Inner-arm Dynein Research Articles

Clockwise Translocation of Microtubules by Flagellar Inner-Arm Dyneins In Vitro

Cilia and flagella are equipped with multiple species of dyneins that have diverse motor properties. To assess the properties of various axonemal dyneins of Chlamydomonas, in vitro microtubule translocation by isolated dyneins was examined with and without flow of the medium. With one inner-arm dynein species, dynein c, most microtubules became aligned parallel to the flow and translocated downstream after the onset of flow. When the flow was stopped, the gliding direction was gradually randomized. In contrast, with inner-arm dyneins d and g, microtubules tended to translocate at a shallow right angle to the flow. When the flow was stopped, each microtubule turned to the right, making a curved track. The clockwise translocation was not accompanied by lateral displacement, indicating that these dyneins generate torque that bends the microtubule. The torque generated by these dyneins in the axoneme may modulate the relative orientation between adjacent doublet microtubules and lead to more efficient functioning of total dyneins.

2P-193 ストークコイルドコイルが内腕ダイニンによるトルクの発生を引き起こす(分子モーター(2),第46回日本生物物理学会年会)

2P-193 ストークコイルドコイルが内腕ダイニンによるトルクの発生を引き起こす(分子モーター(2),第46回日本生物物理学会年会)

2P-194 クラミドモナス鞭毛の根元に局在する新規内腕ダイニンの同定(分子モーター(2),第46回日本生物物理学会年会)

2P-194 クラミドモナス鞭毛の根元に局在する新規内腕ダイニンの同定(分子モーター(2),第46回日本生物物理学会年会)

Novel 44-Kilodalton Subunit of Axonemal Dynein Conserved from Chlamydomonas to Mammals

Cilia and flagella have multiple dyneins in their inner and outer arms. Chlamydomonas inner-arm dynein contains at least seven major subspecies (dynein a to dynein g), of which all but dynein f (also called dynein I1) are the single-headed type that are composed of a single heavy chain, actin, and either centrin or a 28-kDa protein (p28). Dynein d was found to associate with two additional proteins of 38 kDa (p38) and 44 kDa (p44). Following the characterization of the p38 protein (R. Yamamoto, H. A. Yanagisawa, T. Yagi, and R. Kamiya, FEBS Lett. 580:6357-6360, 2006), we have identified p44 as a novel component of dynein d by using an immunoprecipitation approach. p44 is present along the length of the axonemes and is diminished, but not absent, in the ida4 and ida5 mutants, both lacking this dynein. In the ida5 axoneme, p44 and p38 appear to form a complex, suggesting that they constitute the docking site of dynein d on the outer doublet. p44 has potential homologues in other ciliated organisms. For example, the mouse homologue of p44, NYD-SP14, was found to be strongly expressed in tissues with motile cilia and flagella. These results suggest that inner-arm dynein d and its subunit organization are widely conserved.

Disquinesia ciliar: diagnóstico ultraestructural, evolución clínica y alternativas de tratamiento

Ciliary dyskinesia (CD) is a low incidence genetic illness, that presents with a wide clinical spectrum. Also, there are transitory conditions that present with ciliary anomalies, secondary to infectious diseases of the airways. To descube clinical and ultrastructural findings and clinical and therapeutic evolution of these patients. Retrospective review of medical records and electron microscopy findings of 33 patients (aged 1 to 21 years, 14 females) with ultrastructural diagnosis of CD. To obtain follow up information, a telephone survey was done. In 30 patients (90%) the inner dynein arm (IDA) was absent in 50 or more percent of the cilia. Twenty two (66%) had absence of the outer dynein arm. Before diagnosis of CD, 19 patients (57%) presented recurrent otitis media, 25 patients (77%), three or more episodes of rhinosinusitis and 18 patients (56%) had recurrent pneumonia. Middle ear ventilation tubes were placed in 19 patients (57%), and during its use, 12 (68%) remained without othorrea. Sixteen patients (48%) with recurrent episodes of rhinosinusitis required adenoidectomy Seven (21%) required a functional endoscopic sinus surgery (FESS), and 6 (86%) improved after FESS. Our patients with CD presented recurrent infections in different airway locations. In those with a diagnosis of CD and recurrent otológica! and rhinosinusal infections, IDA was absent in a high percentage of cilia. FESS and the use of ventilation tubes may have a beneficial role in a subgroup of patients with CD.

Dyneins Across Eukaryotes: A Comparative Genomic Analysis

Dyneins are large minus-end-directed microtubule motors. Each dynein contains at least one dynein heavy chain (DHC) and a variable number of intermediate chains (IC), light intermediate chains (LIC) and light chains (LC). Here, we used genome sequence data from 24 diverse eukaryotes to assess the distribution of DHCs, ICs, LICs and LCs across Eukaryota. Phylogenetic inference identified nine DHC families (two cytoplasmic and seven axonemal) and six IC families (one cytoplasmic). We confirm that dyneins have been lost from higher plants and show that this is most likely because of a single loss of cytoplasmic dynein 1 from the ancestor of Rhodophyta and Viridiplantae, followed by lineage-specific losses of other families. Independent losses in Entamoeba mean that at least three extant eukaryotic lineages are entirely devoid of dyneins. Cytoplasmic dynein 2 is associated with intraflagellar transport (IFT), but in two chromalveolate organisms, we find an IFT footprint without the retrograde motor. The distribution of one family of outer-arm dyneins accounts for 2-headed or 3-headed outer-arm ultrastructures observed in different organisms. One diatom species builds motile axonemes without any inner-arm dyneins (IAD), and the unexpected conservation of IAD I1 in non-flagellate algae and LC8 (DYNLL1/2) in all lineages reveals a surprising fluidity to dynein function.

2P174 クラミドモナス鞭毛における3種類の新規内腕ダイニン重鎖の同定(分子モーター,ポスター発表,第45回日本生物物理学会年会)

2P174 クラミドモナス鞭毛における3種類の新規内腕ダイニン重鎖の同定(分子モーター,ポスター発表,第45回日本生物物理学会年会)

2P173 クラミドモナス ダイニンI1(ダイニンf)2個の重鎖は異なる特性を持つ(分子モーター,ポスター発表,第45回日本生物物理学会年会)

2P173 クラミドモナス ダイニンI1(ダイニンf)2個の重鎖は異なる特性を持つ(分子モーター,ポスター発表,第45回日本生物物理学会年会)

2P176 溶液潅流による外力存在下でのクラミドモナス軸糸内腕ダイニンによる微小管滑走速度の変化(分子モーター,ポスター発表,第45回日本生物物理学会年会)

2P176 溶液潅流による外力存在下でのクラミドモナス軸糸内腕ダイニンによる微小管滑走速度の変化(分子モーター,ポスター発表,第45回日本生物物理学会年会)

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.

Functional binding of inner-arm dyneins with demembranated flagella ofChlamydomonas mutants

Experiments were carried out to see if isolated inner arm dyneins could functionally combine with axonemes lacking them. High-salt extract from the axoneme of Chlamydomonas oda1 mutant lacking outer-arm dynein was added to the demembranated cell models of ida1oda1 lacking inner arm dynein f (dynein I1) and outer arm dynein. After incubation, the originally paralyzed ida1oda1 axonemes recovered the ability to beat in the presence of ATP. A similar good motility recovery after incubation with crude oda1 extract was observed in ida9oda2 lacking outer arm and inner arm dynein c, and partial recovery in ida4oda1 lacking outer arm and inner arm species a, c, and d. These observations indicate that dynein f and dynein c can functionally bind with mutant axonemes lacking them. A method for combining isolated inner arm dyneins with axonemes in a functionally active manner should provide a powerful experimental tool with which to study the mechanism of beating.

2P199 Conformational Changes of Inner-Arm Dynein f from Chlamydomonas Coupled with Phosphorylation of Its Intermediated Chain IC138. III(37. Molecular motor (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

2P199 Conformational Changes of Inner-Arm Dynein f from Chlamydomonas Coupled with Phosphorylation of Its Intermediated Chain IC138. III(37. Molecular motor (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

1P283 Identification of two novel inner-arm dynein heavy chains in Chlamydomonas flagella(9. Molecular motor (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

1P283 Identification of two novel inner-arm dynein heavy chains in Chlamydomonas flagella(9. Molecular motor (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Phototactic activity inChlamydomonas'non-phototactic' mutants deficient in Ca2+-dependent control of flagellar dominance or in inner-arm dynein

In the mechanism underlying the phototactic behavior of Chlamydomonas, Ca(2+) has been thought to control the dominance between the two flagella so as to steer the cell to correct directions. A newly isolated mutant, lsp1, that displays weak phototaxis was found to be defective in this Ca(2+)-dependent shift in flagellar dominance; in demembranated and reactivated cell models, the trans flagellum (the flagellum farthest from the eyespot) beat more strongly than the other (the cis flagellum) in about half of the cells regardless of the Ca(2+) concentration between <10(-9) M and 10(-6) M, a range over which wild-type cell models display switching of flagellar dominance. This is unexpected because ptx1, another mutant that is also deficient in flagellar dominance control, has been reported to lack phototactic ability. We therefore re-examined ptx1 and another reportedly non-phototactic mutant, ida1, which lacks inner arm dynein subspecies f (also called I1). Both were found to retain reduced phototactic abilities. These results indicate that both Ca(2+)-dependent flagellar dominance control and inner-arm dynein subspecies f are important for phototaxis, but are not absolutely necessary. Analysis of the flagellar beat frequency in lsp1 cell models showed that both of the flagella beat at the frequency of the cis flagellum in wild type. In addition, lsp1 and ptx1 were found to be deficient in determining the sign of phototactic migration. Hence, the Ca(2+)-dependent flagellar dominance control detected in demembranated cells might be involved in the determination of the sign of phototaxis. The gene responsible for the lsp1 mutation was identified by phenotype rescue experiments and found to have sequences for phosphorylation.

Mutations in genes encoding inner arm dynein heavy chains inTetrahymena thermophila lead to axonemal hypersensitivity to Ca2+

Calcium-dependent ciliary reversals are seen in ciliated protozoans such as Tetrahymena in response to depolarizing stimuli, but the axonemal mechanisms responsible for this response are not well understood. The model is that the outer arm dyneins (OADs) control the beating frequency while the inner arm dyneins (IADs) regulate ciliary waveform. Since ciliary reversal is a type of waveform change, the model would predict that IAD mutations could affect ciliary reversal. We have used gene disruption techniques to generate several behavioral mutants of Tetrahymena with functional disruptions of various IADs. One such mutant, called KO-6, is missing I1 (the two-headed IAD) and is unable to show ciliary reversals in response to any stimuli due to a loss of axonemal Ca2+ sensitivity [Eur J Cell Biol 80 (2001) 486-497; Cell Motil Cytoskeleton 53 (2002) 281-288.]. In contrast, disruption of 3 one-headed IADs [Liu et al., Cell Motil Cytoskeleton 59 (2004), 201-214] produced mutants, which showed over-responsiveness in bioassays measuring either their depolarization-induced avoiding reactions (AR) in Na+ and Ba2+ solutions or their duration of backward swimming (continuous ciliary reversal or CCR) in K+ solutions. Detergent-extracted and reactivated mutants also showed increased probabilities of CCR at lower Ca2+ concentrations suggesting that the behavioral over-responsiveness of these three mutants in vivo is due to increased axonemal Ca2+ sensitivity. Our data suggest the possibility that the one-headed IADs and the two-headed IAD act antagonistically in vivo and that loss of any one of the one-headed IADs leads to behavioral over-responsiveness due to less resistance to I1-induced reversals.

3P190 138K中間鎖のリン酸化によるクラミドモナス内腕ダイニンfの構造変化Il(分子モーター))

3P190 138K中間鎖のリン酸化によるクラミドモナス内腕ダイニンfの構造変化Il(分子モーター))

Slow ADP-dependent acceleration of microtubule translocation produced by an axonemal dynein

Dynein has four nucleotide binding sites, of which the functional significance is unknown except for the single catalytic site. To obtain clues to the function of non-catalytic nucleotide binding, we examined the effect of ADP on the in vitro motility of Chlamydomonas inner-arm dynein species ‘a’. Upon continuous perfusion with ATP and ADP, microtubules glided on a dynein-coated glass surface with a velocity that gradually increased over a few minutes. The velocity increased faster at higher ADP concentrations. These results suggest that this dynein is activated by nucleotide binding to regulatory site(s) through an extremely slow process.

A Tektin Homologue Is Decreased inChlamydomonasMutants Lacking an Axonemal Inner-Arm Dynein

In ciliary and flagellar axonemes, various discrete structures such as inner and outer dynein arms are regularly arranged on the outer doublet microtubules. Little is known about the basis for their regular arrangement. In this study, proteins involved in the attachment of inner-arm dyneins were searched by a microtubule overlay assay on Chlamydomonas mutant axonemes. A 58-kDa protein (p58) was found approximately 80% diminished in the mutants ida6 and pf3, both lacking one (species e) of the seven inner-arm species (a-g). Analysis of its cDNA indicated that p58 is homologous to tektin, a protein that was originally found in sea urchin and thought to be crucial for the longitudinal periodicity of the doublet microtubule. Unlike sea urchin tektin, which is a component of protofilament ribbons that occur after Sarkosyl treatment of axonemes, p58 was not contained in similar Sarkosyl-resistant ribbons from Chlamydomonas axonemes. Immunofluorescence microscopy showed that p58 was localized uniformly along the axoneme and on the basal body. The p58 signal was reduced in ida6 and pf3. These results suggest that a reduced amount of p58 is sufficient for the production of outer doublets, whereas an additional amount of it is involved in inner-arm dynein attachment.

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.

3P198 クラミドモナス鞭毛内腕ダイニンcの微小管の負電荷領域から受ける影響および微小管に沿った一次元ブラウン運動(分子モーター)

3P198 クラミドモナス鞭毛内腕ダイニンcの微小管の負電荷領域から受ける影響および微小管に沿った一次元ブラウン運動(分子モーター)