%0 Generic
%A Scheiderer, Jan Lukas
%C Heidelberg
%D 2023
%F heidok:32764
%R 10.11588/heidok.00032764
%T Kinesin-1 stepping dissected with MINFLUX and MINSTED tracking – Spontaneous blinkers for live-cell MINFLUX imaging
%U https://archiv.ub.uni-heidelberg.de/volltextserver/32764/
%X The fluorescence-based nanoscopy methods MINFLUX and MINSTED are currently revolutionizing the field of imaging and single molecule tracking by achieving molecular spatial precision with low photon numbers. Using a polymer-based in vitro assay with strongly reduced fluorescence background, I identified MINFLUX-compatible spontaneously blinking fluorophores by quantifying their blinking properties. Due to the spontaneous blinkers being live-cell compatible and having few milliseconds short on-events, I expect them to advance the imaging field by drastically accelerating MINFLUX measurements. The main focus of this work, however, is the application of the nanoscopy methods MINFLUX and MINSTED for tracking of the motor protein kinesin-1. Requiring an only ~1 nm small fluorophore as label, they are inherently less artefact-prone than established techniques which require the attachment of comparatively large beads for a similar spatio-temporal resolution. With an improved interferometric MINFLUX approach, we successfully resolved regular steps and substeps of the kinesin-1 stalk and heads. By discovering that ATP binds to the motor in the one-head-bound state and is hydrolyzed in the two-head-bound state, we aim to solve a long-standing controversy in the field. Furthermore, we deduced that when the rear head of kinesin-1 detaches from the microtubule, it rotates around its front into a rightward-displaced unbound state. In conjunction with an observed stalk rotation, I concluded the motor to walk in a symmetric hand-over-hand fashion. Finally, we successfully resolved the stepping of kinesin-1 with MINSTED, confirming many findings from the MINFLUX experiments and observing motor sidestepping and protofilament switching.  These findings will prove helpful in developing treatments for diseases linked to malfunction of kinesins. Beyond that, this thesis establishes MINFLUX and MINSTED for the tracking of dynamic biological processes on the single molecule level.