Light: Science & Applications - Localization of single molecules with structured illumination and structured detection

Extracting quantitative information on biomolecular oligomerisation with high resolution remains a significant challenge. Here, the authors propose SPINNA, a framework that compares nearest-neighbour ...

Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes to be affected in neural diseases. Monitoring this process, and in particular synaptic vesicle recycl...

STAR Protocols is an open access, peer-reviewed journal from Cell Press. We offer structured, transparent, accessible, and repeatable step-by-step experimental and computational protocols from all are...

Neurons critically depend on long-distance transport orchestrated by motor proteins walking over their highly asymmetric microtubule cytoskeleton. These microtubules are organized uniformly in axons w...

Communications Biology - A MINFLUX tracking study of a truncated kinesin-1 mutant in gently fixed primary rat hippocampal neurons revealed 8 nm substeps and a rotation of its head during the...

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 Statement S.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.