Resolving kinesin stepping: one head at a time
Posted on: 11 October 2019
Preprint posted on 28 May 2019
Article now published in Life Science Alliance at http://dx.doi.org/10.26508/lsa.201900456
The power of dual colour FIONA technique to track the stepping of KLP11 and KLP20 heteromeric motors
Selected by Ben Craske, Gaetan Dias Mirandela, Thibault Legal and Toni McHugh
Categories: biochemistry, cell biology, molecular biology
Context:
Kinesins are essential molecular motors responsible for the transport of biological materials such as melanosomes or chromosomes on microtubules. Kinesin proteins are commonly known to walk toward the plus-end of microtubules via their two motor domains. The binding/dissociation of each of the motor domains to the microtubule is tightly mediated by ATP binding/hydrolysis. Some of the kinesin-2 molecular motors form heterotrimeric complexes composed of two different motor domains. In this case, the consistency of a “run” is questionable: whether each of the motor domains steps and resides on the microtubule in the same way or behaves differently remains to be unraveled. Additionally, another level of complexity has to be taken into account: the auto-inhibition effect mediated by the tail. The tail of kinesins is known to play an important role in the regulation of the motor activity. However, how the tail is regulating each of the motor domains’ activity remains debatable in the case of hetero-oligomeric states of certain kinesin-2 motors.
Therefore, to shed light on these questions, the authors used dual-color fluorescence imaging with one nanometer accuracy (dcFIONA) in order to track independently each of the motor domains, KLP11 and KLP20, of a heterotrimeric kinesin-2 protein from C. elegans.
Key findings:
In this work, Willi L. Steep and Zeynep Ökten used a heterodimeric processive variant of the heterotrimeric kinesin-2 from C.elegans: The variant, eeKLP11/KLP20, contains activating mutations in the stalk of the molecular motor reducing the auto-inhibition effect of the tail (Figure 1).
First, the authors demonstrated that, under limited concentration of ATP, they were able to measure, within nanometer resolution, the stepping and dwell times of two different motor domains at the same time after labelling each of them individually. The step length of the dual-labelled Kinesin-2 remains unchanged in light of the single labelled one suggesting that the dual-tagging process does not affect the kinesin motor, thus validating the dual-labelling approach undertaken.
For the dwell-time of each motor domain, a double exponential distribution is expected when measured on a single-labelled motor. In fact, this is attesting of the ATP-waiting time for the label motor and hidden step of the second motor domain. Hence, by measuring the two motor domains simultaneously, the authors expected to observe a single exponential for each of the motor domains (assuming that the hidden step of the second motor domain will be known here). Surprisingly, the authors were able to observe a single and double exponential distribution for KLP20 and KLP11 respectively (Figure 2).
Therefore, this result indicates for the first time that a second rate limiting step exists for eeKLP11 additionally to the ATP binding one. The authors demonstrated that this finding was consistent regardless of the tag or fluorophore employed during the study.
A hypothesis regarding the second rate-limiting step by the authors was the asymmetric auto-inhibition of the Kinesin-2 mediated by both the position of KLP11 and by the tail. The authors have previously shown that the auto-inhibition is reduced if the motor domains are swapped in relation to the tails for a KLP11-20/KLP20-11 with non-modified tails. The new technique used now allowed them to study the direct influence of the tail on the heads’ activity in the “EE” and WT version of the motor.
To assess this, the authors investigate the effect of the tail on the late dwell time for the “stalk variant” eeKLP11/20 compared to the wtKLP11/20 under saturated and excess concentration of ATP. The authors observed a decrease of 1.6 fold between the variant and WT which was corresponding to the decreased speed of the WT in ATP excess condition. Together with the previous experiment, this result provides the first evidence of an asymmetric effect of the tail of kinesin-2 on the stepping from an in vitro perspective.
doi: https://doi.org/10.1242/prelights.14427
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