Kinesin rotates unidirectionally and generates torque while walking on microtubules

Ramaiya, Avin; Roy, Basudev; Bugiel, Michael; Schäffer, Erik

doi:10.1073/pnas.1706985114

Cited by 90 publications

(125 citation statements)

References 44 publications

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“…The GeNTOP calibration showed that we achieved the optical-trap spring constant-the trap stiffness κ-necessary for kinesin picotensiometry employing microspheres. 6,9,[12][13][14] Also, for the used laser power, the trap stiffness quantitatively agreed with a Mie theory calculation based on the dielectric properties of germanium at the infrared trapping laser wavelength (see methods). Thus, GeNTOPs had indeed the expected very high refractive index of 4.34.…”

Section: Germanium Nanospheres For Ultraprecision Spatiotemporal Meassupporting

confidence: 68%

“…[1][2][3] However, many such processes cannot be measured at their native spatiotemporal resolution but only under conditionsfor example, low nucleotide concentrations-at which the mechanochemistry is slowed down and might be different. 6 For example, the benchmark, 3.4-Å-DNA-base-pair-sized steps of the RNA polymerase, naturally operating on a millisecond time scale, could only be resolved on a second time scale. 7 The inherent trade-off between temporal and spatial precision and the resolution limit itself are quantified by the product δ √ τ that has a constant value, with the lower limit hardly depending on the experiment.…”

mentioning

confidence: 99%

“…8 During an ATP hydrolysis cycle, kinesin motors advance by 8 nm along microtubules against forces of several piconewtons via a rotational hand-over-hand mechanism. 6,9 While consensus develops on how kinesin motors work 10,11 important details remain unclear. For example, it is controversial whether intermediate mechanical steps in the hydrolysis cycle exist and can support load.…”

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confidence: 99%

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Germanium nanospheres for ultraresolution picotensiometry of kinesin motors

Sudhakar

Abdosamadi

Jachowski

et al. 2020

Preprint

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Force spectroscopy on single molecular machines generating piconewton forces is often performed using optical tweezers.1–3 Since optical forces scale with the trapped particle volume, piconewton force measurements require micron-sized probes practically limiting the spatiotemporal resolution.1,2,4,5 Here, we have overcome this limit by developing high-refractive index germanium nanospheres as ultraresolution trapping probes. Using these probes, we have dissected the molecular motion of the cytoskeletal motor kinesin-1 that transports vesicles along microtubule filaments. With a superior spatiotemporal resolution, we have resolved a controversy unifying its stepping and detachment behavior. We found that single motors took 4-nm-center-of-mass steps with alternating force dependence of their dwell times. At maximum force, motors did not detach but switched to a weakly bound state. In this state, motors slid on the microtubule with 8-nm steps on a microsecond time scale. Kinesins remained in this intermediate slip state before either truly detaching or reengaging in directed motion. Surprisingly, reengagement and, thus, rescue of directed motion occurred in about 80 percent of events. Such rescue events suggest that macroscopically observed run lengths of individual motors are concatenations and rescues need to be accounted for to understand long-range transport. Furthermore, teams of motors involved in transport may be synchronized through the weakly bound slip state. Apart from ultraresolution optical trapping, germanium nanospheres are promising candidates for applications ranging from nanophotonics to energy storage.

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Section: Germanium Nanospheres For Ultraprecision Spatiotemporal Meassupporting

confidence: 68%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Germanium nanospheres for ultraresolution picotensiometry of kinesin motors

Sudhakar

Abdosamadi

Jachowski

et al. 2020

Preprint

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“…However, the forward scattered light detects very little of this motion. We suspect that this happens due to the nature of the signals in the forward and backward directions [13,14], the forward scatter being an interference between unscattered light and the scattered light, while the backscattered one being interference between that reflected from the interface and that scattered off the particle. In the case of low amplitude oscillations, the interface oscillates while the particle remains quite unaffected, such that the backscattered detection senses it while the forward scatter does not.…”

Section: Resultsmentioning

confidence: 99%

Detection of self-generated nanowaves on the interface of an evaporating sessile water droplet

Bhatt¹,

Vaippully²,

Kharbanda³

et al. 2019

Opt. Express

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Evaporating sessile droplets have been known to exhibit oscillations on the air-liquid interface. These are generally over millimeter scales. Using a novel approach, we are able to measure surface height changes of 500 nm amplitude using optical trapping of a set of microscopic particles at the interface, particularly when the vertical thickness of the droplet reduces to less than 50 µm. We find that at the later stages of the droplet evaporation, particularly when the convection currents become large, the top air-water interface starts to spontaneously oscillate vertically as a function of time in consistency with predictions. We also detect travelling wave trains moving in the azimuthal direction of the drop surface which are consistent with hydrothermal waves at a different combination of Reynolds, Prandtl and Evaporation than previously observed. This is the first time that wavetrains have been observed in water, being extremely challenging to detect both interferometrically and with infra-red cameras. We also find that such waves apply a force parallel to the interface along the propagation direction.

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“…In that conformation, both heads couldn't attach themselves to microtubule lattice at the same time 19 . Considering the fact that the 3kin structure is not accompanied by a bound tail domain that limits free movement 21 and the fact that kinesin is known to rotate round stalk axis 22,23 , we have no reason to doubt its validity. The structure has been analyzed with DomainFinder application 24 .…”

Section: Methodsmentioning

confidence: 99%

Delineating elastic properties of kinesin linker and their sensitivity to point mutations

Świątek

Gudowska–Nowak

2020

Sci Rep

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We analyze free energy estimators from simulation trials mimicking single-molecule pulling experiments on a neck linker of a kinesin motor. For that purpose, we have performed a version of steered molecular dynamics (SMD) calculations. the sample trajectories have been analyzed to derive distribution of work done on the system. In order to induce stretching of the linker, we have applied a constant pulling force to the molecule and allowed for a subsequent relaxation of its structure. the use of fluctuation relations (FR) relevant to non-equilibrium systems subject to thermal fluctuations allows us to assess the difference in free energy between stretched and relaxed conformations. To further understand effects of potential mutations on elastic properties of the linker, we have performed similar in silico studies on a structure formed of a polyalanine sequence (Ala-only) and on three other structures, created by substituting selected types of amino acid residues in the linker's sequence with alanine (Ala) ones. the results of SMD simulations indicate a crucial role played by the Asparagine (Asn) and Lysine (Lys) residues in controlling stretching and relaxation properties of the linker domain of the motor.The motor proteins' ability to generate movement creates a situation, where it is possible to treat different parts of one molecule as linked but functionally separate objects, that possess an ability to move at different times and speeds 1 . That makes the field of motor proteins a desirable testing ground for application of various theoretical models, that aim to filter the inherent complexity of biological systems 2-5 . Among the motors, members of the kinesin protein superfamily are consistently used throughout the years in research focusing on molecular motors' mechanical properties, both in vitro 2 and in silico 2,4-6 .A conformational change of a region within kinesin has been shown to be associated with its movement along microtubule 7 . This region has been labeled as neck linker. The label refers to a concise (less than 20 amino acids length) amino acid sequence in a single kinesin head that acts as a bridge between α-6 helix in coiled-coil dimerization domain and α-7 helix in the core motor domain, respectively 8 . While its exact length as well as the placement within sequence of its N-and C-termini are not set in stone 9 , some of its residue patterns are present across all kinesin families, while neck linker sequences within a single family are very similar 8 . All this suggest that parts of neck linker region have been conserved, which makes neck linker a non-random, specific sequence. An on-going accumulation of experimental data evidence suggests that a transition of the neck linker from a disordered (random coil) state to an ordered (β-sheet) conformation is a key factor in determining a mechanism of force-generation that is a crucial element of molecular motors' ability to move along microtubules 7,8 . Attempts at substituting neck linker with a peptide of a different sequence, as well as exte...

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Kinesin rotates unidirectionally and generates torque while walking on microtubules

Cited by 90 publications

References 44 publications

Germanium nanospheres for ultraresolution picotensiometry of kinesin motors

Germanium nanospheres for ultraresolution picotensiometry of kinesin motors

Detection of self-generated nanowaves on the interface of an evaporating sessile water droplet

Delineating elastic properties of kinesin linker and their sensitivity to point mutations

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