Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins

Coupling of Rotation and Catalysis in F1-ATPase Revealed by Single-Molecule Imaging and Manipulation

SummaryF1-ATPase is a rotary molecular motor that proceeds in 120° steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F1 that phosphate release drives the last 40° of the 120° step, and that the 40° rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F1 is forced to rotate slowly, that release of Cy3-ADP occurs at ∼240° after it is bound as Cy3-ATP at 0°. This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete

コウイキ ジュウガイ ボウシ サク ノ カイコウブ ガ ニホンジカ シンニュウ ボウシ ニ オヨボス エイキョウ

広域で設置される獣害防止柵では道路や河川などと交わる場所に柵のない開口部が生じる。本研究ではニホンジカの高密度生息地域である神奈川県丹沢山系に設置された広域獣害防止柵(52km)を対象に,開口部の存在がシカ侵入防止に及ぼす影響について調査した。開口部は2.0箇所/kmの割合で存在しており,道路と河川による開口部が多くほぼ同数(各0.8箇所/km)を占めた。開口部のシカ通過頻度は,開口部幅3.0m以下で有意に低くなった。広域柵には倒木や野生動物の働きによって生じた破損開口部も存在したが,野生動物由来の破損部を通過するシカは少なかった。柵の山側から農地に侵入してくるシカは,開口部1箇所あたり0.24頭/日と推定された。山から里への移動は日没後に,里から山への移動は早朝に活発となった。すなわち,農地への侵入は夜間に行われており,侵入したシカは柵から最遠2kmの農地にまで侵入していた。しかし日没後と早朝に開口部を逆方向に通過する個体も少なからずみられ,それぞれ前者の37%および30%を占めた。これは昼間に人里側のヤブに潜む個体と考えられた。獣害防止のためには人里のヤブ刈り払いが重要といえる。Regional wildlife control fences are often interrupted by roads and streams. To investigate the negative impacts of these openings for efficiency of fences, a sensor camera survey was conducted at a sika deer fence that was constructed along the foot of the Tanzawa Mountains in Kanagawa Prefecture over 52km. Openings were found at the ratio of about 2.0 per kilometer. The frequency of gaps by road and human trails was 0.8/km, and 0.8/km by streams. Passing frequency of the deer became low at narrow openings (<3 m). Although there were holes that were created by mid-size animals, the passing frequency of the deer for those holes was low. Daily invading frequency of the deer from mountainside to farmland through the fence was estimated to be 0.24 deer/km, indicating the effectiveness of the fence. During the nighttime, the deer invaded farmlands over a distance of two km from the edge of the mountain woodland. The deer indicated a bimodal nocturnal activity pattern. Many of them passed through gaps from mountain side to farmland around 17 : 00-20 : 00, and returned to the mountain side around 4 : 00-6 : 00. However, about one-third of the deer tended to move reversely. As the latter deer seem to be hiding in the bushes at farmland side during daytime, clearing of those bushes is necessary for removing such deer

Asymmetry of inner dynein arms and inter-doublet links in Chlamydomonas flagella

Although the widely shared “9 + 2” structure of axonemes is thought to be highly symmetrical, axonemes show asymmetrical bending during planar and conical motion. In this study, using electron cryotomography and single particle averaging, we demonstrate an asymmetrical molecular arrangement of proteins binding to the nine microtubule doublets in Chlamydomonas reinhardtii flagella. The eight inner arm dynein heavy chains regulate and determine flagellar waveform. Among these, one heavy chain (dynein c) is missing on one microtubule doublet (this doublet also lacks the outer dynein arm), and another dynein heavy chain (dynein b or g) is missing on the adjacent doublet. Some dynein heavy chains either show an abnormal conformation or were replaced by other proteins, possibly minor dyneins. In addition to nexin, there are two additional linkages between specific pairs of doublets. Interestingly, all these exceptional arrangements take place on doublets on opposite sides of the axoneme, suggesting that the transverse functional asymmetry of the axoneme causes an in-plane bending motion

Force-Generating Mechanism of Axonemal Dynein in Solo and Ensemble

In eukaryotic cilia and flagella, various types of axonemal dyneins orchestrate their distinct functions to generate oscillatory bending of axonemes. The force-generating mechanism of dyneins has recently been well elucidated, mainly in cytoplasmic dyneins, thanks to progress in single-molecule measurements, X-ray crystallography, and advanced electron microscopy. These techniques have shed light on several important questions concerning what conformational changes accompany ATP hydrolysis and whether multiple motor domains are coordinated in the movements of dynein. However, due to the lack of a proper expression system for axonemal dyneins, no atomic coordinates of the entire motor domain of axonemal dynein have been reported. Therefore, a substantial amount of knowledge on the molecular architecture of axonemal dynein has been derived from electron microscopic observations on dynein arms in axonemes or on isolated axonemal dynein molecules. This review describes our current knowledge and perspectives of the force-generating mechanism of axonemal dyneins in solo and in ensemble

Different motilities of microtubules driven by kinesin-1 and kinesin-14 motors patterned on nanopillars

モータータンパク質は種類により協働性が異なることを発見 --分子を自在に並べる技術により生体分子モーターの協働性を計測--. 京都大学プレスリリース. 2020-01-23.How're your cells' motors running?. 京都大学プレスリリース. 2020-01-23.Kinesin is a motor protein that plays important roles in a variety of cellular functions. In vivo, multiple kinesin molecules are bound to cargo and work as a team to produce larger forces or higher speeds than a single kinesin. However, the coordination of kinesins remains poorly understood because of the experimental difficulty in controlling the number and arrangement of kinesins, which are considered to affect their coordination. Here, we report that both the number and spacing significantly influence the velocity of microtubules driven by nonprocessive kinesin-14 (Ncd), whereas neither the number nor the spacing changes the velocity in the case of highly processive kinesin-1. This result was realized by the optimum nanopatterning method of kinesins that enables immobilization of a single kinesin on a nanopillar. Our proposed method enables us to study the individual effects of the number and spacing of motors on the collective dynamics of multiple motors