Inner arm dynein 1 is essential for Ca++-dependent ciliary reversals in Tetrahymena thermophila

Cilia in many organisms undergo a phenomenon called ciliary reversal during which the cilia reverse the beat direction, and the cell swims backwards. Ciliary reversal is typically caused by a depolarizing stimulus that ultimately leads to a rise in intraciliary Ca++ levels. It is this increase in intraciliary Ca++ that triggers ciliary reversal. However, the mechanism by which an increase in intraciliary Ca++ causes ciliary reversal is not known. We have previously mutated the DYH6 gene of Tetrahymena thermophila by targeted gene knockout and shown that the knockout mutants (KO6 mutants) are missing inner arm dynein 1 (I1). In this study, we show that KO6 mutants do not swim backward in response to depolarizing stimuli. In addition to being unable to swim backwards, KO6 mutants swim forward at approximately one half the velocity of wild-type cells. However, the ciliary beat frequency in KO6 mutants is indistinguishable from that of wild-type cells, suggesting that the slow forward swimming of KO6 mutants is caused by an altered waveform rather than an altered beat frequency. Live KO6 cells are also able to increase and decrease their swim speeds in response to stimuli, suggesting that some aspects of their swim speed regulation mechanisms are intact. Detergent-permeabilized KO6 mutants fail to undergo Ca++-dependent ciliary reversals and do not show Ca++-dependent changes in swim speed after MgATP reactivation, indicating that the axonemal machinery required for these responses is insensitive to Ca++ in KO6 mutants. We conclude that Tetrahymena inner arm dynein 1 is not only an essential part of the Ca++-dependent ciliary reversal mechanism but it also may contribute to Ca++-dependent changes in swim speed and to the formation of normal waveform during forward swimming.     

Differential light chain assembly influences outer arm dynein motor function

Tctex1 and Tctex2 were originally described as potential distorters/sterility factors in the non-Mendelian transmission of t-haplotypes in mice. These proteins have since been identified as subunits of cytoplasmic and/or axonemal dyneins. Within the Chlamydomonas flagellum, Tctex1 is a subunit of inner arm I1. We have now identified a second Tctex1-related protein (here termed LC9) in Chlamydomonas. LC9 copurifies with outer arm dynein in sucrose density gradients and is missing only in those strains completely lacking this motor. Zero-length cross-linking of purified outer arm dynein indicates that LC9 interacts directly with both the IC1 and IC2 intermediate chains. Immunoblot analysis revealed that LC2, LC6, and LC9 are missing in an IC2 mutant strain (oda6-r88) that can assemble outer arms but exhibits significantly reduced flagellar beat frequency. This defect is unlikely to be due to lack of LC6, because an LC6 null mutant (oda13) exhibits only a minor swimming abnormality. Using an LC2 null mutant (oda12-1), we find that although some outer arm dynein components assemble in the absence of LC2, they are nonfunctional. In contrast, dyneins from oda6-r88, which also lack LC2, retain some activity. Furthermore, we observed a synthetic assembly defect in an oda6-r88 oda12-1 double mutant. These data suggest that LC2, LC6, and LC9 have different roles in outer arm assembly and are required for wild-type motor function in the Chlamydomonas flagellum.     

The dynein heavy chain gene family in Tetrahymena thermophila

The dynein ATPases are a family of motor enzymes that drive microtubule sliding in cilia and flagella and contribute to microtubule-based transport inside cells. The multi-dynein hypothesis makes two predictions: 1) Axonemes contain multiple dynein heavy chain (DHC) isoforms, each encoded by a different gene; 2) Each isoform performs a specific role in ciliary beating. We used PCR-based techniques to clone thirteen different DHC sequences from Tetrahymena genomic DNA. All thirteen genes appeared to be expressed in growing cells. Comparisons of the deduced amino acid sequences of the thirteen DHCs with other known DHCs suggested that we have cloned three outer arm DHCs, two cytoplasmic DHCs, and eight inner arm DHCs.     

Properties of the full-length heavy chains of Tetrahymena ciliary outer arm dynein separated by urea treatment

An important challenge is to understand the functional specialization of dynein heavy chains. The ciliary outer arm dynein from Tetrahymena thermophila is a heterotrimer of three heavy chains, called alpha, beta and gamma. In order to dissect the contributions of the individual heavy chains, we used controlled urea treatment to dissociate Tetrahymena outer arm dynein into a 19S beta/gamma dimer and a 14S alpha heavy chain. The three heavy chains remained full-length and retained MgATPase activity. The beta/gamma dimer bound microtubules in an ATP-sensitive fashion. The isolated alpha heavy chain also bound microtubules, but this binding was not reversed by ATP. The 19S beta/gamma dimer and the 14S alpha heavy chain could be reconstituted into 22S dynein. The intact 22S dynein, the 19S beta/gamma dimer, and the reconstituted dynein all produced microtubule gliding motility. In contrast, the separated alpha heavy chain did not produce movement under a variety of conditions. The intact 22S dynein produced movement that was discontinuous and slower than the movement produced by the 19S dimer. We conclude that the three heavy chains of Tetrahymena outer arm dynein are functionally specialized. The alpha heavy chain may be responsible for the structural binding of dynein to the outer doublet A-tubule and/or the positioning of the beta/gamma motor domains near the surface of the microtubule track.     

Structure and function of outer dynein arm intermediate and light chain complex

The outer dynein arm (ODA) is a molecular complex that drives the beating motion of cilia/flagella. Chlamydomonas ODA is composed of three heavy chains (HCs), two ICs, and 11 light chains (LCs). Although the three-dimensional (3D) structure of the whole ODA complex has been investigated, the 3D configurations of the ICs and LCs are largely unknown. Here we identified the 3D positions of the two ICs and three LCs using cryo–electron tomography and structural labeling. We found that these ICs and LCs were all localized at the root of the outer-inner dynein (OID) linker, designated the ODA-Beak complex. Of interest, the coiled-coil domain of IC2 extended from the ODA-Beak to the outer surface of ODA. Furthermore, we investigated the molecular mechanisms of how the OID linker transmits signals to the ODA-Beak, by manipulating the interaction within the OID linker using a chemically induced dimerization system. We showed that the cross-linking of the OID linker strongly suppresses flagellar motility in vivo. These results suggest that the ICs and LCs of the ODA form the ODA-Beak, which may be involved in mechanosignaling from the OID linker to the HCs.     

A temperature-sensitive mutation affecting synthesis of outer arm dyneins in Tetrahymena thermophila.

We have characterized a novel, temperature-sensitive mutation affecting motility in Tetrahymena thermophila. Mutants grew and divided normally at the restrictive temperature (38 degrees C), but became nonmotile. Scanning electron microscopic analysis indicated that nonmotile mutants contained the normal number of cilia and that the cilia were of normal length. Transmission electron microscopic analysis indicated that axonemes isolated from nonmotile mutants lacked outer dynein arms, so the mutation was named oad 1 (outer arm deficient). Motile mutants shifted to 38 degrees C under conditions that prevent cell growth and division (starvation) remained motile suggesting that once assembled into axonemes at the permissive temperature (28 degrees C) the outer arm dyneins remain functional at 38 degrees C. Starved, deciliated mutants regenerated a full complement of functional cilia at 38 degrees C, indicating that the mechanism that incorporates the outer arm dynein into developing axonemes is not affected by the oad 1 mutation. Starved, nonmotile mutants regained motility when shifted back to 28 degrees C, but not when incubated with cycloheximide. We interpret these results to rule out the hypothesis that the oad 1 mutation affects the site on the microtubules to which the outer arm dyneins bind. Axonemes isolated from mutants grown for one generation at 38 degrees C had a mean of 6.0 outer arm dyneins, and axonemes isolated from mutants grown for two generations at 38 degrees C had a mean of 3.2 outer arm dyneins. Taken together, these results indicate that the oad 1 mutation affects the synthesis of outer arm dyneins in Tetrahymena.     

Recombination of ciliary dynein of Tetrahymena with the outer fibers.

Recombination of ciliary dyneins of Tetrahymena pyriformis with the outer fibers was investigated using turbidimetry, co-sedimentation analysis and electron microscopy. As reported by Gibbons, 30S dynein could recombine with the outer fibers, while 14S dynein did to so a lesser extent. At acidic pH, however, most of the 14S dynein was also rebound to the outer fibers. When an excess of crude dynein fraction was added to the outer fiber fraction at pH 8.2, electron microscopic observations showed that the outer doublet microtubules were decorated not only with arms but also with other electron-dense materials. On the other hand, when crude dynein fraction was mixed with the outer fibers in an appropriate quantity, only arms were reconstituted at the regular positions of A-subfibers. ATP had an inhibitory effect on the recombination of dynein with the outer fibers.     

An outer arm dynein light chain acts in a conformational switch for flagellar motility

A system distinct from the central pair–radial spoke complex was proposed to control outer arm dynein function in response to alterations in the mechanical state of the flagellum. In this study, we examine the role of a Chlamydomonas reinhardtii outer arm dynein light chain that associates with the motor domain of the γ heavy chain (HC). We demonstrate that expression of mutant forms of LC1 yield dominant-negative effects on swimming velocity, as the flagella continually beat out of phase and stall near or at the power/recovery stroke switchpoint. Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme. Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain. We propose that this γ HC–LC1–microtubule ternary complex functions as a conformational switch to control outer arm activity.     

Dephosphorylation of inner arm 1 is associated with ciliary reversals in Tetrahymena thermophila

In many organisms, depolarizing stimuli cause an increase in intraciliary Ca2+, which results in reversal of ciliary beat direction and backward swimming. The mechanism by which an increase in intraciliary Ca2+ causes ciliary reversal is not known. Here we show that Tetrahymena cells treated with okadaic acid or cantharidin to inhibit protein phosphatases do not swim backwards in response to depolarizing stimuli. We also show that both okadaic acid and cantharidin inhibit backward swimming in reactivated, extracted cell models treated with Ca2+. In contrast, treatment of whole cells or extracted cell models with protein kinase inhibitors has no effect on backward swimming. These results suggest that a component of the axonemal machinery is dephosphorylated during ciliary reversal. The phosphorylation state of inner arm dynein 1 (I1) was determined before and after cells were exposed to depolarizing conditions that induce ciliary reversal. An I1 intermediate chain is phosphorylated in forward swimming cells but is dephosphorylated in cells treated with a depolarizing stimulus. Our results suggest that dephosphorylation of Tetrahymena inner arm dynein 1 may be an essential part of the mechanism of ciliary reversal in response to increased intraciliary Ca2+.     

A Chlamydomonas outer arm dynein mutant missing the alpha heavy chain

A novel Chlamydomonas flagellar mutant (oda-11) missing the alpha heavy chain of outer arm dynein but retaining the beta and gamma heavy chains was isolated. Restriction fragment length polymorphism analysis with an alpha heavy chain locus genomic probe indicated that the oda-11 mutation was genetically linked with the structural gene of the alpha heavy chain. In cross-section electron micrographs, the oda-11 axoneme lacked the outermost appendage of the outer arm, indicating that the alpha heavy chain should be located in this region in the wild-type outer arm. This mutant swam at 119 microns/s at 25 degrees C, i.e., at an intermediate speed between those of wild type (194 microns/s) and of oda-1 (62 microns/s), a mutant missing the entire outer dynein arm. The flagellar beat frequency (approximately 50 Hz) was also between those of wild type (approximately 60 Hz) and oda-1 (approximately 26 Hz). These results indicate that the outer dynein arm of Chlamydomonas can be assembled without the alpha heavy chain, and that the outer arm missing the alpha heavy chain retains partial function.     

Tctex2-related outer arm dynein light chain is phosphorylated at activation of sperm motility

When the motility of sperm is activated, only one light chain of flagellar outer arm dynein is phosphorylated in many organisms. We show here that the light chain to be phosphorylated was shown to be light chain 2 (LC2) in rainbow trout and chum salmon sperm and LC1 in sea urchin sperm. Molecular analyses of the phosphorylated light chains from sperm flagella of the salmonid fishes and sea urchin revealed that the light chains are homologs of the mouse t complex-encoded protein Tctex2, which is one of the putative t complex distorters. These results suggest that mouse Tctex2 might also be a light chain of flagellar outer arm dynein and that the abortive phosphorylation of Tctex2/outer arm dynein light chain might be related to the less progressive movement of sperm.     

Structure and mass of mammalian respiratory ciliary outer arm 19S dynein

Mammalian respiratory ciliary outer arm dyneins isolated as the major ATPase peak migrating at 19S on sucrose density gradients were examined by transmission electron microscopy of negatively stained samples and scanning transmission electron microscopy of unstained samples. The predominant discrete particle structure observed was composed of two globular heads apparently connected by amorphous or indistinct material. The heads were either circular or slightly elliptical of mean 13 +/- 1 X 10 +/- 2 nm dimensions. The mass of this structure averaged 1.22 +/- 0.34 million daltons with the individual globular heads averaging 310 +/- 77 kilodaltons (kD). Negative staining revealed that one or both of the globular heads often contained a central accumulation of stain measuring 2.5 +/- 1 nm across. A second type of structure, appearing with lesser frequency in the 19S fraction than in the unfractionated dynein preparation loaded onto the sucrose gradient, was a single globular head of 13 +/- 1 X 10 +/- 2 nm often with 2 +/- 1 nm centrally accumulated stain and with or without an appendage. This one-headed particle thus resembled one-half of the two-headed particle. Mass measurements were lower, however, for isolated, single globular heads, averaging 220 +/- 111 kD. A third type of particle observed was a ring-like structure with 4 +/- 1 nm centrally accumulated stain and without appendages. The ring structure was slightly larger in diameter, 14 +/- 1 nm, and had a greater peripheral accumulation of negative stain than either of the one- or two-headed particles, suggesting that it was not derived therefrom.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Chlamydomonas outer arm dynein mutant with a truncated beta heavy chain

A new allele of the Chlamydomonas oda4 flagellar mutant (oda4-s7) possessing abnormal outer dynein arms was isolated. Unlike the previously described oda4 axoneme lacking all three (alpha, beta, and gamma) outer-arm dynein heavy chains, the oda4-s7 axoneme contains the alpha and gamma heavy chains and a novel peptide with a molecular mass of approximately 160 kD. The peptide reacts with a mAb (18 beta B) that recognizes an epitope on the NH2-terminal part of the beta heavy chain. These observations indicate that this mutant has a truncated beta heavy chain, and that the NH2-terminal part of the beta heavy chain is important for the stable assembly of the outer arms. In averaged electron microscopic images of outer arms from cross sections of axonemes, the mutant outer arm lacks its mid-portion, producing a forked appearance. Together with our previous finding that the mutant oda11 lacks the alpha heavy chain and the outermost portion of the arm (Sakakibara, H., D. R. Mitchell, and R. Kamiya. 1991. J. Cell Biol. 113:615-622), this result defines the approximate locations of the three outer arm heavy chains in the axonemal cross section. The swimming velocity of oda4-s7 is 65 +/- 8 microns/s, close to that of oda4 which lacks the entire outer arm (62 +/- 8 microns/s) but significantly lower than the velocities of wild type (194 +/- 23 microns/s) and oda11 (119 +/- 17 microns/s). Thus, the lack of the beta heavy chain impairs outer-arm function more seriously than does the lack of the alpha heavy chain, suggesting that the alpha and beta chains play different roles in outer arm function.     

Substructure of the outer dynein arm

The substructure of the outer dynein arm has been analyzed in quick-frozen deep-etch replicas of Tetrahymena and Chlamydomonas axonemes. Each arm is found to be composed of five morphologically discrete components: an elliptical head; two spherical feet; a slender stalk; and an interdynein linker. The feet make contact with the A microtubule of each doublet; the stalk contacts the B microtubule; the head lies between the feet and stalk; and the linker associates each arm with its neighbor. The spatial relationships between these five components are found to be distinctly different in rigor (ATP-depleted) versus relaxed (ATP- or vanadate plus ATP-treated) axonemes, and the stalk appears to alter its affinity for the B microtubule in the relaxed state. Images of living cilia attached to Tetrahymena cells show that the relaxed configuration is adopted in vivo. We relate our observations to morphological and experimental studies reported by others and propose several models that suggest how this newly described dynein morphology may relate to dynein function.     

Localization of an intermediate chain of outer arm dynein by immunoelectron microscopy

We have used immunoelectron microscopy to determine the location of an intermediate chain in the isolated outer arm dynein from Chlamydomonas flagella. When the purified alpha beta dimer of the outer arm was incubated with antibodies recognizing two distinct epitopes on its 69-kDa intermediate chain and then negatively stained and examined by electron microscopy, both antibodies appeared to have bound to the base of the Y-shaped stem that connects the two heads of the particle. These results indicate that this intermediate chain is located at the base of the stem. Inasmuch as this polypeptide is tightly associated with the 78-kDa intermediate chain and several light chains in an intermediate chain-light chain complex, it is likely that this entire assemblage is located at the base of the particle. Thus, these polypeptides are in a potentially important position with regard to the ATP-insensitive (structural end) binding of dynein to microtubules and to dynein-dynein interactions within the axoneme.     

Outer dynein arm light chain 1 is essential for controlling the ciliary response to cyclic AMP in Paramecium tetraurelia

The individual role of the outer dynein arm light chains in the molecular mechanisms of ciliary movements in response to second messengers, such as Ca(2+) and cyclic nucleotides, is unclear. We examined the role of the gene termed the outer dynein arm light chain 1 (LC1) gene of Paramecium tetraurelia (ODAL1), a homologue of the outer dynein arm LC1 gene of Chlamydomonas reinhardtii, in ciliary movements by RNA interference (RNAi) using a feeding method. The ODAL1-silenced (ODAL1-RNAi) cells swam slowly, and their swimming velocity did not increase in response to membrane-hyperpolarizing stimuli. Ciliary movements on the cortical sheets of ODAL1-RNAi cells revealed that the ciliary beat frequency was significantly lower than that of control cells in the presence of ≥ 1 mM Mg(2+)-ATP. In addition, the ciliary orientation of ODAL1-RNAi cells did not change in response to cyclic AMP (cAMP). A 29-kDa protein phosphorylated in a cAMP-dependent manner in the control cells disappeared in the axoneme of ODAL1-RNAi cells. These results indicate that ODAL1 is essential for controlling the ciliary response by cAMP-dependent phosphorylation.     

ATP-dependent structural changes of the outer dynein arm in Tetrahymena cilia: a freeze-etch replica study

With the rapid-freeze, deep-etch replica technique, the structural conformations of outer dynein arms in demembranated cilia from Tetrahymena were analyzed under two different conditions, i.e., in the absence of ATP and in the presence of ATP and vanadate. In the absence of ATP, the lateral view of axonemes was characterized by the egg- shaped outer dynein arms, which showed a slightly baseward tilt with a mean inclination of 11.1 degrees +/- 3.4 degrees SD from the perpendicular to the doublet microtubules. On the other hand, in the presence of 1 mM ATP and 100 microM vanadate, the outer arms were extended and slender and showed an increased baseward tilt with a mean inclination of 31.6 degrees +/- 4.9 degrees SD. In ATP-activated axonemes, these two types of arms coexisted, each type occurring in groups along one row of outer arms. These findings strongly suggest that the interdoublet sliding is caused by dynamic structural changes of dynein arms that follow the hydrolysis of ATP.