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以MINFLUX表征的动力蛋白的步进动力学。

Stepping dynamics of dynein characterized by MINFLUX.

作者信息

Slivka Joseph, Gleave Emma, Wijewardena Devinda P, Canty John T, Selvin Paul R, Carter Andrew P, Yildiz Ahmet

机构信息

Department of Physics, University of California at Berkeley, Berkeley CA 94720 USA.

Medical Research Council Laboratory of Molecular Biology, Division of Structural Studies, Francis Crick Avenue, Cambridge, CB2 0QH, UK.

出版信息

bioRxiv. 2024 Jul 18:2024.07.16.603667. doi: 10.1101/2024.07.16.603667.

DOI:10.1101/2024.07.16.603667
PMID:39071311
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11275781/
Abstract

Cytoplasmic dynein is a dimeric motor that drives minus-end directed transport on microtubules (MTs). To couple ATP hydrolysis to a mechanical step, a dynein monomer must be released from the MT before undergoing a conformational change that generates a bias towards the minus end. However, the dynamics of dynein stepping have been poorly characterized by tracking flexible regions of the motor with limited resolution. Here, we developed a cysteine-light mutant of yeast dynein and site-specifically labeled its MT-binding domain in vitro. MINFLUX tracking at sub-millisecond resolution revealed that dynein hydrolyzes one ATP per step and takes multitudes of 8 nm steps at physiological ATP. Steps are preceded by the transient movement towards the plus end. We propose that these backward "dips" correspond to MT release and subsequent diffusion of the stepping monomer around its MT-bound partner before taking a minus-end-directed conformational change of its linker. Our results reveal the order of sub-millisecond events that result in a productive step of dynein.

摘要

细胞质动力蛋白是一种二聚体马达蛋白，驱动微管（MT）上的负端定向运输。为了将ATP水解与机械步骤耦合，动力蛋白单体必须在经历构象变化之前从微管上释放，该构象变化会产生向负端的偏向。然而，通过以有限分辨率追踪马达蛋白的柔性区域，动力蛋白步移的动力学特征尚不明确。在这里，我们开发了一种酵母动力蛋白的半胱氨酸轻突变体，并在体外对其微管结合结构域进行了位点特异性标记。亚毫秒分辨率的MINFLUX追踪显示，动力蛋白每步水解一个ATP，并在生理ATP条件下进行多个8纳米的步移。步移之前会有向正端的短暂移动。我们认为这些向后的“下降”对应于微管释放以及步移单体在其与微管结合的伙伴周围的后续扩散，然后其连接体发生向负端的构象变化。我们的结果揭示了导致动力蛋白有效步移的亚毫秒级事件的顺序。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/1557d662dec5/nihpp-2024.07.16.603667v1-f0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/843c5520ac1c/nihpp-2024.07.16.603667v1-f0005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/156a73045a3a/nihpp-2024.07.16.603667v1-f0006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/382a47a9522d/nihpp-2024.07.16.603667v1-f0007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/a38bcbc0876f/nihpp-2024.07.16.603667v1-f0008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/42d8fe2c4885/nihpp-2024.07.16.603667v1-f0009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/b0349bfd8b57/nihpp-2024.07.16.603667v1-f0010.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/a9dd71a92252/nihpp-2024.07.16.603667v1-f0011.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/bc53f50be430/nihpp-2024.07.16.603667v1-f0012.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/a240589d4750/nihpp-2024.07.16.603667v1-f0013.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/e88abb26c9dc/nihpp-2024.07.16.603667v1-f0001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/b14831f7df40/nihpp-2024.07.16.603667v1-f0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/f2e95e43367e/nihpp-2024.07.16.603667v1-f0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/1557d662dec5/nihpp-2024.07.16.603667v1-f0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/843c5520ac1c/nihpp-2024.07.16.603667v1-f0005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/156a73045a3a/nihpp-2024.07.16.603667v1-f0006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/382a47a9522d/nihpp-2024.07.16.603667v1-f0007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/a38bcbc0876f/nihpp-2024.07.16.603667v1-f0008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/42d8fe2c4885/nihpp-2024.07.16.603667v1-f0009.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/b0349bfd8b57/nihpp-2024.07.16.603667v1-f0010.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/a9dd71a92252/nihpp-2024.07.16.603667v1-f0011.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/bc53f50be430/nihpp-2024.07.16.603667v1-f0012.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/a240589d4750/nihpp-2024.07.16.603667v1-f0013.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/e88abb26c9dc/nihpp-2024.07.16.603667v1-f0001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/b14831f7df40/nihpp-2024.07.16.603667v1-f0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/f2e95e43367e/nihpp-2024.07.16.603667v1-f0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/243a/11275781/1557d662dec5/nihpp-2024.07.16.603667v1-f0004.jpg

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本文引用的文献

1
The mechanochemical cycle of reactive full-length human dynein 1.活性全长人动力蛋白1的机械化学循环
Nat Struct Mol Biol. 2025 Apr 22. doi: 10.1038/s41594-025-01543-3.
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MINFLUX reveals dynein stepping in live neurons.MINFLUX 揭示了活神经元中的动力蛋白的步进运动。
Proc Natl Acad Sci U S A. 2024 Sep 17;121(38):e2412241121. doi: 10.1073/pnas.2412241121. Epub 2024 Sep 10.
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Nanometer-resolution tracking of single cargo reveals dynein motor mechanisms.单货物纳米分辨率追踪揭示动力蛋白运动机制。
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Nde1 promotes Lis1-mediated activation of dynein.Nde1 促进 Lis1 介导的动力蛋白激活。
Nat Commun. 2023 Nov 9;14(1):7221. doi: 10.1038/s41467-023-42907-x.
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Direct observation of motor protein stepping in living cells using MINFLUX.利用 MINFLUX 在活细胞中直接观察马达蛋白的运动。
Science. 2023 Mar 10;379(6636):1010-1015. doi: 10.1126/science.ade2676. Epub 2023 Mar 9.
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MINFLUX dissects the unimpeded walking of kinesin-1.MINFLUX剖析驱动蛋白-1的自由行走。
Science. 2023 Mar 10;379(6636):1004-1010. doi: 10.1126/science.ade2650. Epub 2023 Mar 9.
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Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility.三色单分子成像揭示了动力蛋白在运动过程中的构象动力学。
Proc Natl Acad Sci U S A. 2021 Aug 3;118(31). doi: 10.1073/pnas.2101391118.
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Structure and Mechanics of Dynein Motors.动力蛋白的结构与力学性质。
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Dynein Harnesses Active Fluctuations of Microtubules for Faster Movement.动力蛋白利用微管的主动涨落实现更快移动。
Nat Phys. 2020;16:312-316. doi: 10.1038/s41567-019-0757-4. Epub 2020 Jan 20.
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MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells.MINFLUX 纳米显微镜可在细胞中实现 3D 多色纳米分辨率。
Nat Methods. 2020 Feb;17(2):217-224. doi: 10.1038/s41592-019-0688-0. Epub 2020 Jan 13.