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Resolving kinesin stepping: one head at a time

Willi L. Stepp, Zeynep Ökten

Preprint posted on 28 May 2019 https://doi.org/10.1101/651281

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

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.

Tags: fiona, heteromeric kinesin-2, molecular motors

Posted on: 11 October 2019

doi: https://doi.org/10.1242/prelights.14427

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Author's response

Willi L. Stepp and Dr Zeynep Ökten shared

Would it be possible to fine tune the ATP concentration to quantitatively assess the second rate limiting step described by this approach?

Unfortunately, with this experimental setup, the range of ATP concentrations that allows us to measure dcFIONA data is rather limited. A lower ATP concentration sets high demands on the stability of the setup (the microscope, but also the microtubules etc). A higher ATP concentration would demand for faster imaging which is challenging when imaging with single dyes. Qdots are no option here, as all that we tested bind unspecifically to our kinesin-2s.

We instead measured the stepping of a head-swapped KLP11-20/20-11 and show, that both speed and KLP11 dwell time are inbetween the wtKLP and eeKLP. Thus confirming, that this second rate is indeed linked to the auto-inhibition of the motor.

Are dwell times of each of the motor domains of a homodimeric kinesin distributed as a single exponential population?

There has been one study on a double labelled homodimeric myosin-V before. Unfortunately, there seems to be no information on the dwell-times of this motor. Maybe the authors could comment on this question.

Warshaw DM, Kennedy GG, Work SS, Krementsova EB, Beck S, Trybus KM (2005) Differential labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity. Biophys J 88: L30–L32.

Is this second rate limiting reaction a conserved mechanism across kinesin-2 protein family?

Kinesin-2 is a motor that was very specifically evolved for the function in cilia. Also, there are very few other motors that show heteromeric activity. While we do not have any information on the function of this second rate-limiting step in the KLP11 stepping cycle, we think that it might be linked to one of the specific mechanisms for IFT. This would suggest a high probability for the mechanism to be also present in other kinesin-2 motors.

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