Mechanics of single kinesin molecules measured by optical trapping nanometry

Abstract

We have analyzed the mechanics of individual kinesin molecules by optical trapping nanometry. A kinesin molecule was adsorbed onto a latex bead, which was captured by an optical trap and brought into contact with an axoneme that was bound to a glass surface. The displacement of kinesin during force generation was determined by measuring the position of the beads with nanometer accuracy. As the displacement of kinesin was attenuated because of the compliance of the kinesin-to-bead and kinesin-to-microtubule linkages, the compliance was monitored during force generation and was used to correct the displacement of kinesin. Thus the velocity and the unitary steps could be obtained accurately over a wide force range. The force-velocity curves were linear from 0 to a maximum force at 10 microM and 1 mM ATP, and the maximum force was approximately 7 pN, which is larger by approximately 30% than values previously reported. Kinesin exhibited forward and occasionally backward stepwise displacements with a size of approximately 8 nm. The histograms of step dwell time show a monotonic decrease with time. Model calculations indicate that each kinesin head steps by 16-nm, whereas kinesin molecule steps by 8-nm.

Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

“…The stall force under load for kinesin was measured to be around 5 -7pN [204,376,244]. Contradictory results have been found for the stall force of dynein.…”

“…Conversely, some informations on the mechanochemical cycle can be extracted from experimental measurements of the variations of stepping rates under various conditions [204]. As these processes are stochastic (and the stochasticity is indeed included in the models), not only the mean value of the motor displacement but also the distribution of the fluctuations can bring some information on the mechanochemical cycle, for example to estimate the number of states in the cycle [42].…”

Emergence of flagellar beating from the collective behavior of individual ATP-powered dyneins

“…4b. The dyneins are modeled as periodic force generators with an inherent duty ratio governed by the ATP hydrolysis cycle [23,24]. We assume that the dyneins will linearly increase the sliding force during their active phase (governed by t a ) until the stall force F stall is reached, after which the dynein detaches and the force drops to zero during the recovery stroke (waiting phase) of the dyneins (governed by t w ), see Figs.…”

“…2, making the dyneins periodic force generators [23], where the time period of the force generation is governed by the ATP hydrolysis cycle and depends on the concentration of ATP in the solution [23,24].…”

A modified active Brownian dynamics model using asymmetric energy conversion and its application to the molecular motor system

“…Step size is known to be independent of the ATP concentration and external load and is about 8 nm, which corresponds to the axial length of one tubulin-dimer, the building block of the microtubule [1,[8][9][10][11][12][13][14][15][16][17]. In spite of the extensive single-molecule studies, how kinesin functions to generate the force for discrete translation on a single protofilament of the microtubule is not well understood, leaving many questions on, for instance, substeps, backsteps, and fraction of power stroke and diffusion to each step [10,[18][19][20][21][22][23].…”