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Published on: January 14, 2018
MINFLUX reveals dynein stepping in live neurons
Jonas M Schleske1, Jasmine Hubrich1, Jan Otto Wirth1
1Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg 69120, Germany.
Abstract:
Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety of cargoes, performing successive nanometer-sized steps within milliseconds. Due to the limited spatiotemporal precision of established methods for molecular tracking, current knowledge of dynein stepping is essentially limited to slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization to directly track CRISPR/Cas9-tagged endogenous dynein with nanometer/millisecond precision in living primary neurons. We show that endogenous dynein primarily takes 8 nm steps, including frequent sideways steps but few backward steps. Strikingly, the majority of direction reversals between retrograde and anterograde movement occurred on the time scale of single steps (16 ms), suggesting a rapid regulatory reversal mechanism. Tug-of-war-like behavior during pauses or reversals was unexpectedly rare. By analyzing the dwell time between steps, we concluded that a single rate-limiting process underlies the dynein stepping mechanism, likely arising from just one adenosine 5'-triphosphate hydrolysis event being required during each step. Our study underscores the power of MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells.
Insights
Dynein motor proteins move cargo within cells using 8 nm steps and frequently change direction rapidly, within milliseconds. This study reveals dynein
Area of Science:
- Cellular Biology
- Molecular Motors
- Neuroscience
Background:
- Dynein is crucial for retrograde intracellular transport, moving various cargoes in discrete steps.
- Previous studies on dynein stepping were limited by low spatiotemporal resolution, often requiring in vitro conditions.
- Understanding dynein's precise movements in vivo is essential for comprehending intracellular transport dynamics.
Purpose of the Study:
- To investigate the stepping mechanism and directionality of endogenous dynein in living neurons with unprecedented precision.
- To characterize the kinetics and dynamics of dynein motor activity in its native cellular environment.
- To explore the regulatory mechanisms governing dynein's movement and direction reversals.
Main Methods:
- Utilized CRISPR/Cas9 gene editing to tag endogenous dynein for high-precision tracking.
- Employed MINFLUX super-resolution microscopy for nanometer/millisecond spatiotemporal resolution.
- Tracked dynein movement in living primary neurons.
Main Results:
- Endogenous dynein primarily moves in 8 nm steps, with frequent sideways and rare backward movements.
- Direction reversals between retrograde and anterograde transport occur rapidly, on the timescale of single steps (16 ms).
- Tug-of-war interactions during pauses or reversals were infrequent, and a single rate-limiting step, likely involving ATP hydrolysis, governs dynein's mechanism.
Conclusions:
- MINFLUX localization provides critical insights into the spatiotemporal dynamics of protein function in living cells.
- Dynein exhibits rapid directionality switching and a unified stepping mechanism, likely driven by single ATP hydrolysis events.
- This research refines our understanding of intracellular transport and molecular motor regulation.
