Cargando…

Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking

Cytoplasmic dynein is a dimeric motor protein which processively moves along microtubule. Its motor domain (head) hydrolyzes ATP and induces conformational changes of linker, stalk, and microtubule binding domain (MTBD) to trigger stepping motion. Here we applied scattering imaging of gold nanoparti...

Descripción completa

Detalles Bibliográficos
Autores principales: Ando, Jun, Shima, Tomohiro, Kanazawa, Riko, Shimo-Kon, Rieko, Nakamura, Akihiko, Yamamoto, Mayuko, Kon, Takahide, Iino, Ryota
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978368/
https://www.ncbi.nlm.nih.gov/pubmed/31974448
http://dx.doi.org/10.1038/s41598-020-58070-y
_version_ 1783490684144058368
author Ando, Jun
Shima, Tomohiro
Kanazawa, Riko
Shimo-Kon, Rieko
Nakamura, Akihiko
Yamamoto, Mayuko
Kon, Takahide
Iino, Ryota
author_facet Ando, Jun
Shima, Tomohiro
Kanazawa, Riko
Shimo-Kon, Rieko
Nakamura, Akihiko
Yamamoto, Mayuko
Kon, Takahide
Iino, Ryota
author_sort Ando, Jun
collection PubMed
description Cytoplasmic dynein is a dimeric motor protein which processively moves along microtubule. Its motor domain (head) hydrolyzes ATP and induces conformational changes of linker, stalk, and microtubule binding domain (MTBD) to trigger stepping motion. Here we applied scattering imaging of gold nanoparticle (AuNP) to visualize load-free stepping motion of processive dynein. We observed artificially-dimerized chimeric dynein, which has the head, linker, and stalk from Dictyostelium discoideum cytoplasmic dynein and the MTBD from human axonemal dynein, whose structure has been well-studied by cryo-electron microscopy. One head of a dimer was labeled with 30 nm AuNP, and stepping motions were observed with 100 μs time resolution and sub-nanometer localization precision at physiologically-relevant 1 mM ATP. We found 8 nm forward and backward steps and 5 nm side steps, consistent with on- and off-axes pitches of binding cleft between αβ-tubulin dimers on the microtubule. Probability of the forward step was 1.8 times higher than that of the backward step, and similar to those of the side steps. One-head bound states were not clearly observed, and the steps were limited by a single rate constant. Our results indicate dynein mainly moves with biased small stepping motion in which only backward steps are slightly suppressed.
format Online
Article
Text
id pubmed-6978368
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Nature Publishing Group UK
record_format MEDLINE/PubMed
spelling pubmed-69783682020-01-30 Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking Ando, Jun Shima, Tomohiro Kanazawa, Riko Shimo-Kon, Rieko Nakamura, Akihiko Yamamoto, Mayuko Kon, Takahide Iino, Ryota Sci Rep Article Cytoplasmic dynein is a dimeric motor protein which processively moves along microtubule. Its motor domain (head) hydrolyzes ATP and induces conformational changes of linker, stalk, and microtubule binding domain (MTBD) to trigger stepping motion. Here we applied scattering imaging of gold nanoparticle (AuNP) to visualize load-free stepping motion of processive dynein. We observed artificially-dimerized chimeric dynein, which has the head, linker, and stalk from Dictyostelium discoideum cytoplasmic dynein and the MTBD from human axonemal dynein, whose structure has been well-studied by cryo-electron microscopy. One head of a dimer was labeled with 30 nm AuNP, and stepping motions were observed with 100 μs time resolution and sub-nanometer localization precision at physiologically-relevant 1 mM ATP. We found 8 nm forward and backward steps and 5 nm side steps, consistent with on- and off-axes pitches of binding cleft between αβ-tubulin dimers on the microtubule. Probability of the forward step was 1.8 times higher than that of the backward step, and similar to those of the side steps. One-head bound states were not clearly observed, and the steps were limited by a single rate constant. Our results indicate dynein mainly moves with biased small stepping motion in which only backward steps are slightly suppressed. Nature Publishing Group UK 2020-01-23 /pmc/articles/PMC6978368/ /pubmed/31974448 http://dx.doi.org/10.1038/s41598-020-58070-y Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Ando, Jun
Shima, Tomohiro
Kanazawa, Riko
Shimo-Kon, Rieko
Nakamura, Akihiko
Yamamoto, Mayuko
Kon, Takahide
Iino, Ryota
Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking
title Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking
title_full Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking
title_fullStr Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking
title_full_unstemmed Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking
title_short Small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking
title_sort small stepping motion of processive dynein revealed by load-free high-speed single-particle tracking
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978368/
https://www.ncbi.nlm.nih.gov/pubmed/31974448
http://dx.doi.org/10.1038/s41598-020-58070-y
work_keys_str_mv AT andojun smallsteppingmotionofprocessivedyneinrevealedbyloadfreehighspeedsingleparticletracking
AT shimatomohiro smallsteppingmotionofprocessivedyneinrevealedbyloadfreehighspeedsingleparticletracking
AT kanazawariko smallsteppingmotionofprocessivedyneinrevealedbyloadfreehighspeedsingleparticletracking
AT shimokonrieko smallsteppingmotionofprocessivedyneinrevealedbyloadfreehighspeedsingleparticletracking
AT nakamuraakihiko smallsteppingmotionofprocessivedyneinrevealedbyloadfreehighspeedsingleparticletracking
AT yamamotomayuko smallsteppingmotionofprocessivedyneinrevealedbyloadfreehighspeedsingleparticletracking
AT kontakahide smallsteppingmotionofprocessivedyneinrevealedbyloadfreehighspeedsingleparticletracking
AT iinoryota smallsteppingmotionofprocessivedyneinrevealedbyloadfreehighspeedsingleparticletracking