22S and 12S soluble dyneins isolated from the cytosol also have similar Km and Vmax values as well as vanadate photocleavage patterns and temperature optima. A mAb raised against the soluble 22S dynein reacted with the 22S ciliary dyneins but not the 12S axonemal or the 19S cytoplasmic dynein. All isolated dyneins supported similar microtubule gliding rates but had different ionic requirements for the translocation buffer. These results suggest that: (i) the two soluble 22S and 12S dyneins are precursor molecules of the ciliary dyneins, (ii) the subunits of the outer arm dynein are already assembled in the cytosol as a three-headed bouquet, and (iii) the 22S and 12S soluble dyneins are functional prior to being transported and attached to the axonemes of the cilia.

Animals↗
PubMed2002

Loss of function of axonemal dynein Mdnah5 causes primary ciliary dyskinesia and hydrocephalus.

Primary ciliary dyskinesia (PCD), also known as Kartagener's syndrome, is a human syndrome that results from ciliary dysfunction. This syndrome is characterized by recurrent respiratory infections, situs inversus and infertility. In some cases, hydrocephalus is also observed. We have characterized an insertional mutation in a mouse axonemal dynein heavy chain gene (Mdnah5) that reproduces most of the classical features of PCD, including recurrent respiratory infections, situs inversus and ciliary immotility. These mice also suffer from hydrocephalus and die perinatally. Electron microscopic studies demonstrate the loss of axonemal outer arms. These results show that mutations in Mdnah5 are a primary cause of PCD and provide direct evidence that mutations in an axonemal dynein can cause hydrocephalus. Mutations in the human DNAH5 have recently been identified in PCD patients. Comparison of the mouse model and the human data suggests that the degree of ciliary dysfunction is causally related to the severity of human PCD, particularly the presence of hydrocephalus.

Animals↗
PubMed2002

The T complex distorter 2 candidate gene, Dnahc8, encodes at least two testis-specific axonemal dynein heavy chains that differ extensively at their amino and carboxyl termini.

Homozygosity for the t haplotype allele of the testis-specifically expressed axonemal dynein heavy chain (axDHC) gene, Dnahc8, has been linked to male sterility resulting from aberrant sperm motility. However, the near absence of Dnahc8 expression has been associated with male sterility resulting from an early breakdown in sperm flagellar development. Although axDHCs are integral participants in flagellar motility, a role in flagellar morphogenesis has never been attributed to a member of this highly conserved gene family. To gain a better understanding of this presumed novel role for Dnahc8, we have studied the organization and expression of full-length Dnahc8(+) and Dnahc8(t) transcripts. Our results demonstrate the existence of at least two alternatively spliced, testis-specific Dnahc8 mRNAs transcribed from both the + and t alleles. A highly expressed isoform encodes a protein with significant homology nearly throughout to the gamma heavy chain of the Chlamydomonas axonemal outer arm dynein, while a more poorly expressed isoform codes for a protein whose sequence diverges significantly from that of other axDHCs at both its N and C termini. While in situ hybridization studies demonstrate that both mRNA species accumulate exclusively in mid to late spermatocytes, each isoform shows spatial independence. Additional experiments demonstrate the existence of a testis-expressed mRNA with no significant open reading frame, a portion of which is antisense to the 5'-untranslated region of the highly divergent Dnahc8 isoform. The cumulative data imply that Dnahc8 may have acquired functional plasticity in the testis through the tightly controlled expression of both typical and unusual isoforms.

Amino Acid Sequence↗
PubMed1995

Mechanochemical aspects of axonemal dynein activity studied by in vitro microtubule translocation.

We have determined the relationship between microtubule length and translocation velocity from recordings of bovine brain microtubules translocating over a Paramecium 22S dynein substratum in an in vitro assay chamber. For comparison with untreated samples, the 22S dynein has been subjected to detergent and/or to pretreatments that induce phosphorylation of an associated 29 kDa light chain. Control and treated dyneins have been used at the same densities in the translocation assays. In any given condition, translocation velocity (v) shows an initial increase with microtubule length (L) and then reaches a plateau. This situation may be represented by a hyperbola of the general form v = aL/(L+b), which is formally analogous to the Briggs-Haldane relationship, which we have used to interpret our data. The results indicate that the maximum translocation velocity Vo(= a) is increased by pretreatment, whereas the length constant KL(= b), which corresponds to Km, does not change with pretreatment, implying that the mechanochemical properties of the pretreated dyneins differ from those of control dyneins. The conclusion that KL is constant for defined in vitro assays rules out the possibility that the velocity changes seen are caused by changes in geometry in the translocation assays or by the numbers of dyneins or dynein heads needed to produce maximal translocational velocity. From our analysis, we determine that f, the fraction of cycle time during which the dynein is in the force-generating state, is small--roughly 0.01, comparable to the f determined previously for heavy meromyosin. The practical limits of these mechanochemical changes imply that the maximum possible ciliary beat frequency is about 120 Hz, and that in the physiological range of 5-60 Hz, beat frequency could be controlled by varying the numbers of phosphorylated outer arm dyneins along an axonemal microtubule.

Animals↗
PubMed2001

Axonemal dynein intermediate-chain gene (DNAI1) mutations result in situs inversus and primary ciliary dyskinesia (Kartagener syndrome).

Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS).

Alleles↗
PubMed2004

Dysfunction of axonemal dynein heavy chain Mdnah5 inhibits ependymal flow and reveals a novel mechanism for hydrocephalus formation.

Motility of unicellular organisms occurred early in evolution with the emergence of cilia and flagella. In vertebrates, motile cilia are required for numerous functions such as clearance of the airways and determination of left-right body asymmetry. Ependymal cells lining the brain ventricles also carry motile cilia, but their biological function has remained obscure. Here, we show that ependymal cilia generate a laminar flow of cerebrospinal fluid through the cerebral aqueduct, which we term as 'ependymal flow'. The axonemal dynein heavy chain gene Mdnah5 is specifically expressed in ependymal cells, and is essential for ultrastructural and functional integrity of ependymal cilia. In Mdnah5-mutant mice, lack of ependymal flow causes closure of the aqueduct and subsequent formation of triventricular hydrocephalus during early postnatal brain development. The higher incidence of aqueduct stenosis and hydrocephalus formation in patients with ciliary defects proves the relevance of this novel mechanism in humans.

Animals↗
PubMed2000

Stochastic simulation of processive and oscillatory sliding using a two-headed model for axonemal dynein.

Computer simulations have been used in an attempt to understand experimental observations of processive and oscillatory sliding by one or a few axonemal dyneins. A simple two-headed model has been examined using stochastic simulation methods. To explain the experimental observations, the model must be capable of taking backward steps, as well as forward steps, and there must be hysteresis in switching between forward or backward stepping. When the effects of Brownian movement on motor strain are included, it is not possible to obtain oscillations as regular as the experimental records by using motor strain to regulate switching between forward or backward stepping.

Biological Clocks↗
PubMedDate not supplied

New axonemal dynein heavy chains from Tetrahymena thermophila.

Two dyneins can be extracted from Tetrahymena ciliary axonemes. The 22S dynein contains three heavy chains (HC), sediments at 22S in a sucrose gradient, and makes up the outer arms. The 14S dynein contains two to six HCs, sediments at 14S, and is thought to contribute to formation of the inner arms. We have identified two large proteins that are extracted from Tetrahymena axonemes with high salt and that sediment together at approximately 18S. The two large proteins cleave when subjected to UV light in the presence of ATP and vanadate, suggesting both proteins are dynein HC. Antibodies against one of the 18S HCs do not recognize 22S dynein HCs. Antibodies to 22S dynein HC do not bind appreciably to 18S dynein photocleavage fragments. Taken together, these results indicate that the large proteins that sediment at 18S are axonemal dynein heavy chains.

Animals↗
PubMed2005

An axonemal dynein particularly important for flagellar movement at high viscosity. Implications from a new Chlamydomonas mutant deficient in the dynein heavy chain gene DHC9.

Ciliary and flagellar axonemes contain multiple inner arm dyneins of which the functional difference is largely unknown. In this study, a Chlamydomonas mutant, ida9, lacking inner arm dynein c was isolated and shown to carry a mutation in the DHC9 dynein heavy chain gene. The cDNA sequence of DHC9 was determined, and its information was used to show that >80% of it is lost in the mutant. Electron microscopy and image analysis showed that the ida9 axoneme lacked electron density near the base of the S2 radial spoke, indicating that dynein c localizes to this site. The mutant ida9 swam only slightly slower than the wild type in normal media. However, swimming velocity was greatly reduced when medium viscosity was modestly increased. Thus, dynein c in wild type axonemes must produce a significant force when flagella are beating in viscous media. Because motility analyses in vitro have shown that dynein c is the fastest among all the inner arm dyneins, we can regard this dynein as a fast yet powerful motor.

Amino Acid Sequence↗