Kinesin-1, -2, and -3 motors use family-specific mechanochemical strategies to effectively compete with dynein during bidirectional transport
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Evaluation Summary:
In their study, Gicking et al. study the physical properties of artificial complexes composed of the dynein-dynactin-BicD2 (DDB) complex linked to one of three classes of kinesins (1, 2, or 3) via a DNA scaffold. They find that all three kinesins can move to the plus-end of microtubules when coupled to the DDB complex. This is surprising because motors in the kinesin-2 and kinesin-3 families have been shown to have a higher load sensitivity. However, the authors show that the faster reattachment kinetics of these motors compensate for their faster detachment rates under load. This work is relevant to both the biophysics field for advancing knowledge in fundamental science, and in the neuroscience field since disruption of neuronal transport leads to a variety of neurodegenerative diseases.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Bidirectional cargo transport in neurons requires competing activity of motors from the kinesin-1, -2, and -3 superfamilies against cytoplasmic dynein-1. Previous studies demonstrated that when kinesin-1 attached to dynein-dynactin-BicD2 (DDB) complex, the tethered motors move slowly with a slight plus-end bias, suggesting kinesin-1 overpowers DDB but DDB generates a substantial hindering load. Compared to kinesin-1, motors from the kinesin-2 and -3 families display a higher sensitivity to load in single-molecule assays and are thus predicted to be overpowered by dynein complexes in cargo transport. To test this prediction, we used a DNA scaffold to pair DDB with members of the kinesin-1, -2, and -3 families to recreate bidirectional transport in vitro, and tracked the motor pairs using two-channel TIRF microscopy. Unexpectedly, we find that when both kinesin and dynein are engaged and stepping on the microtubule, kinesin-1, -2, and -3 motors are able to effectively withstand hindering loads generated by DDB. Stochastic stepping simulations reveal that kinesin-2 and -3 motors compensate for their faster detachment rates under load with faster reattachment kinetics. The similar performance between the three kinesin transport families highlights how motor kinetics play critical roles in balancing forces between kinesin and dynein, and emphasizes the importance of motor regulation by cargo adaptors, regulatory proteins, and the microtubule track for tuning the speed and directionality of cargo transport in cells.
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Evaluation Summary:
In their study, Gicking et al. study the physical properties of artificial complexes composed of the dynein-dynactin-BicD2 (DDB) complex linked to one of three classes of kinesins (1, 2, or 3) via a DNA scaffold. They find that all three kinesins can move to the plus-end of microtubules when coupled to the DDB complex. This is surprising because motors in the kinesin-2 and kinesin-3 families have been shown to have a higher load sensitivity. However, the authors show that the faster reattachment kinetics of these motors compensate for their faster detachment rates under load. This work is relevant to both the biophysics field for advancing knowledge in fundamental science, and in the neuroscience field since disruption of neuronal transport leads to a variety of neurodegenerative diseases.
(This preprint has been …
Evaluation Summary:
In their study, Gicking et al. study the physical properties of artificial complexes composed of the dynein-dynactin-BicD2 (DDB) complex linked to one of three classes of kinesins (1, 2, or 3) via a DNA scaffold. They find that all three kinesins can move to the plus-end of microtubules when coupled to the DDB complex. This is surprising because motors in the kinesin-2 and kinesin-3 families have been shown to have a higher load sensitivity. However, the authors show that the faster reattachment kinetics of these motors compensate for their faster detachment rates under load. This work is relevant to both the biophysics field for advancing knowledge in fundamental science, and in the neuroscience field since disruption of neuronal transport leads to a variety of neurodegenerative diseases.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors use a model system to investigate how three classes of kinesins (1, 2 and 3) interact with the dynein-dynactin-truncated BicD2 complex when coupled via a DNA scaffold. Complexes with kinesin 1 have been shown to have a plus-end bias, but unexpectedly the authors show that this is also true for kinesins 2 and 3 despite these motors having a higher load sensitivity. The authors reconcile this finding by showing via simulations that faster reattachment kinetics compensate for faster detachment rates under load. They conclude that motor kinetics is another important feature in understanding both the velocity and directionality that cargo is transported.
This is the first study directly comparing three classes of constitutively active kinesin motors versus DDB in a controlled fashion, which is a …
Reviewer #1 (Public Review):
The authors use a model system to investigate how three classes of kinesins (1, 2 and 3) interact with the dynein-dynactin-truncated BicD2 complex when coupled via a DNA scaffold. Complexes with kinesin 1 have been shown to have a plus-end bias, but unexpectedly the authors show that this is also true for kinesins 2 and 3 despite these motors having a higher load sensitivity. The authors reconcile this finding by showing via simulations that faster reattachment kinetics compensate for faster detachment rates under load. They conclude that motor kinetics is another important feature in understanding both the velocity and directionality that cargo is transported.
This is the first study directly comparing three classes of constitutively active kinesin motors versus DDB in a controlled fashion, which is a strength of this study. The caveat is that these results may require modification when dynein and kinesin are coupled via an activating adaptor rather than DNA. However, the studies in the current manuscript are a required prerequisite, as different activating adaptors would be needed for the different classes of kinesin, thus introducing another variable into how the two classes of motors interact. Moreover, results from these studies can be used as a platform for further investigation of the effect of MAPs, regulatory proteins, and PTMs of the MT on model bidirectional complexes.
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Reviewer #2 (Public Review):
The authors present an intriguing study regarding bidirectional transport of cargoes by kinesin and dynein. They examine the mechanistic details of the "paradox of codependence" between kinesin and dynein motility by coupling dynein-dynactin-BicD2 (DDB) and kinesins -1, -2, and -3 via a DNA linker and observing motility using TIRF. Kinesins from different families with differing single molecule motility properties were predicted to differentially affect bidirectional transport, but all three kinesins are able to effectively withstand hindering loads from DDB. Experimental results extracted from unloaded motility assays were compared to simulations to understand the kinesin family-specific implications on observed transport properties.
The DNA-linked motor assay is innovative and appropriate for this work. …
Reviewer #2 (Public Review):
The authors present an intriguing study regarding bidirectional transport of cargoes by kinesin and dynein. They examine the mechanistic details of the "paradox of codependence" between kinesin and dynein motility by coupling dynein-dynactin-BicD2 (DDB) and kinesins -1, -2, and -3 via a DNA linker and observing motility using TIRF. Kinesins from different families with differing single molecule motility properties were predicted to differentially affect bidirectional transport, but all three kinesins are able to effectively withstand hindering loads from DDB. Experimental results extracted from unloaded motility assays were compared to simulations to understand the kinesin family-specific implications on observed transport properties.
The DNA-linked motor assay is innovative and appropriate for this work. Parameters extracted from the unloaded TIRF motility assays are appropriate, well-justified, and support the authors' conclusions. Coupling experiments with theory, as well as previous work, also gives the working mechanism a substantial foundation. The study is rigorous, well-written, and will be of interest to a wide biophysical audience.
The authors should consider whether the DNA linker length and stiffness may affect the proposed cooperation between dynein and kinesin. Motor spacing and linker stiffness have been shown in other systems to affect motor coupling. Further, while the authors report on shortcomings of optical trapping assays and their effect on detachment rates (which are important for the cooperation mechanism here), trapping experiments could help clarify their proposed mechanisms for how kinesin -1, -2, and -3 (which behave differently as single molecules) can all withstand dynein's "tug-of-war."
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Reviewer #3 (Public Review):
Gicking et al. analyze the relation between DDB (retrograde motor complex) and different members of kinesin family. The authors directly linked DDB and kinesin-1, -2 and -3 using DNA linker. Consistent with previous force measurement, kinesin-1 can dominate DDB. On the other hand, it is an unexpected and interesting observation that kinesin-2 and -3 can withstand loads by DDB because these motors are sensitive to load and easily detach from microtubules under loaded conditions in optical tweezers experiments. The authors performed computer simulations and suggested that fast detachment in kinesin-2 and -3 can be antagonized by fast reattachment. The work will impact thinking about physical properties of kinesins under loaded conditions.
Weaknesses:
(1) To show DDB-kinesin-2 relation, the authors analyzed …Reviewer #3 (Public Review):
Gicking et al. analyze the relation between DDB (retrograde motor complex) and different members of kinesin family. The authors directly linked DDB and kinesin-1, -2 and -3 using DNA linker. Consistent with previous force measurement, kinesin-1 can dominate DDB. On the other hand, it is an unexpected and interesting observation that kinesin-2 and -3 can withstand loads by DDB because these motors are sensitive to load and easily detach from microtubules under loaded conditions in optical tweezers experiments. The authors performed computer simulations and suggested that fast detachment in kinesin-2 and -3 can be antagonized by fast reattachment. The work will impact thinking about physical properties of kinesins under loaded conditions.
Weaknesses:
(1) To show DDB-kinesin-2 relation, the authors analyzed KIF3A/KIF3A homodimers. This reviewer does not think KIF3A/KIF3A homodimers represent kinesin-2. The motor domain of kinesin-2 is a heterodimer composed of KIF3A and KIF3B in the cell. The authors have previously shown that properties of KIF3A/KIF3A homodimer are different from those of KIF3A/KIF3B (Andreasson et al., Curr Biol., 2015).(2) While these in vitro results are interesting, physiological meaning of these findings is not very clear.
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