Inner-arm Dynein Research Articles

鞭毛内腕ダイニン運動性のADPによるゆっくりとした活性化

鞭毛内腕ダイニン運動性のADPによるゆっくりとした活性化

138K中間鎖のリン酸化によるクラミドモナス両腕ダイニンfの構造変化

138K中間鎖のリン酸化によるクラミドモナス両腕ダイニンfの構造変化

Rescue of a Chlamydomonas inner-arm-dynein-deficient mutant by electroporation-mediated delivery of recombinant p28 light chain.

We have recently shown that rabbit actin can be introduced by electroporation into the Chlamydomonas ida5 mutant lacking conventional actin and rescue its mutant phenotype [Hayashi et al., 2001: Cell Motil. Cytoskeleton 49:146-153]. In this study, we explored the possibility of using electroporation for functional assay of a recombinant protein. The p28 light chain of inner-arm dyneins was expressed in Escherichia coli, purified to homogeneity, and introduced by electroporation into a non-motile mutant ida4oda6 that lacks it. Because this protein was insoluble in the low ionic strength solution used in the previous study, electroporation was performed at physiological ionic strength in the presence of Ca(2+). Most cells shed their flagella after electroporation. Reflagellation took place within 3 h and up to 30% of the cells became motile, indicating that the introduced p28 retained its functional activity. Fluorescently-labeled p28 was equally effective; in this case fluorescence was observed along the flagella. The presence of Ca(2+) and deflagellation appeared to be important for efficient protein delivery, because a triple mutant with the fa1 mutation deficient in the flagellar shedding mechanism recovered motility only very poorly. Similar results were obtained with other combinations of recombinant proteins and mutants. This study thus demonstrates the feasibility of using electroporation for activity assays of recombinant proteins.

Functional diversity of axonemal dyneins as studied in Chlamydomonas mutants

Cilia and flagella of most organisms are equipped with two kinds of motor protein complex, the inner and outer dynein arms. The two arms were previously thought to be similar to each other, but recent studies using Chlamydomonas mutants indicate that they differ significantly in subunit structure and arrangement within the axoneme. For example, whereas the outer dynein arm exists as a single protein complex containing three heavy chains, the inner dynein arm comprises seven different subspecies each containing one or two discrete heavy chains. Furthermore, the two kinds of arms appear to differ in function also. Most strikingly, our studies suggest that inner-arm dynein, but not outer-arm dynein, is under the control of the central pair microtubules and radial spokes. The axoneme thus appears to be equipped with two rather distinct systems for beating: one involving inner-arm dyneins, the central pair and radial spokes, and the other involving outer-arm dynein alone.

Mechanical Properties of a Single-Headed Processive Motor, Inner-Arm Dynein Subspecies-c of ChlamydomonasStudied at the Single Molecule Level.

Dynein from inner arms of Chlamydomonasflagella contains sevendistinct subspecies, a through g. Several lines of evidence suggest thesesubspecies play important roles in generating flagellar beating and thatthe different subspecies are functionally diverse. To evaluate theirroles and diversity, the mechanical properties of subspecies-c, which isa single-headed motor, were examined using optical trap nanometry. Apolystyrene bead coated with a small number of subspecies-c moleculeswas captured with the optical trap and brought into contact with amicrotubule fixed to a coverslip. Movements of the bead were measured bya quadrant photodiode sensor with sub-nanometer- and millisecond-resolution.Beads carrying a single active subspecies-c molecule moved processivelyalong the microtubules in 8-nm steps but slipped backwards under highloads. Force-velocity relationships of single subspecies-c molecules werealmost linear and the shapes of the normalized curves at 5 μM and 100μM ATP were similar. These results indicate that dynein subspecies-cfunctions in a very different way from conventional motor proteins, suchas myosin and kinesin, and has properties that could produceself-oscillation in vivo.

1N1600 138K中間鎖をリン酸化したクラミドモナス内腕ダイニンf(I1ダイニン)のin vitro運動アッセイ(12.細胞生物学的課題,一般演題,日本生物物理学会第40回年会)

1N1600 138K中間鎖をリン酸化したクラミドモナス内腕ダイニンf(I1ダイニン)のin vitro運動アッセイ(12.細胞生物学的課題,一般演題,日本生物物理学会第40回年会)

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.

Recovery of flagellar dynein function in a Chlamydomonas actin/dynein‐deficient mutant upon introduction of muscle actin by electroporation

Flagellar and ciliary inner-arm dyneins contain actin as a subunit; however, the function of this actin subunit remains unknown. As a first step toward experimental manipulation of actin in dynein, we developed a method for introducing exogenous actin into Chlamydomonas cells by electroporation. A non-motile mutant, ida5oda1, lacking inner-arm dyneins due to the absence of conventional actin, was electroporated in the presence of rabbit skeletal muscle actin. About 20% of the electroporated cells recovered motility under optimal conditions. In addition, by taking advantage of their phototactic behavior, the rescued cells could be concentrated. Motility was also recovered with fluorescently labeled actin; in this case, axonemes became fluorescent after electroporation, suggesting that actin was in fact incorporated as a dynein subunit. The feasibility of incorporating a substantial amount of macromolecules by electroporation will be useful not only for studying actin function, but also for a variety of studies using Chlamydomonas in which no efficient methods have been developed for expressing or introducing foreign proteins and other macromolecules.

3P154アデニンヌクレオチドに依存した鞭毛内腕ダイニンのモーター活性変化

3P154アデニンヌクレオチドに依存した鞭毛内腕ダイニンのモーター活性変化

A Novel-Type Processive Motor, Inner-Arm Dynein Subspecies c of Chlamydomonas Flagella.

Inner-arm dynein subspecies c (dynein c) of Chlamydomonas flagella is the single-headed dynein which is capable of generating force on microtubules. We recently found that dynein c is a processive motor: a single molecule of dynein c is required to move a microtubule more than 1 μm. However, the duty ratio of dynein c, i.e. the proportion of strongly attached to total ATPase cycle, was estimated to be ca. 14%. This feature is distinct from that of kinesin which is known to be a two-headed processive motor with the higher duty ratio.

Vigorous beating of Chlamydomonas axonemes lacking central pair/radial spoke structures in the presence of salts and organic compounds.

Flagella of Chlamydomonas mutants lacking the central pair of microtubules or radial spokes do not beat; however, axonemes isolated from these mutants were found to display vigorous bending movements in the presence of ATP and various salts, sugars, alcohols, and other organic compounds. For example, about 15% of the total axonemes isolated from pf18, a mutant lacking the central pair, displayed beating in the presence of 10 mM MgSO(4) and 0.2 mM ATP at about 22 Hz, while none beat with the same concentration of ATP and < or = 5 mM or > or = 25 mM MgSO(4). The beat frequency and waveform of beating pf18 axonemes were similar to those of wild type axonemes beating under the same conditions. Similarly, 10-50% of the axonemes beat in the presence of 0.5 M sucrose, 2.0 M glycerol, or 1.7 M[10% (v/v)] ethanol. The appearance of motility did not correlate with the change in axonemal ATPase; however, these substances at those concentrations commonly increased the amplitude of nanometer-scale oscillation (hyper-oscillation) in pf18 axonemes, as well as the extent of ATP-induced sliding disintegration of protease-treated axonemes. Axonemes of double mutants lacking both the central pair and various subspecies of inner-arm dynein also beat at increased MgSO(4) concentrations, but axonemes lacking outer-arm dynein in addition to the central pair did not beat. These and other observations suggest that small molecules perturb the regulation of microtubule sliding through some change in water activity or osmotic stress. Axonemes must have an intrinsic ability to beat without the central pair/radial spokes under a variety of non-physiological solution conditions, as long as the outer dynein arms are present. Apparently, the major function of the central pair/radial spoke structures is to restore this activity under physiological conditions.

ADP-dependent microtubule translocation by flagellar inner-arm dyneins.

Previous studies have shown that the motility of flagellar and ciliary axonemes in many organisms are influenced by the concentration of both ATP and ADP. Detergent-extracted cell models of Chlamydomonas oda1, a mutant lacking flagellar outer-arm dynein, displayed slightly lower flagellar beating frequencies when reactivated with ATP in the presence of an ATP-regenerating system, composed of creatine phosphate and creatine phosphokinase, than when reactivated with ATP alone. Thus, presence of a low concentration of ADP may somehow stimulate axonemal motility. To see if this motility stimulation is due to a direct effect on dynein, we analyzed the effect of ADP on the in vitro microtubule translocation caused by isolated inner-arm dyneins in the presence of ATP. Of the seven inner-arm dyneins (species a-g) fractionated by ion-exchange chromatography, most species translocated microtubules at faster speed in the presence of 0.1 mM ATP and 0.1 mM ADP than in the presence of 0.1 mM ATP alone. Most notably, species a and e did not translocate microtubules at all in the presence of the ATP-regenerating system, indicating that a trace amount of ADP is necessary for their motility. This regulation may be effected through binding of ADP to some of the four nucleotide binding sites in each dynein heavy chain.

Vigorous beating ofChlamydomonas axonemes lacking central pair/radial spoke structures in the presence of salts and organic compounds

Flagella of Chlamydomonas mutants lacking the central pair of microtubules or radial spokes do not beat; however, axonemes isolated from these mutants were found to display vigorous bending movements in the presence of ATP and various salts, sugars, alcohols, and other organic compounds. For example, about 15% of the total axonemes isolated from pf18, a mutant lacking the central pair, displayed beating in the presence of 10 mM MgSO(4) and 0.2 mM ATP at about 22 Hz, while none beat with the same concentration of ATP and < or = 5 mM or > or = 25 mM MgSO(4). The beat frequency and waveform of beating pf18 axonemes were similar to those of wild type axonemes beating under the same conditions. Similarly, 10-50% of the axonemes beat in the presence of 0.5 M sucrose, 2.0 M glycerol, or 1.7 M[10% (v/v)] ethanol. The appearance of motility did not correlate with the change in axonemal ATPase; however, these substances at those concentrations commonly increased the amplitude of nanometer-scale oscillation (hyper-oscillation) in pf18 axonemes, as well as the extent of ATP-induced sliding disintegration of protease-treated axonemes. Axonemes of double mutants lacking both the central pair and various subspecies of inner-arm dynein also beat at increased MgSO(4) concentrations, but axonemes lacking outer-arm dynein in addition to the central pair did not beat. These and other observations suggest that small molecules perturb the regulation of microtubule sliding through some change in water activity or osmotic stress. Axonemes must have an intrinsic ability to beat without the central pair/radial spokes under a variety of non-physiological solution conditions, as long as the outer dynein arms are present. Apparently, the major function of the central pair/radial spoke structures is to restore this activity under physiological conditions.

Regulation of outer-arm-dynein activity by phosphorylation and control of ciliary beat frequency

Ciliary beating is empowered by a mechanochemical enzyme, dynein, which appears as two rows of projections on doublet microtubules. While inner-arm dyneins modulate beat form, outer-arm dynein empowers ciliary beat and sets beat frequency. Beat frequency is controlled via phosphorylation of outer-arm dynein. UsingParamecium tetraurelia as model system, we have previously identified a regulatory light chain of outer-arm dynein (22S dynein), Mr29 (p29), whose phosphorylation is cAMP-dependent. The phosphorylation state of the p29 in 22 S dynein determines in vitro microtubule translocation velocity. Although in vitro phosphorylation of p29 takes place in a short time, the percent change ist significantly less than the percent change in dynein activation, or in ciliary beat frequency. A potential mechanism that explains how a few activated dyneins can change ciliary beating is discussed.

Inner-arm dynein c of Chlamydomonas flagella is a single-headed processive motor.

Axonemal dyneins are force-generating ATPases that produce movement of eukaryotic cilia and flagella. Several studies indicate that inner-arm dyneins mainly produce bending moments in flagella and that these motors have inherent oscillations in force and motility. Processive motors such as kinesins have high duty ratios of attached to total ATPase cycle (attached plus detached) times compared to sliding motors such as myosin. Here we provide evidence that subspecies-c, a single-headed axonemal inner-arm dynein, is processive but has a low duty ratio. Ultrastructurally it is similar to other dyneins, with a single globular head, long stem and a slender stalk that attaches to microtubules. In vitro studies of microtubules sliding over surfaces coated with subspecies-c at low densities (measured by single-molecule fluorescence) show that a single molecule is sufficient to move a microtubule more than 1 microm at 0.7 microm s(-1). When many motors interact the velocity is 5.1 microm s(-1), fitting a duty ratio of 0.14. Using optical trap nanometry, we show that beads carrying a single subspecies-c motor move processively along the microtubules in 8-nm steps but slip backwards under high loads. These results indicate that dynein subspecies-c functions in a very different way from conventional motor proteins, and has properties that could produce self-oscillation in vivo.

Assessment of inner dynein arm structure and possible function in ciliary and flagellar axonemes.

The construction and assessment of a three-dimensional computer-generated model of inner dynein arms on a 96-nm repeat unit of an axonemal doublet is described. The model is based on published electron micrographs of axonemes from Tetrahymena cilia and eel sperm, which were prepared using several different techniques: negative stain, freeze etch, and thin section. The inner arm structure is represented as three inner dynein arm complexes containing four inner dynein arms (IDAs), three dyads, and one single-headed arm, each capable of bridging the interdoublet gap. The IDA structures in the model have been correlated with the domains containing dynein heavy-chain isoforms mapped by several authors using genetic analyses of Chlamydomonas mutants. The model is consistent with micrographic evidence from axonemes of cilia and flagella from other organisms that led previously to conflicting structural interpretations. In this reconciling interpretation, the different alignments of the IDAs relative to the corresponding outer dynein arms observed in micrographs of differently prepared samples, result from the IDAs being arrested at different stages of their cycles of activity in each preparation. By interpolating between these positions of arrest, cycles of activity are proposed for each of the IDAs during which the arms attach to the neighbouring doublet microtubule and drive it tipwards.

Direct measurement of inter-doublet elasticity in flagellar axonemes.

The outer doublet microtubules in ciliary and flagellar axonemes are presumed to be connected with each other by elastic links called the inter-doublet links or the nexin links, but it is not known whether there actually are such elastic links. In this study, to detect the elasticity of the putative inter-doublet links, shear force was applied to Chlamydomonas axonemes with a fine glass needle and the longitudinal elasticity was determined from the deflection of the needle. Wild-type axonemes underwent a high-frequency, nanometer-scale vibration in the presence of ATP. When longitudinal shear force was applied, the average position of the needle tip attached to the axoneme moved linearly with the force applied, yielding an estimate of spring constant of 2.0 (S.D.: 0.8) pN/nm for 1 microm of axoneme. This value did not change in the presence of vanadate, i.e., when dynein does not form strong cross bridges. In contrast, it was at least five times larger when ATP was absent, i.e., when dynein forms strong cross bridges. The measured elasticity did not significantly differ in various mutant axonemes lacking the central-pair microtubules, a subset of inner-arm dynein, outer-arm dynein, or the radial spokes, although it was somewhat smaller in the latter two mutants. It was also observed that the shear displacement in an axoneme in the presence of ATP often took place in a stepwise manner. This suggests that the inter-doublet links can reversibly detach from and reattach to the outer doublets in a cooperative manner. This study thus provides the first direct measure of the elasticity of inter-doublet links and also demonstrates its dynamic nature.

Recovery of flagellar inner-arm dynein and the fertilization tubule in Chlamydomonas ida5 mutant by transformation with actin genes.

The ida5 mutant of Chlamydomonas, first isolated as a mutant lacking a subset of axonemal inner-arm dyneins, has recently been shown to lack conventional actin owing to a serious mutation in its gene. It lacks inner-arm dyneins probably because actin is an essential subunit for their assembly. In addition, male gametes of ida5 are unable to produce the fertilization tubule, a structure that contains a core of actin filament bundles. To establish that those observed deficiencies are solely attributable to the loss of actin, and to provide a basis for future studies on the actin function in this organism, we examined in this study whether transformation of this mutant with cloned actin genes can rescue the mutant phenotypes. Cotransformation of the double mutant ida5arg2 with the wild-type actin gene and arginino-succinate lyase gene that suppresses the arg2 mutation yielded several transformants that displayed increased motility. All of them were found to have acquired the introduced actin gene in the genome and the product actin in the flagella, and regained the missing inner-arm dyneins and wild-type motility. In addition, most transformants also became able to grow the fertilization tubule when mating reaction was induced. In addition to the wild-type actin gene, we also used a chimeric actin gene in which the N-terminal 12 amino-acid sequence of Chlamydomonas actin was replaced by that of the greatly divergent Tetrahymena actin. Transformants with this gene also resulted in recovery of inner-arm dynein and 70-80% of the wild-type level of motility. These results established that the lack of inner-arm dynein and the fertilization tubule in ida5 are consequences of its loss of conventional actin. Furthermore, they demonstrate that Chlamydomonas offers an excellent experimental system with which to study the structure-function relationship of actin by means of mutant analysis.

Chlamydomonas inner-arm dynein mutant, ida5, has a mutation in an actin-encoding gene.

Chlamydomonas flagellar inner-arm dynein consists of seven subspecies (a-g), of which all but f contain actin as subunits. The mutant ida5 and a new strain, ida5-t, lack four subspecies (a, c, d, and e). These mutants were found to have mutations in the conventional actin gene, such that its product is totally lost; ida5 has a single-base deletion that results in a stop codon at a position about two-thirds from the 5' end of the coding region, and ida5-t lacks a large portion of the entire actin gene. Two-dimensional gel electrophoresis patterns of the axonemes and inner-arm subspecies b and g of ida5 lacked the spot of actin (isoelectric point [pI] = approximately 5.3) but had two novel spots with pIs of approximately 5.6 and approximately 5.7 instead. Western blot with different kinds of anti-actin antibodies suggested that the proteins responsible for the two novel spots and conventional actin are different but share some antigenicity. Since Chlamydomonas has been shown to have only a single copy of the conventional actin gene, it is likely that the novel spots in ida5 and ida5-t originated from another gene(s) that codes for a novel actin-like protein(s) (NAP), which has hitherto been undetected in wild-type cells. These mutants retain the two inner-arm subspecies b and g, in addition to f, possibly because NAP can functionally substitute for the actin in these subspecies while they cannot in other subspecies. The net growth rate of ida5 and ida5-t cells did not differ from that of wild type, but the mating efficiency was greatly reduced. This defect was apparently caused by deficient growth of the fertilization tubule. These results suggest that NAP can carry out some, but not all, functions performed by conventional actin in the cytoplasm and raise the possibility that Chlamydomonas can live without ordinary actin.

Induction of temporary beating in paralyzed flagella of Chlamydomonas mutants by application of external force.

To help understand the mechanism by which the sliding movement of outer-doublet microtubules in cilia and flagella is converted into bending waves, we examined the effect of mechanical force imposed on the flagella of Chlamydomonas mutants lacking the central pair or multiple dyneins. These mutants were almost completely nonmotile under normal conditions. A bend was produced in a flagellum either by holding a cell with a micropipette and quickly moving it with a piezoelectric actuator; or by pushing a flagellum with a microneedle. After removal of the external force, mutants lacking the central pair (pf18 and pf19) displayed beating at irregular intervals of > 1 second for one to several cycles. Similarly, a double mutant (ida2ida4) lacking four species of inner-arm dynein displayed beating at intervals of > 0.1 second for up to 80 cycles. However, paralyzed flagella of double mutants that lack the outer dynein arm in addition to the central pair or the inner dynein arm did not show cyclical movements upon application of external force. These results indicate that the central pair and the inner dynein arm are important for both stable bend formation at the base and efficient bend propagation along the flagellar length. They also suggest that the outer dynein arm, and not the inner dynein arm, enables the flagellar axoneme to propagate bends independently of the central pair. We propose that the axoneme is equipped with two independent motor systems for oscillatory movements: an outer-arm system controlled by the axonemal mechanical state independently of the central pair/radial spoke system, and an inner-arm system controlled by both the axonemal mechanical state and the central pair/radial spokes.