TY  - GEN
ID  - heidok35697
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/35697/
A1  - Schleske, Jonas Michael
AV  - public
KW  - MINFLUX
KW  -  Optical Nanoscopy
KW  -  Live Neurons
KW  -  CRISPR/Cas9
KW  -  Motorprotein
KW  -  Tracking
TI  - MINFLUX Tracking of Endogenous Dynein in Live Neurons
N2  - MINFLUX represents a novel class of optical nanoscopy techniques, enabling tracking measurements with nanometer/millisecond spatiotemporal precision by requiring substantially fewer detected photons than established methods for molecular tracking. By leveraging this photonefficient precision, MINFLUX permitted the use of a comparatively small fluorescent label to directly observe the nanometer-sized steps of the molecular motor dynein in living primary neurons at physiological adenosine 5?-triphosphate (ATP) concentrations, which was previously only feasible in vitro and at markedly decelerated conditions. To tag dynein endogenously at different sites, CRISPR/Cas9 genome editing was implemented, and the successful tagging was confirmed through the utilization of a self-built widefield microscope. During MINFLUX tracking measurements, reversals within bidirectional axonal transport were observed to occur on a time scale corresponding to individual steps, indicating the presence of a rapid reversal mechanism that is distinct from a stochastic tug-of-war-like behavior. The analysis of the millisecond-short dwell times between consecutive steps revealed that the mechanochemical cycle is single-rate limited, indicating that dynein consumes one ATP to perform a step. Having established MINFLUX tracking of endogenous dynein in live neurons, it is anticipated that MINFLUX will facilitate further minimally invasive studies of rapid and undisturbed protein dynamics in live cells.
CY  - Heidelberg
Y1  - 2024///
ER  -