Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s42003-023-05393-x

Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.11410/kenbikyo.58.2_60

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

α- and β-tubulin have an unstructured glutamate-rich region at their C-terminal tails (CTT). The function of this region in cilia/flagella is still unclear, except that glutamates in CTT act as the sites for posttranslational modifications that affect ciliary motility. A unicellular alga Chlamydomonas possesses only two a-tubulin genes and two b-tubulin genes, each pair encoding an identical protein. This simple gene organization may enable a complete replacement of the wild-type tubulin with its mutated version. Here, using CRISPR/Cas9, we generated mutants expressing tubulins with modified CTTs. We found that the mutant whose four glutamate residues in the α-tubulin CTT have been replaced by alanine almost completely lacked polyglutamylated tubulin and displayed paralyzed cilia. In contrast, the mutant lacking the glutamate-rich region of the β-tubulin CTT assembled short cilia without the central apparatus. This phenotype is similar to the mutants harboring a mutation in a subunit of katanin, whose function has been shown to depend on the b-tubulin CTT. Therefore, our study reveals distinct and important roles of α- and β-tubulin CTT in the formation and function of cilia.

DOI： 10.1242/jcs.261070

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.21203/rs.3.rs-3080731/v1

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1101/2023.02.14.528553

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cold Spring Harbor Laboratory  

DOI： 10.7554/eLife.79990

Language：English   Publishing type：(MISC) Institution technical report and pre-print, etc.   Publisher：Cold Spring Harbor Laboratory  

<title>Abstract</title>The unicellular alga <italic>Chlamydomonas reinhardtii</italic> assembles flagella after pH shock-induced deflagellation, in which process the cell vigorously synthesizes flagellar proteins. Previously, newly synthesized flagellar proteins were chased using radioisotopes. Here, we have developed an alternative, non-radioactive method using the surface sensing of translation (SUnSET) assay, an assay that takes advantage of the incorporation of the antibiotic puromycin into newly synthesized peptides. Just after deflagellation, puromycin-labeled proteins increased dramatically in the cytoplasm and newly assembled flagella. Axonemal incorporation of newly synthesized proteins occurred from the distal tip of the flagellum. Cycloheximide, a protein synthesis inhibitor, almost completely prevented the cellular and flagellar incorporation of puromycin, although, as reported previously, it allowed reassembly of half-length flagella from the cytoplasmic “flagellar precursor” proteins. In contrast to wild-type cells, a cycloheximide-resistant mutant <italic>act2</italic> produced nearly full-length flagella containing puromycin-labeled proteins even in the presence of cycloheximide. These and other results demonstrate that the SUnSET method serves as a powerful tool for visualizing flagellar incorporation of nascent proteins in <italic>Chlamydomonas.</italic>

DOI： 10.1101/2022.01.27.478094

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Cilia are essential organelles required for cell signaling and motility. Nearly all motile cilia have a "9+2" axoneme composed of 9 outer doublet microtubules plus 2 central microtubules; the central microtubules together with their projections is termed the central apparatus (CA). In Chlamydomonas reinhardtii, a model organism for studying cilia, 30 proteins are known CA components, and ∼36 more are predicted to be CA proteins. Among the candidate CA proteins is the highly conserved FAP70, which also has been reported to be associated with the doublet microtubules. Here we determined by super-resolution structured illumination microscopy that FAP70 is located exclusively in the CA, and show by cryo-electron microscopy that its N-terminus is located at the base of the CA's C2a projection. We also found that fap70-1 mutant axonemes lack most of the C2a projection. Mass spectrometry revealed that fap70-1 axonemes lack not only FAP70 but two other conserved candidate CA proteins, FAP65 and FAP147. Finally, FAP65 and FAP147 co-immunoprecipitated with HA-tagged FAP70. Taken together, these data identify FAP70, FAP65, and FAP147 as the first defining components of the C2a projection.

DOI： 10.1242/jcs.258540

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

<italic>Caenorhabditis elegans</italic> is used as a model system to understand the neural basis of behavior, but application of caged compounds to manipulate and monitor the neural activity is hampered by the innate photophobic response of the nematode to short-wavelength light or by the low temporal resolution of photocontrol. Here, we develop boron dipyrromethene (BODIPY)-derived caged compounds that release bioactive phenol derivatives upon illumination in the yellow wavelength range. We show that activation of the transient receptor potential vanilloid 1 (TRPV1) cation channel by spatially targeted optical uncaging of the TRPV1 agonist <italic>N</italic>-vanillylnonanamide at 580 nm modulates neural activity. Further, neuronal activation by illumination-induced uncaging enables optical control of the behavior of freely moving <italic>C. elegans</italic> without inducing a photophobic response and without crosstalk between uncaging and simultaneous fluorescence monitoring of neural activity.

DOI： 10.1073/pnas.2009634118

PubMed

Other Link： https://syndication.highwire.org/content/doi/10.1073/pnas.2009634118

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>
The Z-disc forms a boundary between sarcomeres, which constitute structural and functional units of striated muscle tissue. Actin filaments from adjacent sarcomeres are cross-bridged by α-actinin in the Z-disc, allowing transmission of tension across the myofibril. Despite decades of studies, the 3D structure of Z-disc has remained elusive due to the limited resolution of conventional electron microscopy. Here, we observed porcine cardiac myofibrils using cryo-electron tomography and reconstructed the 3D structures of the actin-actinin cross-bridging complexes within the Z-discs in relaxed and activated states. We found that the α-actinin dimers showed contraction-dependent swinging and sliding motions in response to a global twist in the F-actin lattice. Our observation suggests that the actin-actinin complex constitutes a molecular lattice spring, which maintains the integrity of the Z-disc during the muscle contraction cycle.

DOI： 10.1038/s42003-020-01321-5

PubMed

Other Link： http://www.nature.com/articles/s42003-020-01321-5

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1101/2020.04.14.041897

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Corresponding author   Language：Japanese   Publishing type：Research paper (international conference proceedings)  

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)  

KIF1A is a kinesin family motor involved in the axonal transport of synaptic vesicle precursors (SVPs) along microtubules (MTs). In humans, more than 10 point mutations in KIF1A are associated with the motor neuron disease hereditary spastic paraplegia (SPG). However, not all of these mutations appear to inhibit the motility of the KIF1A motor, and thus a cogent molecular explanation for how KIF1A mutations lead to neuropathy is not available. In this study, we established in vitro motility assays with purified full-length human KIF1A and found that KIF1A mutations associated with the hereditary SPG lead to hyperactivation of KIF1A motility. Introduction of the corresponding mutations into the Caenorhabditis elegans KIF1A homolog unc-104 revealed abnormal accumulation of SVPs at the tips of axons and increased anterograde axonal transport of SVPs. Our data reveal that hyperactivation of kinesin motor activity, rather than its loss of function, is a cause of motor neuron disease in humans.

DOI： 10.1073/pnas.1905690116

PubMed

Authorship：Corresponding author   Language：Japanese   Publishing type：Research paper (international conference proceedings)  

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Cell Biology  

DOI： 10.1091/mbc.E17-11-0689

Scopus

PubMed

Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese  

Authorship：Corresponding author   Language：Japanese   Publishing type：Research paper (international conference proceedings)  

J-GLOBAL

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1091/mbc.E17-05-0285

Web of Science

PubMed

Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/jmicro/dfx018

Web of Science

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Biophysical Society of Japan  

Existence of cellular structures with specific size and length raises a fundamental question in biology: how do cells measure length? One conceptual answer to this question is a molecular ruler, but examples of such rulers in eukaryotes have been lacking. We recently identified a molecular ruler in eukaryotic cilia and flagella using genetic screening and cryo-electron tomography. We demonstrated that FAP59 and FAP172 form a 96-nm-long complex in Chlamydomonas flagella and the absence of the complex disrupted 96-nm repeats of axonemes. Furthermore, lengthening of the FAP59/172 complex resulted in extension of the repeats up to 128 nm.

DOI： 10.2142/biophys.55.250

CiNii Books

Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Eukaryotic flagella and cilia can exhibit planar and non-planar beating, and the mechanism controlling these beating patterns is not well understood. Chlamydomonas reinhardtii flagella beat in approximately the same plane with either an asymmetric ciliary-type or symmetric flagellar-type waveform. Each B-tubule of the number 1, 5 and 6 doublets of the flagellar axoneme possesses a beak-like structure. The number 5 and 6 beak structures are implicated in conversion of ciliary motion into flagellar motion. Here, we show that in a null mutant of Bug22, the asymmetric ciliary waveform is converted into a three-dimensional (non-planar) symmetric flagellar waveform. Bug22 is localized to approximately the proximal half to two-thirds of the flagellum, similar to localization of beak-like structures. However, as shown by immunogold labeling, Bug22 associates with axonemal microtubules without apparent preference for any particular doublets. Interestingly, bug22 mutants lack all beaklike structures. We propose that one function of Bug22 is to regulate the anchoring of the beak-like structures to the doublet microtubules and confine flagellar beating to a plane.

DOI： 10.1242/jcs.140723

Web of Science

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Rockefeller University Press  

DOI： 10.1083/jcb.200609038

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2004.05.111

Web of Science

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Poliovirus (PV), when injected intramuscularly into the calf, is incorporated into the sciatic nerve and causes an initial paralysis of the inoculated limb in transgenic mice carrying the human PV receptor (hPVR/CD155) gene. Here, we demonstrated by using an immunoelectron microscope that PV particles exist on vesicle structures in nerve terminals of neuromuscular junctions. We also demonstrated in glutathione S-transferase pull-down experiments that the dynein light chain, Tctex-1, interacts directly with the cytoplasmic domain of hPVR. In the axons of differentiated rat PC12 cells transfected with expression vectors for hPVRs, vesicles composed of PV and hPVRalpha, as well as a mutant hPVRalpha (hPVRMalpha) that had a reduced ability to bind Tctex-1, co-localized with Tctex-1. However, vesicles containing PV, dextran, and hPVRalpha had only retrograde motion, while those containing PV, dextran, and hPVRMalpha had anterograde or retrograde motion. Topical application of the antimicrotubule agent vinblastine to the sciatic nerve reduced the amount of virus transported from the calf to the spinal cord. These results suggest that direct efficient interaction between the cytoplasmic domain and Tctex-1 is essential for the efficient retrograde transport of PV-containing vesicles along microtubules in vivo.

DOI： 10.1128/JVI.78.13.7186-7198.2004

Web of Science

PubMed

Language：English   Presentation type：Oral presentation(invited, special)  

Event date： 2022.5

Language：Japanese   Presentation type：Oral presentation(general)  

Event date： 2021.6

Language：English   Presentation type：Oral presentation(invited, special)  

Event date： 2021.3

Language：English   Presentation type：Oral presentation(invited, special)  

Event date： 2020.5

Language：English   Presentation type：Oral presentation(invited, special)  

Language：Japanese  

Event date： 2019.12

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Event date： 2019.6

Language：English   Presentation type：Oral presentation(invited, special)  

Event date： 2019.6

Language：English   Presentation type：Oral presentation(invited, special)  

Event date： 2018.11

Language：English   Presentation type：Oral presentation(invited, special)  

Event date： 2018.8

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Venue：信州大学  

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Event date： 2018.3

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Language：Japanese  

Event date： 2017.11

Language：English   Presentation type：Oral presentation(invited, special)  

Language：Japanese  

Language：English   Presentation type：Oral presentation(invited, special)  

Language：English  

Language：English  

Language：Japanese  

Language：Japanese