ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance

A new mutant strain of Chlamydomonas, ptx1, has been identified which is defective in phototaxis. This strain swims with a rate and straightness of path comparable with that of wild-type cells, and retains the photoshock response. Thus, the mutation does not cause any gross defects in swimming ability or photoreception, and appears to be specific for phototaxis. Calcium is required for phototaxis in wild- type cells, and causes a concentration-dependent shift in flagellar dominance in reactivated, demembranated cell models. ptx1-reactivated models are defective in this calcium-dependent shift in flagellar dominance. This indicates that the mutation affects one or more components of the calcium-dependent axonemal regulatory system, and that this system mediates phototaxis. The reduction or absence of two 75-kD axonemal proteins correlates with the nonphototactic phenotype. Axonemal fractionation studies, and analysis of axonemes from mutant strains with known structural defects, failed to reveal the structural localization of the 75-kD proteins within the axoneme. The proteins are not components of the outer dynein arms, two of the three types of inner dynein arms, the radial spokes, or the central pair complex. Because changes in flagellar motility ultimately require the regulation of dynein activity, cell models from mutant strains defective in specific dynein arms were reactivated at various calcium concentrations. Mutants lacking the outer arms, or the I1 or I2 inner dynein arms, retain the wild-type calcium-dependent shift in flagellar dominance. Therefore, none of these arms are the sole mediators of phototaxis.     

Direction of force generated by the inner row of dynein arms on flagellar microtubules

Our goal was to determine the direction of force generation of the inner dynein arms in flagellar axonemes. We developed an efficient means of extracting the outer row of dynein arms in demembranated sperm tail axonemes, leaving the inner row of dynein arms structurally and functionally intact. Sperm tail axonemes depleted of outer arms beat at half the beat frequency of sperm tails with intact arms over a wide range of ATP concentrations. The isolated, outer arm-depleted axonemes were induced to undergo microtubule sliding in the presence of ATP and trypsin. Electron microscopic analysis of the relative direction of microtubule sliding (see Sale, W. S. and P. Satir, 1977, Proc. Natl. Acad. Sci. USA, 74:2045-2049) revealed that the doublet microtubule with the row of inner dynein arms, doublet N, always moved by sliding toward the proximal end of the axoneme relative to doublet N + 1. Therefore, the inner arms generate force such that doublet N pushes doublet N + 1 tipward. This is the same direction of microtubule sliding induced by ATP and trypsin in axonemes having both inner and outer dynein arms. The implications of this result for the mechanism of ciliary bending and utility in functional definition of cytoplasmic dyneins are discussed.     

Mutations at twelve independent loci result in absence of outer dynein arms in Chylamydomonas reinhardtii

35 strains of Chlamydomonas mutant missing the entire outer dynein arm were isolated by screening slow-swimming phenotypes. They comprised 10 independent genetic loci (odal-10) including those of previously isolated mutants oda38 and pf28. The 10 loci were distinct from pf13 and pf22, loci for nonmotile mutants missing the outer arm. These results indicate that at least 12 genes are responsible for the assembly of the outer dynein arms. There were no mutants lacking partial structures of the outer arm, suggesting that lack of a single component results in failure of assembly of entire outer arms. Temporary dikaryons derived from mating of two different oda strains often, but not always, recovered the wild-type motility within 2 h of mating. Hence, outer arms can be transported and attached to the outer doublets independently of flagellar growth.     

Functional recombination of dynein 1 with demembranated sea urchin sperm partially extracted with KC1.

Demembranated sea urchin sperm were extracted with 0.5 M KCl as described earlier and reactivated in a solution containing 1 mM ATP. Their flagellar beat frequency was approximately 13 Hz, while that of standard reactivated sperm which had not been extracted with KCl was approximately 31 Hz at 23°C. Addition of soluble dynein 1 caused a gradual increase in the flagellar beat frequency to approximately 25 Hz after 10 min at room temperature. This restoration of frequency occurred in the absence or presence of ATP. Examination by electron microscopy showed that, whereas KCl-extracted sperm were lacking the majority of the outer arms on the doublet tubules, they had regained most of their outer arms following incubation with soluble dynein 1.     

Flagellar quiescence in Chlamydomonas: Characterization and defective quiescence in cells carrying sup-pf-1 and sup-pf-2 outer dynein arm mutations

Chlamydomonas reinhardtii can use their flagella for two distinct types of movement: swimming through liquid or gliding on a solid substrate. Cells switching from swimming to gliding motility undergo a reversible flagellar quiescence. This phenomenon appears to involve the outer dynein arms, since mutants having altered outer arm beta and gamma dyneins (sup-pf-1 and sup-pf-2) show a diminished ability to quiesce. Sup-pf-1 and sup-pf-2 were originally isolated as gain-of-function mutations that suppress the flagellar paralysis resulting from radial spoke or central pair defects. Defective quiescence is also a gain-of-function phenomenon, as cells completely lacking outer arm heavy chains show a normal quiescence phenotype. These data suggest that regulation of outer arm dynein activity is essential for flagellar quiescence and furthermore that regulation of quiescence involves a signal transduction pathway that shares elements with the radial spoke/central pair system.     

The reactivation of demembranated human spermatozoa lacking outer dynein arms is independent of pH

The effect of pH, Mg-ATP, and free calcium on activity of the inner dynein arm was investigated using demembranated human spermatozoa lacking the outer dynein arms (LODA). The results were compared with those obtained for demembranated-reactivated normal spermatozoa to evaluate the functional properties of the inner and outer dynein arms in axonemal motility. The reactivation of Triton X-100–demembranated LODA spermatozoa was analysed at various pHs and concentrations of Mg-ATP and calcium using video recordings. The percentage of reactivated LODA spermatozoa as a function of Mg-ATP concentration was not dependent on pH, whereas reactivation of normal human spermatozoa is pH dependent. This suggests that there may be a pH-dependent regulatory mechanism associated with the outer dynein arms. A delay in the principal bend propagation of normal and LODA reactivated cells was found at pH 7.1. This disappeared at pH 7.8 in normal but not in LODA populations. This suggests a role for outer dynein arms in the initiation of the propagation of flagellar bends at alkaline pH. The level of LODA and normal sperm reactivation both depended on the calcium concentration in the medium. At lower free calcium concentrations, the reactivation level and beat frequency of reactivated cells were higher. Our results suggest a functional difference between outer and inner dynein arms of human spermatozoa based on a differential pH sensitivity. Moreover, calcium seems to exert its regulatory action elsewhere than on the outer dynein arms. Mol. Reprod. Dev. 49:416–425, 1998. © 1998 Wiley-Liss, Inc.     

Three members of the LC8/DYNLL family are required for outer arm dynein motor function

The highly conserved LC8/DYNLL family proteins were originally identified in axonemal dyneins and subsequently found to function in multiple enzyme systems. Genomic analysis uncovered a third member (LC10) of this protein class in Chlamydomonas. The LC10 protein is extracted from flagellar axonemes with 0.6 M NaCl and cofractionates with the outer dynein arm in sucrose density gradients. Furthermore, LC10 is specifically missing only from axonemes of those strains that fail to assemble outer dynein arms. Previously, the oda12-1 insertional allele was shown to lack the Tctex2-related dynein light chain LC2. The LC10 gene is located approximately 2 kb from that of LC2 and is also completely missing from this mutant but not from oda12-2, which lacks only the 3' end of the LC2 gene. Although oda12-1 cells assemble outer arms that lack only LC2 and LC10, this strain exhibits a flagellar beat frequency that is consistently less than that observed for strains that fail to assemble the entire outer arm and docking complex (e.g., oda1). These results support a key regulatory role for the intermediate chain/light chain complex that is an integral and highly conserved feature of all oligomeric dynein motors.     

Three-dimensional structures of the flagellar dynein-microtubule complex by cryoelectron microscopy

The outer dynein arms (ODAs) of the flagellar axoneme generate forces needed for flagellar beating. Elucidation of the mechanisms underlying the chemomechanical energy conversion by the dynein arms and their orchestrated movement in cilia/flagella is of great importance, but the nucleotide-dependent three-dimensional (3D) movement of dynein has not yet been observed. In this study, we establish a new method for reconstructing the 3D structure of the in vitro reconstituted ODA-microtubule complex and visualize nucleotide-dependent conformational changes using cryoelectron microscopy and image analysis. As the complex went from the rigor state to the relaxed state, the head domain of the beta heavy chain shifted by 3.7 nm toward the B tubule and inclined 44 degrees inwards. These observations suggest that there is a mechanism that converts head movement into the axonemal sliding motion.     

ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery

Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.     

Geometric Clutch model version 3: the role of the inner and outer arm dyneins in the ciliary beat

The Geometric Clutch model of ciliary and flagellar beating uses the transverse force (t-force) that develops between the outer doublets of the axoneme as the regulator for activating and deactivating the dynein motors and organizing the flagellar beat. The version of the model described here adds detail to the formulations used in the two previous versions as follows: (1) In place of two opposing sets of dyneins, the new model has four sets of dyneins, corresponding to two sets on each side of the axoneme acting in series. (2) The four sets of dyneins are each subdivided into two ranks representing inner and outer arm dyneins. (3) The force produced by each dynein is governed by a force-velocity relationship that is independently specified for the inner and outer arms. Consistent with the original model, the new version of the Geometric Clutch model can simulate both the effective and recovery stroke phases of the ciliary beat using a single uniform algorithm. In addition, the new version can operate with the outer arms disabled. Under this condition, the simulation exhibits a beat pattern similar to the original but the beat frequency is reduced to approximately one third. These results are contingent on using force-velocity relationships for the inner and outer arms similar to those described by Brokaw [1999: Cell Motil. Cytoskeleton 42:134-148], where the inner arms contribute most of the driving force at low shear velocities. This constitutes the first examination of the effects of the force-velocity characteristics of dynein on a cilia-like beat in a theoretical framework.     

Transport and arrangement of the outer-dynein-arm docking complex in the flagella of Chlamydomonas mutants that lack outer dynein arms

The outer dynein arms of Chlamydomonas flagella are attached to a precise site on the outer doublet microtubules and repeat at a regular interval of 24 nm. This binding is mediated by the outer dynein arm docking complex (ODA-DC), which is composed of three protein subunits. In this study, antibodies against the 83- and 62-kD subunits (DC83 and DC62) of the ODA-DC were used to analyze its state of association with outer arm components within the cytoplasm, and its localization in the axonemes of oda mutants. Immunoprecipitation indicates that DC83 and DC62 are preassembled within the cytoplasm, but that they are not associated with outer arm dynein. Both proteins are lost or greatly diminished in oda1 and oda3, mutants in the structural genes of DC62 and DC83, respectively, demonstrating that their association is necessary for their stable presence in the cytoplasm. Immunoelectron microscopy indicates that DC83 repeats at 24-nm intervals along the length of the doublet microtubules of oda6, which lacks outer arms; thus, outer arm periodicity may be determined by the ODA-DC. Flagellar regeneration and temporary dikaryon experiments indicate that the ODA-DC can be rapidly transported into the flagellum and assembled on the doublet microtubules independently of the outer arms and independently of flagellar growth. Unexpectedly, the intensity of ODA-DC labeling decreased toward the distal ends of axonemes of oda6 but not wild-type cells, suggesting that the outer arms reciprocally contribute to the assembly/stability of the ODA-DC.     

Inner arm dynein ATPase fraction of sea urchin sperm flagella causes active sliding of axonemal outer doublet microtubule.

In order to clarify the role of the inner arms of the axoneme in sperm flagellar movement, we prepared an ATPase fraction (12S) from the outer arm-depleted axonemes of sea urchin sperm flagella. When both arm-depleted axonemes were incubated with the 12S ATPase, they exhibited the sliding disintegration of outer doublet microtubules. Electron microscopy revealed that the ATPase rebound to the original inner arm sites of the axoneme. Therefore, it is quite likely that the 12S ATPase is one of the components of the inner arms. We referred to it as "inner arm dynein".     

Structural conformation of the ciliary ATPase dynein.

The ATPase dynein forms part of a mechanoohemically active complex responsible for the sliding filament mechanism of ciliary and flagellar motion. Extraction of demembranated cilia from the lamellibranch mollusc Unio or the protozoan Tetrahymena by 0.5 m-KCl solubilizes the outer rows of dynein and, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, releases the A form (Mr 360,000) of dynein into solution. Negative contrast electron microscopy of the solubilized dynein fraction reveals an homogeneous array of 93 Å particles that we identify as the ATPase dynein in its monomeric form. Because of the method of dynein extraction, the conformation of the molecule, and the size and shape of the outer arms in situ, we suggest that monomeric dynein is only one part of a larger, non-covalently joined molecular complex that forms the entire arm. When KCl-extracted axonemes are viewed by negative contrast but prior to fractionation of the dynein, individual arms can be seen that comprise three to four of the 93 Å subunits, thus suggesting that each arm is a multisubunit polymer of dynein or dynein-like molecules.     

Partial purification and characterization of dynein adenosine triphosphatase from bovine sperm

Outer dynein arm polypeptides that possess Mg+2-adenosine triphosphatase (ATPase) activity have been extracted from the flagellar axonemes of demembranated bovine sperm. Electron microscopy of intact and salt-extracted sperm demonstrates a relatively selective removal of the outer dynein arms. The salt extract contains a specific ATPase activity of 55 nmoles inorganic phosphate (Pi)/min/mg protein. Sucrose density gradient centrifugation of this extract results in a 6-fold increase in specific activity of ATPase (333 nmole/Pi/min/mg protein), which sediments as a single 13S peak. Concomitant with the increase in specific activity, there is enrichment of three high molecular weight polypeptides (Mr greater than 300,000) characteristic of dynein heavy chains. ATPase activities in the initial extract and in the 13S peak are inhibited by concentrations of vanadate and erythro-9-[3-2-(hydroxynonyl)]adenine similar to those that inhibit ATPase activity in sea urchin sperm dynein. These findings indicate that outer arm dynein ATPase can be extracted and partially purified from bovine sperm.     

Ultrastructure of the flagellar apparatus inPyramimonas octopus (Prasinophyceae)

Summary While green algae usually lack one of the outer dynein arms in the axoneme, flagella of the octoflagellated prasinophytePyramimonas octopus possess dynein arms on all peripheral doublets. The outer dynein arm on doublet no. 1 is modified, and additional structures are associated with doublets no. 2 and 6. The flagellar scales are asymmetrically arranged. Thus the two rows of thick flagellar hairscales are displaced towards doublet no. 6,i.e., in the direction of the effective stroke of each flagellum. The underlayer of small scales includes two nearly opposite double rows scales, arranged in the longitudinal direction of the flagellum. The hairscales emerge from these rows. The double rows are separated on one side by 9, on the other by 11 rows of helically arranged scales. The central pair of microtubules twists, but the axoneme itself (represented by the 9 peripheral doublets), does not seem to rotate. The flagella are arranged in two groups, showing modified 180° rotational symmetry. The effective strokes of the two central flagella are exactly opposite, while the other flagella beat in six intermediate directions.     

DC3, the 21-kDa subunit of the outer dynein arm-docking complex (ODA-DC), is a novel EF-hand protein important for assembly of both the outer arm and the ODA-DC

The outer dynein arm-docking complex (ODA-DC) is a microtubule-associated structure that targets the outer dynein arm to its binding site on the flagellar axoneme (Takada et al. 2002. Mol. Biol. Cell 13, 1015-1029). The ODA-DC of Chlamydomonas contains three proteins, referred to as DC1, DC2, and DC3. We here report the isolation and sequencing of genomic and full-length cDNA clones encoding DC3. The sequence predicts a 21,341 Da protein with four EF-hands that is a member of the CTER (calmodulin, troponin C, essential and regulatory myosin light chains) group and is most closely related to a predicted protein from Plasmodium. The DC3 gene, termed ODA14, is intronless. Chlamydomonas mutants that lack DC3 exhibit slow, jerky swimming because of loss of some but not all outer dynein arms. Some outer doublet microtubules without arms had a "partial" docking complex, indicating that DC1 and DC2 can assemble in the absence of DC3. In contrast, DC3 cannot assemble in the absence of DC1 or DC2. Transformation of a DC3-deletion strain with the wild-type DC3 gene rescued both the motility phenotype and the structural defect, whereas a mutated DC3 gene was incompetent to rescue. The results indicate that DC3 is important for both outer arm and ODA-DC assembly.     

Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity

Cilia/flagella are conserved organelles that generate fluid flow in eukaryotes. The bending motion of flagella requires concerted activity of dynein motors. Although it has been reported that the central pair apparatus (CP) and radial spokes (RSs) are important for flagellar motility, the molecular mechanism underlying CP- and RS-mediated dynein regulation has not been identified. In this paper, we identified nonspecific intermolecular collision between CP and RS as one of the regulatory mechanisms for flagellar motility. By combining cryoelectron tomography and motility analyses of Chlamydomonas reinhardtii flagella, we show that binding of streptavidin to RS heads paralyzed flagella. Moreover, the motility defect in a CP projection mutant could be rescued by the addition of exogenous protein tags on RS heads. Genetic experiments demonstrated that outer dynein arms are the major downstream effectors of CP- and RS-mediated regulation of flagellar motility. These results suggest that mechanosignaling between CP and RS regulates dynein activity in eukaryotic flagella.     

Structural and functional reconstitution of inner dynein arms in Chlamydomonas flagellar axonemes

The inner row of dynein arms contains three dynein subforms. Each is distinct in composition and location in flagellar axonemes. To begin investigating the specificity of inner dynein arm assembly, we assessed the capability of isolated inner arm dynein subforms to rebind to their appropriate positions on axonemal doublet microtubules by recombining them with either mutant or extracted axonemes missing some or all dyneins. Densitometry of Coomassie blue-stained polyacrylamide gels revealed that for each inner dynein arm subform, binding to axonemes was saturable and stoichiometric. Using structural markers of position and polarity, electron microscopy confirmed that subforms bound to the correct inner arm position. Inner arms did not bind to outer arm or inappropriate inner arm positions despite the availability of sites. These and previous observations implicate specialized tubulin isoforms or nontubulin proteins in designation of specific inner dynein arm binding sites. Further, microtubule sliding velocities were restored to dynein-depleted axonemes upon rebinding of the missing inner arm subtypes as evaluated by an ATP-induced microtubule sliding disintegration assay. Therefore, not only were the inner arm dynein subforms able to identify and bind to the correct location on doublet microtubules but they bound in a functionally active conformation.     

Functional analysis of an individual IFT protein: IFT46 is required for transport of outer dynein arms into flagella

Intraflagellar transport (IFT), which is the bidirectional movement of particles within flagella, is required for flagellar assembly. IFT particles are composed of approximately 16 proteins, which are organized into complexes A and B. We have cloned Chlamydomonas reinhardtii and mouse IFT46, and show that IFT46 is a highly conserved complex B protein in both organisms. A C. reinhardtii insertional mutant null for IFT46 has short, paralyzed flagella lacking dynein arms and with central pair defects. The mutant has greatly reduced levels of most complex B proteins, indicating that IFT46 is necessary for complex B stability. A partial suppressor mutation restores flagellar length to the ift46 mutant. IFT46 is still absent, but levels of the other IFT particle proteins are largely restored, indicating that complex B is stabilized in the suppressed strain. Axonemal ultrastructure is restored, except that the outer arms are still missing, although outer arm subunits are present in the cytoplasm. Thus, IFT46 is specifically required for transporting outer arms into the flagellum.     

Vanadate-sensitized cleavage of dynein heavy chains by 365-nm irradiation of demembranated sperm flagella and its effect on the flagellar motility

Irradiation of demembranated flagella of sea urchin sperm at 365 nm in the presence of 0.05-1 mM MgATP and 5-10 microM vanadate (Vi) cleaves the alpha and beta heavy chains of the outer arm dynein at the same site and at about the same rate as reported previously for the solubilized dynein (Gibbons, I. R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J. Y., and Gibbons, B. H. (1987) J. Biol. Chem. 262, 2780-2786). The decrease in intact alpha and beta heavy chain material is biphasic, with about 80% being lost with a half-time of 8-10 min, and the remainder more slowly. Five other axonemal polypeptides of Mr greater than 350,000 are lost similarly, concomitant with the appearance of at least 9 new peptides of Mr 150,000-250,000. The motility of irradiated sperm flagella upon subsequent dilution into reactivation medium containing 1 mM ATP and 2.5 mM catechol shows a progressive decrease in flagellar beat frequency for irradiation times that produce up to about 50% cleavage of the dynein heavy chains; more prolonged irradiation causes irreversible loss of motility. Competition between photocleaved and intact outer arm dynein for rebinding to dynein-depleted sperm flagella shows that cleavage has little effect upon the ability for rebinding, although the cleaved dynein partially inhibits subsequent motility. Substitution of MnATP for the MgATP in the irradiation medium prevents the loss of all of the axonemal polypeptides during irradiation for up to 60 min and also protects the potential for subsequent flagellar motility. It is concluded that loss of the five axonemal polypeptides upon irradiation results from a Vi-sensitized photocleavage similar to that which occurs in the alpha and beta heavy chains of outer arm dynein and that these polypeptides represent Vi-inhibitable ATPase subunits of dyneins located in the inner arms and possibly elsewhere in the flagellar axoneme.