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Direct observation of motor protein stepping in living cells using MINFLUX

View ORCID ProfileTakahiro Deguchi, View ORCID ProfileMalina K. Iwanski, View ORCID ProfileEva-Maria Schentarra, View ORCID ProfileChristopher Heidebrecht, Lisa Schmidt, View ORCID ProfileJennifer Heck, View ORCID ProfileTobias Weihs, View ORCID ProfileSebastian Schnorrenberg, View ORCID ProfilePhilipp Hoess, View ORCID ProfileSheng Liu, View ORCID ProfileVeronika Chevyreva, View ORCID ProfileKyung-Min Noh, View ORCID ProfileLukas C. Kapitein, View ORCID ProfileJonas Ries
doi: https://doi.org/10.1101/2022.07.25.500391
Takahiro Deguchi
1Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
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Malina K. Iwanski
2Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands
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  • ORCID record for Malina K. Iwanski
Eva-Maria Schentarra
1Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
3Faculty of Biosciences, University of Heidelberg, Heidelberg, Germany
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Christopher Heidebrecht
1Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
3Faculty of Biosciences, University of Heidelberg, Heidelberg, Germany
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Lisa Schmidt
1Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
3Faculty of Biosciences, University of Heidelberg, Heidelberg, Germany
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Jennifer Heck
4Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
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Tobias Weihs
5Abberior Instruments GmbH, Göttingen, Germany
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Sebastian Schnorrenberg
6EMBL Imaging Centre, European Molecular Biology Laboratory, Heidelberg, Germany
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  • ORCID record for Sebastian Schnorrenberg
Philipp Hoess
1Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
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Sheng Liu
1Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
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Veronika Chevyreva
1Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
7The FIRC Institute of Molecular Oncology, Milano, Italy
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Kyung-Min Noh
4Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
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Lukas C. Kapitein
2Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands
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Jonas Ries
1Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
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  • For correspondence: jonas.ries@embl.de
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Abstract

Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in 2D and 3D using the recently introduced super-resolution technique MINFLUX. This allowed us to resolve the precise stepping motion of the motor protein kinesin-1 as it walks on microtubules in living cells. In addition, nanoscopic tracking of motors on microtubule of fixed cells enabled us to resolve their spatial organization with protofilament resolution. Our approach will enable future in vivo studies of motor protein kinetics in complex environments and super-resolution mapping of dense microtubule arrays, and pave the way towards monitoring functional conformational changes of protein machines at high spatiotemporal resolution in living systems.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted July 25, 2022.
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Direct observation of motor protein stepping in living cells using MINFLUX
Takahiro Deguchi, Malina K. Iwanski, Eva-Maria Schentarra, Christopher Heidebrecht, Lisa Schmidt, Jennifer Heck, Tobias Weihs, Sebastian Schnorrenberg, Philipp Hoess, Sheng Liu, Veronika Chevyreva, Kyung-Min Noh, Lukas C. Kapitein, Jonas Ries
bioRxiv 2022.07.25.500391; doi: https://doi.org/10.1101/2022.07.25.500391
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Direct observation of motor protein stepping in living cells using MINFLUX
Takahiro Deguchi, Malina K. Iwanski, Eva-Maria Schentarra, Christopher Heidebrecht, Lisa Schmidt, Jennifer Heck, Tobias Weihs, Sebastian Schnorrenberg, Philipp Hoess, Sheng Liu, Veronika Chevyreva, Kyung-Min Noh, Lukas C. Kapitein, Jonas Ries
bioRxiv 2022.07.25.500391; doi: https://doi.org/10.1101/2022.07.25.500391

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