鞭毛ダイニン外腕の周期的構築におけるドッキング複合体ODA-DCの機能

学位の種別: 課程博士審査委員会委員 : (主査)東京大学教授 武田 洋幸, 東京大学教授 中野 明彦, 東京大学准教授 廣野 雅文, 東京大学教授 吉川 雅英, 東京工業大学准教授 若林 憲一University of Tokyo(東京大学

Protein–protein interactions between intermediate chains and the docking complex of Chlamydomonas flagellar outer arm dynein

AbstractOuter arm dynein (OAD) is bound to specific loci on outer-doublet-microtubules by interactions at two sites: via intermediate chain 1 (IC1) and the outer dynein arm docking complex (ODA-DC). Studies using Chlamydomonas mutants have suggested that the individual sites have rather weak affinities for microtubules, and therefore strong OAD attachment to microtubules is achieved by their cooperation. To test this idea, we examined interactions between IC1, IC2 (another intermediate chain) and ODA-DC using recombinant proteins. Recombinant IC1 and IC2 were found to form a 1:1 complex, and this complex associated with ODA-DC in vitro. Binding of IC1 to mutant axonemes revealed that there are specific binding sites for IC1. From these data, we propose a novel model of OAD-outer doublet association

Cooperative binding of the outer arm-docking complex underlies the regular arrangement of outer arm dynein in the axoneme

Outer arm dynein (OAD) in cilia and flagella is bound to the outer doublet microtubules every 24 nm. Periodic binding of OADs at specific sites is important for efficient cilia/flagella beating; however, the molecular mechanism that specifies OAD arrangement remains elusive. Studies using the green alga Chlamydomonas reinhardtii have shown that the OAD-docking complex (ODA-DC), a heterotrimeric complex present at the OAD base, functions as the OAD docking site on the doublet. We find that the ODA-DC has an ellipsoidal shape approximately 24 nm in length. In mutant axonemes that lack OAD but retain the ODA-DC, ODA-DC molecules are aligned in an end-to-end manner along the outer doublets. When flagella of a mutant lacking ODA-DCs are supplied with ODA-DCs upon gamete fusion, ODA-DC molecules first bind to the mutant axonemes in the proximal region, and the occupied region gradually extends toward the tip, followed by binding of OADs. This and other results indicate that a cooperative association of the ODA-DC underlies its function as the OAD-docking site and is the determinant of the 24-nm periodicity

Chlamydomonas DYX1C1/PF23 is essential for axonemal assembly and proper morphology of inner dynein arms

Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA) as well as a fraction of the outer dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly

Whole-genome screen identifies diverse pathways that negatively regulate ciliogenesis.

We performed a high-throughput whole-genome RNAi screen to identify novel inhibitors of ciliogenesis in normal and basal breast cancer cells. Our screen uncovered a previously undisclosed, extensive network of genes linking integrin signaling and cellular adhesion to the extracellular matrix (ECM) with inhibition of ciliation in both normal and cancer cells. Surprisingly, a cohort of genes encoding ECM proteins was also identified. We characterized several ciliation inhibitory genes and showed that their silencing was accompanied by altered cytoskeletal organization and induction of ciliation, which restricts cell growth and migration in normal and breast cancer cells. Conversely, supplying an integrin ligand, vitronectin, to the ECM rescued the enhanced ciliation observed on silencing this gene. Aberrant ciliation could also be suppressed through hyperactivation of the YAP/TAZ pathway, indicating a potential mechanistic basis for our findings. Our findings suggest an unanticipated reciprocal relationship between ciliation and cellular adhesion to the ECM and provide a resource that could vastly expand our understanding of controls involving outside-in and inside-out signaling that restrain cilium assembly

Chlamydomonas DYX1C1/PF23 is essential for axonemal assembly and proper morphology of inner dynein arms

Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA) as well as a fraction of the outer dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly

Inner lumen proteins stabilize doublet microtubules in cilia and flagella

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes