Kinesin takes substeps
Simultaneously measuring the nanoscale motion and forces that molecular machines generate provides insights into how they work mechanically to fulfill their cellular function. To study these machines, Sudhakar et al. developed germanium semiconductor nanospheres as probes for so-called optical tweezers. With these high–refractive index nanospheres, they improved the resolution of optical tweezers and discovered that the motor kinesin takes 4-nanometer substeps. Further, instead of detaching from their microtubule track under load, motors slid back on it, enabling rapid reengagement in transport. The new technology will allow investigation of a range of other proteins and their behaviors at nanometer scales.
Science, this issue p. eabd9944
Structured Abstract
INTRODUCTION
Cytoskeletal motors drive many essential mechanical processes inside cells. For example, kinesin motors are key for cell division or vesicle transport. Kinesin-1 transports cargo along microtubules by coupling adenosine 5′-triphosphate (ATP) hydrolysis to perform mechanical work against piconewton loads. This force generation and overall transport distance are limited by motor detachment. However, how kinesins walk and detach is still unclear.
To simultaneously measure the nanoscale motion and forces of molecular machines, optical tweezers are often used. In the tight, mostly infrared laser focus of optical tweezers, small dielectric particles can be trapped and used as handles for sensitive position and force measurements. Because optical forces scale with the particle volume, piconewton force measurements with nanoparticles require a high laser power. This high power leads to excessive heating and precludes biological measurements. Therefore, for biological single-molecule measurements, micrometer-sized probes are used. However, such probes have a large hydrodynamic drag and therefore lack the spatiotemporal resolution to unravel important fast or small details in the mechanochemistry of molecular machines. These details remain hidden in the storm of Brownian motion.
RATIONALE
To overcome this practical resolution limit of optical tweezers and resolve so-far hidden conformational changes of proteins, we sought to compensate the volume scaling of optical trapping forces by the use of probe materials with a very high refractive index and low light absorption. This compensation should allow the use of nanometer-sized probes and the generation of piconewton optical forces without detrimental heating but with improved temporal response and spatial precision. Promising materials include silicon and germanium that become transparent in the near-infrared, with very high refractive indices exceeding 4. However, efficient methods to fabricate such semiconductor nanospheres suitable for optical trapping do not exist.
RESULTS
We developed a solution-based method to synthesize germanium nanospheres. With a diameter of roughly 70 nm, they are about an order of magnitude smaller as compared with commonly used microspheres and still allowed piconewton force measurements. To find out how kinesin works mechanically, we developed an in vitro reconstituted assay. To this end, we coated the nanospheres with a lipid bilayer [the white rim in the transmission electron microscopy (TEM) image] to mimic vesicles and to be roughly their size inside cells. When we bound kinesin-1 to these “vesicles” and measured the interaction of single motors with microtubules under piconewton tension, we discovered that each hydrolysis cycle is broken up into two 4-nm center-of-mass substeps. The durations of these substeps alternated in their force and ATP dependence, with the duration of one of the substeps being nearly independent of both parameters. Furthermore, when subjected to hindering loads, motors never detached from the microtubule. Instead, motors slipped along the microtubule in 8-nm steps on microsecond time scales. These slip steps are consistent with a bond-rupture model that involves protein friction between the motor and its track. Unexpectedly, motors usually did not detach after a slip event but reengaged in motility that rescued cargo transport.
CONCLUSION
Germanium nanospheres are promising for bioimaging, sensing, optoelectronics, nanophotonics, and energy storage. For optical trapping, the nanospheres open a new temporal window by which to uncover hidden dynamics in molecular machines. The direct observation of load-bearing kinesin substeps resolves a long-standing controversy. Slipping and rescues should allow load distribution and synchronization when motors operate in teams. Understanding their mechanochemistry is important for a better understanding of cellular transport and other essential molecular functions of kinesins, with implications, for example, for neurodegenerative diseases and cancer.
Kinesins are molecular machines that transport vesicles along microtubules inside cells. Membrane-coated germanium nanospheres (TEM micrograph, left) improved the spatiotemporal resolution of optical tweezers and allowed the measurement of substeps during the normal kinesin stepping cycle. Under load, kinesins did not detach but slipped along the microtubule, which led to the discovery of rescues for vesicle transport.
Abstract
Kinesin motors are essential for the transport of cellular cargo along microtubules. How the motors step, detach, and cooperate with each other is still unclear. To dissect the molecular motion of kinesin-1, we developed germanium nanospheres as ultraresolution optical trapping probes. We found that single motors took 4-nanometer center-of-mass steps. Furthermore, kinesin-1 never detached from microtubules under hindering load conditions. Instead, it slipped on microtubules in microsecond-long, 8-nanometer steps and remained in this slip state before detaching or reengaging in directed motion. Unexpectedly, reengagement and thus rescue of directed motion was more frequent. Our observations broaden our knowledge on the mechanochemical cycle and slip state of kinesin. This state and rescue need to be accounted for to understand long-range transport by teams of motors.
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February 12, 2021 at 01:59AM
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