Uncovering kinesin dynamics in neurites with MINFLUX

Neurons grow neurites of several tens of micrometers in length, necessitating active transport from the cell body by motor proteins. By tracking fluorophores as minimally invasive labels, MINFLUX is able to quantify the motion of those proteins with nanometer/millisecond resolution. Here we study th...

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Bibliographic Details
Published inbioRxiv
Main Authors Jan Otto Wirth, Schentarra, Eva Maria, Scheiderer, Lukas, Palacios, Victor Macarron, Tarnawski, Miroslaw, Hell, Stefan W
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 16.12.2023
Subjects
Active transport
Axons
Cell body
Fluorophores
Hippocampus
Inventors
Kinesin
Microscopes
Microtubules
Patent applications
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Summary:Neurons grow neurites of several tens of micrometers in length, necessitating active transport from the cell body by motor proteins. By tracking fluorophores as minimally invasive labels, MINFLUX is able to quantify the motion of those proteins with nanometer/millisecond resolution. Here we study the substeps of a truncated kinesin-1 mutant in primary rat hippocampal neurons, which have so far been mainly observed on microtubules polymerized on glass coverslips. A gentle fixation protocol largely maintains the structure and surface modifications of the microtubules in the cell. By analyzing the time between the substeps, we identify the ATP-binding state of kinesin-1 and observe the associated rotation of the kinesin-1 head in neurites. We also observed kinesin-1 switching microtubules mid-walk, highlighting the potential of MINFLUX to study the details of active cellular transport.Competing Interest StatementS.W.H. is inventor on patent applications WO 2013/072273 and WO 2015/097000 filed by the Max Planck Society that cover basic principles and arrangements of MINFLUX, including single-molecule tracking. S.W.H. is inventor on patent application WO 2020/064108 submitted by the Max Planck Society that covers principles and arrangements of the phase/amplitude modulator for shifting the intensity minimum. S.W.H. is a cofounder of the company Abberior Instruments, which commercializes MINFLUX microscopes. The remaining authors declare no competing interests.
DOI:10.1101/2023.12.15.571866