ODA16p, a Chlamydomonas flagellar protein needed for dynein assembly

Dynein motors of cilia and flagella function in the context of the axoneme, a very large network of microtubules and associated proteins. To understand how dyneins assemble and attach to this network, we characterized two Chlamydomonas outer arm dynein assembly (oda) mutants at a new locus, ODA16. Both oda16 mutants display a reduced beat frequency and altered swimming behavior, similar to previously characterized oda mutants, but only a partial loss of axonemal dyneins as shown by both electron microscopy and immunoblots. Motility studies suggest that the remaining outer arm dyneins on oda16 axonemes are functional. The ODA16 locus encodes a 49-kDa WD-repeat domain protein. Homologues were found in mammalian and fly databases, but not in yeast or nematode databases, implying that this protein is only needed in organisms with motile cilia or flagella. The Chlamydomonas ODA16 protein shares 62% identity with its human homologue. Western blot analysis localizes more than 90% of ODA16p to the flagellar matrix. Because wild-type axonemes retain little ODA16p but can be reactivated to a normal beat in vitro, we hypothesize that ODA16p is not an essential dynein subunit, but a protein necessary for dynein transport into the flagellar compartment or assembly onto the axoneme.     

Chlamydomonas flagellar outer row dynein assembly protein ODA7 interacts with both outer row and I1 inner row dyneins

We previously found that a mutation at the ODA7 locus in Chlamydomonas prevents axonemal outer row dynein assembly by blocking association of heavy chains and intermediate chains in the cytoplasm. We have now cloned the ODA7 locus by walking in the Chlamydomonas genome from nearby molecular markers, confirmed the identity of the gene by rescuing the mutant phenotype with genomic clones, and identified the ODA7 gene product as a 58-kDa leucine-rich repeat protein unrelated to outer row dynein LC1. Oda7p is missing from oda7 mutant flagella but is present in flagella of other outer row or inner row dynein assembly mutants. However, Oda7 levels are greatly reduced in flagella that lack both outer row dynein and inner row I1 dynein. Biochemical fractionation and rebinding studies support a model in which Oda7 participates in a previously uncharacterized structural link between inner and outer row dyneins.     

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.     

Discrete PIH proteins function in the cytoplasmic preassembly of different subsets of axonemal dyneins

Axonemal dyneins are preassembled in the cytoplasm before being transported into cilia and flagella. Recently, PF13/KTU, a conserved protein containing a PIH (protein interacting with HSP90) domain, was identified as a protein responsible for dynein preassembly in humans and Chlamydomonas reinhardtii. This protein is involved in the preassembly of outer arm dynein and some inner arm dyneins, possibly as a cofactor of molecular chaperones. However, it is not known which factors function in the preassembly of other inner arm dyneins. Here, we analyzed a novel C. reinhardtii mutant, ida10, and found that another conserved PIH family protein, MOT48, is responsible for the formation of another subset of inner arm dyneins. A variety of organisms with motile cilia and flagella typically have three to four PIH proteins, including potential homologues of MOT48 and PF13/KTU, whereas organisms without them have no, or only one, such protein. These findings raise the possibility that multiple PIH proteins are commonly involved in the preassembly of different subsets of axonemal dyneins.     

The Motility of Axonemal Dynein Is Regulated by the Tubulin Code

Microtubule diversity, arising from the utilization of different tubulin genes and from posttranslational modifications, regulates many cellular processes including cell division, neuronal differentiation and growth, and centriole assembly. In the case of cilia and flagella, multiple cell biological studies show that microtubule diversity is important for axonemal assembly and motility. However, it is not known whether microtubule diversity directly influences the activity of the axonemal dyneins, the motors that drive the beating of the axoneme, nor whether the effects on motility are indirect, perhaps through regulatory pathways upstream of the motors, such as the central pair, radial spokes, or dynein regulatory complex. To test whether microtubule diversity can directly regulate the activity of axonemal dyneins, we asked whether in vitro acetylation or deacetylation of lysine 40 (K40), a major posttranslational modification of α-tubulin, or whether proteolytic cleavage of the C-terminal tail (CTT) of α- and β-tubulin, the location of detyrosination, polyglutamylation, and polyglycylation modifications as well as most of the genetic diversity, can influence the activity of outer arm axonemal dynein in motility assays using purified proteins. By quantifying the motility with displacement-weighted velocity analysis and mathematically modeling the results, we found that K40 acetylation increases and CTTs decrease axonemal dynein motility. These results show that axonemal dynein directly deciphers the tubulin code, which has important implications for eukaryotic ciliary beat regulation.     

Mechanisms of mammalian ciliary motility: Insights from primary ciliary dyskinesia genetics

Motile cilia and flagella are organelles that, historically, have been poorly understood and inadequately investigated. However, cilia play critical roles in fluid clearance in the respiratory system and the brain, and flagella are required for sperm motility. Genetic studies involving human patients and mouse models of primary ciliary dyskinesia over the last decade have uncovered a number of important ciliary proteins and have begun to elucidate the mechanisms underlying ciliary motility. When combined with genetic, biochemical, and cell biological studies in Chlamydomonas reinhardtii, these mammalian genetic analyses begin to reveal the mechanisms by which ciliary motility is regulated.     

Nucleotide-metabolizing enzymes in Chlamydomonas flagella.

Nucleotides have at least two functions in eukaryotic cilia and flagella. ATP, originating in the cells, is utilized for motility by energy-transducing protein(s) called dynein, and the binding of guanine nucleotides to tubulin, and probably certain transformations of the bound nucleotides, are prerequisites for the assembly of microtubules. Besides dynein, which can be solubulized from Chlamydomonas flagella as a heterogeneous, Mg2+ or Ca2+-activated ATPase, we have purified and characterized five other flagellar enzymes involved in nucleotide transformations. A homogeneous, low molecular weight, Ca2+-specific adenosine triphosphatase was isolated, which was inhibited by Mg2+ and was not specific for ATP. This enzyme was not formed by treating purified dynein with proteases. It was absent from extracts of Tetrahymena cilia. Its function might be an auxiliary energy transducer, or in steering or tactic responses. Two species of adenylate kinase were isolated, one of which was much elevated in regenerating flagella; the latter was also present in cell bodies. A large part of flagellar nucleoside diphosphokinase activity could not be solubilized. Two soluble enzyme species were identified, one of which was also present in cell bodies. Since these enzymes are of interest because they might function in microtubule assembly, we studied the extent to which brain nucleoside diphosphokinase co-polymerizes with tubulin purified by repeated cycles of polymerization. Arginine kinase was not detected in Chlamydomonas flagellar extracts.     

The CSC connects three major axonemal complexes involved in dynein regulation

Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ-ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo-electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin-dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility.     

A novel subunit of axonemal dynein conserved among lower and higher eukaryotes

To elucidate the subunit composition of axonemal inner-arm dynein, we examined a 38 kDa protein (p38) co-purified with a Chlamydomonas inner arm subspecies, dynein d. We found it is a novel protein conserved among a variety of organisms with motile cilia and flagella. Immunoprecipitation using specific antibody verified its association with a heavy chain, actin and a previously identified light chain (p28). Unexpectedly, mutant axonemes lacking dynein d and other dyneins retained reduced amounts of p38. This finding suggests that p38 is involved in the docking of dynein d to specific loci.     

Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility

The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a "high-load environment," we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters.     

The CSC is required for complete radial spoke assembly and wild-type ciliary motility

The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and microtubule sliding in vitro demonstrates that the CSC plays a critical role in modulating dynein activity. Our results not only indicate that the CSC is required for spoke assembly and wild-type motility, but also provide evidence for heterogeneity among the radial spokes.     

Turnover of actin in Chlamydomonas flagella detected by fluorescence recovery after photobleaching (FRAP)

Recent indirect observations have suggested that various axonemal proteins in cilia and flagella of live cells undergo turnover independently of shortening or elongation of the axoneme. To gain direct evidence, here we examined using a FRAP (fluorescence recovery after photobleaching) technique whether actin, a subunit of inner arm dynein, is being turned over in Chlamydomonas flagella. Fluorescently labeled rabbit actin was introduced by electroporation into the cells of ida5oda1, a double mutant between oda1 lacking outer arm dynein and ida5 lacking several species of inner arm dyneins due to the absence of a conventional-type actin. In actin-loaded cells, flagella became motile and fluorescent due to incorporation of inner-arm dyneins containing the labeled actin. Cells were sandwiched between an agar layer and a coverslip so as to restrict flagellar movement. After a small portion of a flagellum was photobleached, the fluorescence intensity in the bleached area was monitored with a sensitive video camera. The fluorescence intensity in the photobleached region was found to recover 10-40% of the original level over several tens of minutes without changing its position. The time course and extent of the recovery varied greatly from one cell to another, suggesting that the turnover depends on cellular conditions. Western blot analysis indicated that 70-80% of flagellar actin was associated with the axoneme. Hence this experiment provides direct evidence that an axonemal component undergoes dynamic exchange in stationary flagella.     

The dynein heavy chain family

Dynein is the large molecular motor that translocates to the (-) ends of microtubules. Dynein was first isolated from Tetrahymena cilia four decades ago. The analysis of the primary structure of the dynein heavy chain and the discovery that many organisms express multiple dynein heavy chains have led to two insights. One, dynein, whose motor domain comprises six AAA modules and two potential mechanical levers, generates movement by a mechanism that is fundamentally different than that which underlies the motion of myosin and kinesin. And two, organisms with cilia or flagella express approximately 14 different dynein heavy chain genes, each gene encodes a distinct dynein protein isoform, and each isoform appears to be functionally specialized. Sequence comparisons demonstrate that functionally equivalent isoforms of dynein heavy chains are well conserved across species. Alignments of portions of the motor domain result in seven clusters: (i) cytoplasmic dynein Dyhl; (ii) cytoplasmic dynein Dyh2; (iii) axonemal outer arm dynein alpha; (iv) outer arm dyneins beta and gamma; (v) inner arm dynein 1alpha; (vi) inner arm dynein 1beta; and (vii) a group of apparently single-headed inner arm dyneins. Some of the dynein groups contained more than one representative from a single organism, suggesting that these may be tissue-specific variants.     

The intraflagellar transport machinery of Chlamydomonas reinhardtii

First discovered in the green alga, Chlamydomonas , intraflagellar transport (IFT) is the bidirectional movement of protein particles along the length of eukaryotic cilia and flagella. Composed of ∼16 different proteins, IFT particles are moved out to the distal tip of the organelle by kinesin-II and are brought back to the cell body by cytoplasmic dynein 1b. Mutant analysis of the IFT motor and particle proteins using diverse organisms has revealed a conserved and essential role for IFT in the assembly and maintenance of cilia and flagella. IFT is thought to mediate this assembly through the delivery of axonemal precursors out to the distal tip of the growing organelle. Consistent with this model, the IFT particle proteins are rich in protein–protein binding motifs, suggesting that the particles may act as scaffolds for the binding of multiple cargoes. With most of the IFT proteins now identified at the level of the gene, this review will briefly examine both the structure and function of the IFT machinery of Chlamydomonas reinhardtii .     

The role of the dynein stalk in cytoplasmic and flagellar motility

We have recently identified a microtubule binding domain within the motor protein cytoplasmic dynein. This domain is situated at the end of a slender 10–12 nm projection which corresponds to the stalks previously observed extending from the heads of both axonemal and cytoplasmic dyneins. The stalks also correspond to the B-links observed to connect outer arm axonemal dyneins to the B-microtubules in flagella and constitute the microtubule attachment sites during dynein motility. The stalks contrast strikingly with the polymer attachment domains of the kinesins and myosins which are found on the surface of the motor head. The difference in dynein's structural design raises intriguing questions as to how the stalk functions in force production along microtubules. In this article, we attempt to integrate the myriad of biochemical and EM structural data that has been previously collected regarding dynein with recent molecular findings, in an effort to begin to understand the mechanism of dynein motility. Received: 13 March 1998 / Revised version: 17 April 1998 / Accepted: 17 April 1998     

Association between actin and light chains in Chlamydomonas flagellar inner-arm dyneins

Inner dynein arms in cilia and flagella contain actin as a subunit; however, the function of this actin is totally unknown. Here we performed chemical crosslinking experiments to examine the interaction of actin with other subunits. Six of the seven Chlamydomonas inner-arm dynein species separated by anion-exchange chromatography contain actin and either one of the two previously identified light chains, p28 and centrin, in a mutually exclusive manner. Western blotting of chemically crosslinked dyneins indicated that actin is directly associated with p28 and centrin but not with the dynein heavy chains (HCs). In contrast, p28 and centrin both appeared to interact directly with the N-terminal half of the HCs. Thus it is likely that actin is associated with the heavy chains through p28/centrin. These light chains may well function in the assembly or targeting of the inner arm to the correct axonemal location.     

Late steps in cytoplasmic maturation of assembly-competent axonemal outer arm dynein in Chlamydomonas require interaction of ODA5 and ODA10 in a complex

Axonemal dyneins are multisubunit enzymes that must be preassembled in the cytoplasm, transported into cilia by intraflagellar transport, and bound to specific sites on doublet microtubules, where their activity facilitates microtubule sliding-based motility. Outer dynein arms (ODAs) require assembly factors to assist their preassembly, transport, and attachment to cargo (specific doublet A-tubule sites). In Chlamydomonas, three assembly factors—ODA5, ODA8, and ODA10—show genetic interactions and have been proposed to interact in a complex, but we recently showed that flagellar ODA8 does not copurify with ODA5 or ODA10. Here we show that ODA5 and ODA10 depend on each other for stability and coexist in a complex in both cytoplasmic and flagellar extracts. Immunofluorescence and immuno–electron microscopy reveal that ODA10 in flagella localizes strictly to a proximal region of doublet number 1, which completely lacks ODAs in Chlamydomonas. Studies of the in vitro binding of ODAs to axonemal doublets reveal a role for the ODA5/ODA10 assembly complex in cytoplasmic maturation of ODAs into a form that can bind to doublet microtubules.     

Keeping an eye on I1: I1 dynein as a model for flagellar dynein assembly and regulation

Among the major challenges in understanding ciliary and flagellar motility is to determine how the dynein motors are assembled and localized and how dynein-driven outer doublet microtubule sliding is controlled. Diverse studies, particularly in Chlamydomonas, have determined that the inner arm dynein I1 is targeted to a unique structural position and is critical for regulating the microtubule sliding required for normal ciliary/flagellar bending. As described in this review, I1 dynein offers additional opportunities to determine the principles of assembly and targeting of dyneins to cellular locations and for studying the mechanisms that regulate dynein activity and control of motility by phosphorylation.     

Rotation and translocation of microtubules in vitro induced by dyneins from Tetrahymena cilia

Dynein, the force-generating enzyme that powers the movement of cilia and flagella, has been characterized biochemically, but no simple system has been available for examining its motile properties. Here we describe a quantitative in vitro motility assay in which dynein adsorbed onto a glass surface induces linear translocation of purified bovine microtubules. Using this assay, we show that both 22S and 14S dyneins from Tetrahymena cilia induce movement but have distinct motile properties. A unique property of 14S dynein, which has not been described for other motility proteins, is its ability to generate torque that causes microtubules to rotate during forward translocation. In the axoneme, 14S dynein-induced torque may induce rotation of central-pair microtubules and may play an important role in generating three-dimensional ciliary beating patterns.