Flagellar rhythmic beating is driven by sliding movements between microtubules and axonemal dyneins. Axonemal dynein is a molecular motor and is classified into inner-arm dynein and outer-arm dynein according to their positions in the axoneme. In the green alga Chlamydomonas, the outer-arm dynein comprises a single molecular assembly containing three dynein heavy chains (α,β, and γ DHCs), while the inner-arm dynein comprises seven major and three minor subspecies each containing one or two distinct DHCs. The N-terminal portion of each DHC is called the stem (or tail), and is bound with the intermediate chains (ICs) and most of the light chains (LCs). The proximal region of the stem is the site where the DHCs attach to the A-tubule of the outer doublet microtubule. The C-terminal portion of each DHC consists of six AAA+ domains and a microtubule-binding stalk, and the general organization of this region is conserved in all dynein species. This portion forms a globular head and presents the motor activity.

Previous studies using Chlamydomonas have shown that the outer dynein arm and the inner dynein arm differ in functions and structures. Studies using mutants deficient in the outer-arm and inner-arm dyneins have shown that the outer-arm dynein affects the beat frequency, while the inner-arm dynein has an effect on the amplified waveform. The outer-arm dynein presents simpler species and could be isolated and purified more easily than the inner-arm dynein. Previous studies have been shown that different outer-arm DHCs display strikingly different in vitro motility. It is an interesting question that how these distinct dyneins assemble and coordinate to produce the axonemal beating.

The functional property of each dynein could be a key to understand the mechanism of flagellar beating movement. To achieve a better understanding of the function of each species of dynein, I aimed at isolating novel mutants lacking specific dyneins and analyzing their properties. In part I, I described screening of slow-swimming mutants, and seven novel motility-deficient mutants were isolated successfully. Two mutants, #24 and #34, are deficient in the tubulin polyglutamylation, and would facilitate studies to understanding how tubulin polyglutamylation affects flagellar motility in Chlamydomonas. Another outer-arm mutant, #5 that lacks the motor domain of γ DHC but retains the α and β DHCs, is thought to be valuable for studies on the property and function of the γ DHC. All of seven motility-deficient mutants are expected to yield more important information about the mechanism of axonemal beating.

In part II, I characterized the outer-arm mutant #5 and analyzed functional properties of outer-arm DHCs. The mutant #5 was named oda2-t, because it is an allele of oda2 having a structure mutation in γDHC and lacking the entire outer dynein arm, and presents a truncated γ DHC. This type of mutant was isolated for the first time and has been awaited since oda11 lacking the γ DHC and oda4-s7 lacking γ motor domain were isolated. The mutant oda11 swims slower than wild type, but faster than mutants missing the entire outer arm, such as oda2 and oda4. Thus outer-arm dynein containing only the α and β DHCs can function in the axoneme, and the γ DHC apparently increases the activity of the β or γ DHCs. The mutant oda4-s7, expressing only the N-terminal 160-kDa region of the γ DHC and lacking its motor domain, assembles outer arms with the α and β DHCs, whereas the mutant oda4 deficient in the βDHC gene lacks the entire outer arm. In contrast to oda11, oda4-s7 swims at almost the same speed as oda4. Thus, the γ DHC motor domain appears to be essential for the function of the outer-arm dynein, and outer-arm dyneins containing only the α and β DHCs are almost completely non-functional. The question arises to how significantly the γ DHC contributes to the overall outer-arm function.

Western-blot analysis indicated that oda2-t lacks the γ motor domain, but retains the stem portion of the γ DHC. The structure of the γ DHC in oda2-t was determined by RT-PCR analysis. The result showed that theγ DHC in oda2-t has only 1623 amino acids, containing a 1270 amino-acid sequence from the N-terminal region of the γ DHC and a 315 amino-acid sequence from the C-terminal region of the NIT1 protein, connected through a 38 amino-acid adapter. The molecular mass of the truncated γ DHC in oda2-t calculated from the determined sequence is 185379 Da, including 146931 Da of the N-terminal γ DHC sequence. The difference between oda2 and oda2-t suggests that the stem portion of the γ DHC is essential for the stable assembly of outer dynein arm. A similar difference has been observed between oda4-s7 and oda4, which has shown that the N-terminal portion of the γ DHC is important for stable assembly of the outer arm.

The Chlamydomonas outer-arm dynein is composed of three (α, β and γ) DHCs, two intermediate chains (IC1, IC2) and eleven light chains (LCs). I carried out fractionation of dynein by ion-exchange chromatograpy (HPLC) to examine the outer-arm dynein composition of oda2 t. Analyses of SDS-PAGE and Western showed that oda2 t lacked the γ motor domain and LC1, a light chain that was known to be associated with the γ motor domain, but retained the other compositions of outer-arm dynein. The results also indicated that the truncated γ DHC remains stably associated with the γ dimer, while the LC4, associated with the stem of the γ DHC, is stably associated with the truncated γ DHC in the oda2-t axoneme.

The location of outer-arm DHCs in cross section has been determined using oda11 and oda4-s7. Averaged outer-arm images in cross-section micrographs of the mutant axonemes located the γ DHC at the outer-arm tip, and the γ motor domain at an intermediate position between the base and tip. From the images of the double mutant oda11oda4-s7, the γ motor domain was predicted to localize to the inner lobe of the outer-arm image. In this study, outer-arm images of oda2-t in cross-section electron micrographs were classified and averaged using an image clustering protocol, and analyzed by Student t-test. The result showed that a major density difference between oda2-t and wild type was in the inner lobe of the outer arm. It is consistent with previous prediction, and suggests that the motor domain of the γ DHC is located in this region. In addition to the change in the inner lobe, a wedge-shaped area of density was observed on the outer side of the entire outer arm, as well as the tip area observed in oda4-s7. This difference could be an alteration in orientation of the total arm, which might be caused by the loss of the proximal structure. The mutant oda2-t appears to have a slightly impaired ability to assemble outer dynein arms on the outer doublets (average outer arms per nine outer doublets: oda2-t, 6.7; wild type, 7.9), and a similar reduction in the outer-arm number has also been observed in oda4-s7.

To study the function of the γ motor domain, I compared the swimming velocity and beat frequency in wild type, oda2 (lacking the α, β and γ DHCs), oda11 (lacking the α DHC), oda4-s7 (lacking the β motor domain) and oda2-t (lacking the γ motor domain). The average velocity (standard deviation) for each strain (μm/s) was wild type, 161.6 (9.2); oda11, 120.4 (13.0); oda4-s7, 59.7 (7.4); oda2-t, 87.8 (9.4); oda2, 50.6 (6.6), and the beat frequency was: wild type, 69Hz; oda11, 56Hz; oda4-s7, 34Hz; oda2-t, 50Hz; oda2, 30Hz. These results suggested that the outer-arm dynein lacking the γ motor domain can function to some extent. Previous studies on the outer-arm structure, as well as this study, demonstrated that the γ motor domain is located very close to the microtubule, and may be critically important for the overall structure of the outer arm. Surprisingly, motility analyses indicated the outer arm retains partial function even without the γ motor doamin. From a phenomenological point of view, the β DHC is the most important power generator among the three DHCs of the outer arm.

Chlamydomonas flagella display Ca(2+)-dependent waveform conversion. The axonemes beat in an asymmetrical pattern at Ca(2+) concentrations lower than 10-6M, while they beat in a symmetrical pattern at Ca(2+) concentrations higher than 10-5M. Conversion to the symmetrical pattern is observed in live cells when displaying transient backward swimming upon illumination with intense light. Previous studies have shown mutants lacking the outer-arm dynein did not display the light-induced back swimming. To examine function of three DHCs at high Ca(2+) concentration, I analyzed motilities of reactivated wiled-type and mutant axonemes under 10-4M and 10-8M Ca(2+) conditions. At 10-8M Ca(2+), the average beat frequency (standard deviation) for each strain (Hz) was wild type,75.8 (11.7); oda11, 49.4 (5.4); oda4-s7, 38.4 (3.3); oda2-t, 47.1 (8.7); oda2, 31.0 (3.4). These frequencies are close to the flagellar native frequency. At high Ca(2+) concentration, only <10% of oda2 axonemes beat, and displayed moving straight with a symmetrical waveform of very small amplitude. The average beat frequency (standard deviation) for each strain (Hz) was wild type, 91.7 (14.1); oda11, 31.5 (6.9); oda4-s7, 42.6 (8.1); oda2-t, 19.6 (7.7); oda2, 15.9 (3.8), and the averaged velocity (standard deviation) for each strain (μm/s) was: wild type, 115.1 (24.2); oda11, 9.4 (2.3); oda4-s7, 20.8 (8.6); oda2-t, 10.2 (6.2); oda2, 3.5 (1.4). Axonemes of oda2 beat at 1/6 the wild-type frequency, and the beating was so ineffective that it did not result in efficient propulsion of the axoneme, only moving at very small speed. The motility of oda2-t axonemes was a little higher than that of oda2, but much lower than that of oda4-s7 axonemes. These results suggest that the outer-arm dynein lacking the γ motor domain appears to lose most of the motility at high Ca(2+) concentration. Since LC4, a Ca(2+)-binding light chain, has been suggested to play a role in the Ca(2+)-dependent waveform conversion, I speculate that LC4 possibly controls waveforms through theγ DHC to which it binds, while the γ DHC produces the main power in the reactivated axonemes at high Ca(2+) concentration.

The ATPase activities of the wild-type and mutant axonemes were measured in the presence of 1mM ATP in HMDEK at 25°C. Reactivated axonemes displayed beating for longer than 10 minutes, and checked that the phosphate liberation was linear with time for the initial 5 minutes. The average ATPase activity (standard deviation) for each strain (μmol phosphate /minute /mg axonemes) was wild type, 1.59 (0.08); oda11, 1.31 (0.10); oda4-s7, 0.60 (0.06); oda2-t, 2.03 (0.29); oda2, 0.35 (0.04). The activity was very low in oda2 axonemes that lack the entire outer arm, suggesting that more than 75% of the ATPase activity in wild-type axonemes can be accounted for by the activity of outer-arm dynein. The ATPase activity of oda2-t axonemes was strikingly higher than that of wild type, indicating that theγ DHC suppresses the ATPase activity of the γ dimer in situ. In addition, the ATPase activity of oda4-s7 was markedly lower than those of oda11 and wild type, suggesting the importance of the γ DHC for the high ATPase activity in the axoneme. The observation that oda11 and oda2-t axonemes have high ATPase activities yet display much poorer motility than wild type suggests that these axonemes have defects in mechanochemical coupling, which is likely to be regulated through interactions between the different DHCs or their associated components. The ATPase activities measured at high Ca(2+) concentration showed the same results, and it showed that the ATPase activity of axonemes are not affected by Ca(2+) concentration.

The mutant oda2-t lacking the γ motor domain, as well as oda11 and oda4-s7, implies that outer-arm dyneins with any desired combinations of the three DHCs could be obtained by using these mutants and the double mutants between them. Thus this mutant should greatly advance studies on the structure and functional property of each DHC in outer-arm dynein.

真核生物の鞭毛・繊毛運動は周辺微小管上に配列したタイニン外腕と内腕が微小管間で滑り力を発生することによって発生する。これらのタイニン腕には複数種の巨大な力発生タンパク質、重鎖、が含まれているが、それぞれどのような特性を持ち、どのように協調して働いているかは明らかではない。本研究はそのうちタイニン外腕の各重鎖の機能を、新たに単離したクラミドモナスの変異株を用いて追及したものである。本論文は2部からなり、第1部では、タイニンに異常のある変異株の単離の試みが簡略に述べられ、第2部では第1部の研究で新たに単離された、タイニン外腕γ重鎖だけを欠失した変異株の詳細な解析結果が記されている。

第1部の研究では、突然変異誘発処理を行った約5000株のクローンから、運動性の悪い変異株26株を単離し、7株の新規な変異株を得た。遺伝解析と軸糸タンパク質解析の結果、そのうちの一つoda2-tが、タイニン外腕の3つの重鎖(aβγ)のうち、γ鎖だけを失った変異株であることが見いたされた。これまでα、βの各重鎖を失った変異株は得られていたが、γだけを失ったものは得られていなかった。oda2-tが得られたことにより、外腕3重鎖のすべてについて欠失変異株が揃ったことになる。このことは、タイニン外腕の構造と機能の研究にとってきわめて重要である。

第2部の研究では、まず、oda2-t変異株の遺伝子の解析から、この株がγ重鎖遺伝子全長4486アミノ酸残基のうち、N末の1270残基だけを発現していることを示した。タイニン重鎖のN末側領域は一般に尾部と呼ばれ、軸糸タイニンでは周辺微小管のA小管との結合部位であることが知られている。7重鎖すべてを失った株oda2がタイニン外腕すべてを欠失しているのに対し、oda2-tはα、β鎖と頭部を欠失したγ鎖からなる外腕を持つ。このことは、タイニン重鎖のN末部分が外腕全体の軸糸中での輸送と構築に必要かつ十分であることを示すものである。軸糸タンパク質組成の解析から、oda2-tの外腕では、7重鎖の頭部に結合することが知られている軽鎖LC1は欠失しているが、尾部に結合するLC4は存在することが確認された。また電子顕微鏡像の解析から、タイニン外腕中における7重鎖は、外腕全体の基部付近に局在することが明らかになった。これまで、α、β重鎖欠失変異株の外腕像からγ重鎖の局在が推測されていたが、この変異株の解析はそれを裏付ける結果である。

本研究の最も重要な知見は、外腕3重鎖のそれぞれを欠失した変異株の運動性の比較により、各重鎖の機能が大きく異なることがあきらかになったことである。これまで、α重鎖を失った外腕は一定程度の機能を持つが、β重鎖を失った外腕はほとんど機能できず、それを欠失した変異株は外腕全部を失った変異株とほぼ同じ速度で泳ぐことが知られていた。 γ重鎖が失われた場合については未知数であったが、今回、oda2-t株の遊泳速度はタイニン外腕すべてを欠失した変異株oda2より1.5倍速いことが判明した。すなわち、外腕はγ重鎖を欠失してもある程度機能できるものと結論される。これまでγ重鎖は外腕の機能に必須であると考えられていたので、この結果は意外である。一方、クラミドモナス軸糸はカルシウムイオン濃度が高い条件では対称型の波形による運動を行うことが知られているが、oda2-tはその運動を行うことが困難であることが判明した。このことは。高カルシウム条件におけるタイニン外腕の機能発現には、7重鎖の存在が必要であることを示唆している。カルシウム結合性の軽鎖LCを結合していることと関連があると考えられる。また、一方軸糸のATPaseを比較すると、oda2-t軸糸はカルシウム濃度にかかわらず、野生株軸糸の1.3倍程度の高い値を示した。すなわち、7重鎖は外腕中でその運動性を高めつつ、全体のATP消費を抑える機能を持つものと考えられる。そのような知見が得られたのは、本研究が最初である。

以上のように、本論文で述べられている結果はタイニン外腕の各重鎖が果たす役割に関する重要な新知見を多く含み、今後のタイニンと鞭毛運動機構の研究にとって重要な意味を持つと考えられる。なお、本論文は九州工業大学高崎寛子氏、安永卓生氏、京都大学八木俊樹氏、Connecticut大学Miho Sakato氏、Steve King氏、東京大学中澤友紀氏、神谷 律氏との共同研究であるが、論文提出者が主体となって実験および解析を行ったもので、論文提出者の寄与が十分であると判断する。

したがって、博士(理学)の学位を授与できると認める。