Peer review of: Kinesin-14 motors drive a right-handed helical motion of antiparallel microtubules around (2020)

Reviewers' comments:
Reviewer #1 (Remarks to the Author):
The authors systematically compare the rotational motion of microtubules that Ncd motors
crosslink in vitro. It is well known that several mitotic kinesins such as kinesin-5 and kinesin-14
crosslink two microtubules. An emerging view is that they exhibit off-axis components and
generate the rotation of a microtubule around the long axis of the microtubule as they move on
the other microtubule, suggesting an important role in generating twisting of the mitotic spindle.
Despite its importance of Ncd in the spindle organization, the rotational motion of microtubules
driven by Ncd has not been sufficiently addressed so far. The authors presented a discovery that
Ncd can generate the coiling of microtubule bundles, leading to the generation of chirality in the
mitotic spindle possibly in collaboration with kinesin-5. A weak point of this paper is that the
authors were not able to assess the dependence of the motor density on the helical pitches in 3D
sliding motility assays. However, this point was carefully addressed by 2D microtubule motility
assays performed on a planar glass surface using a FLIC-based technique. This paper will be an
important contribution to the biophysics and cell biology fields, and can also be a useful guide for
those who are interested in applying the 3D measurement of nanometer-scale objects to their own
research fields. The manuscript is well organized and described. I recommend that it be accepted
for publication in Nature Communications.
Additional comments:
- The difference between the current study and the previous work (Nitzsche et al., PNAS 2016)
seems a bit unclear since both papers describe microtubule gliding on Ncd-coated surfaces that
rotated around their longitudinal axes. What is novel in the current paper is the possibility that Ncd
can generate the coiling of microtubule bundles, which is relevant to the recent finding about the
chirality of the mitotic spindle. Although, in principle, this result could be anticipated from the
previous results that Ncd exhibits off-axis components in the microtubule gliding assays, the actual
experimental data about crosslinked microtubule bundles are of critical importance when building a
molecular model of the mitotic spindle assembly.
 - If possible, it might be better to mention briefly the quantitative relationship between the
 size/direction of the powerstroke of Ncd that descrived in the previous study, and the resultant
 rotational motion of microtubules. Although this point might be discussed in the previous study
 (Nitzsche et al., PNAS 2016), the discussion must be too complicated for readers to follow.
Reviewer #2 (Remarks to the Author):
In this paper Mitra et al. have upgraded an in vitro system developed previously by the same
authors (Mitra et al. PNAS 2018) to show that Ncd, a non processive minus end directed motor
protein that has been shown to slide microtubules (Fink et al. Nat Cel Biol, 2009), in a three-
dimensional setting can carry a short stabilised antiparallel microtubule around a longer one
following a helical, right-handed trajectory. Even though this is the first time such a specific setting
has been shown, the amount of previously published data that already shows helical motion of
different kinesin motors (as cited by authors themselves) and observations that spindle is a helical
structure in both human and yeast cells (Novak et al. Nat. Comm 2018; Winey et al. J. Cell Biol
1995; Ding et al. J Cell Biol. 1993) in my opinion do not make this paper in its current state
acceptable for publication in Nature Communication. Even though there is a difference between a
motor protein carrying a quantum dot and several motor proteins carrying a short microtubule, I
was not convinced by the authors’ arguments to see how this work represents a significant jump in
our current understanding of microtubule sliding, especially in the case of the mammalian mitotic
spindle where microtubules of similar lengths seem to coil around each other in order to produce
torque of microtubule bundles. Rotation of a short microtubule around bigger one is not the same
 as twisting of microtubules around each other.
 This being said I believe the work can be accepted for publication with some additional
 experimental data. As I mentioned the biological motivation of the authors seems to be the mitotic
 spindle. In that case, as proposed in the cartoon of the Figure 5F, the authors should do an
 experiment involving two long and fixed microtubules to see whether the motor proteins can exert
 enough force to cause the coiling of two microtubules. This data would in my opinion be much
 more informative for the mitotic spindle context, and would actually mirror what is believed to be
 happening in vivo, where microtubules are fixed at the spindle poles.
 Further comments:
 The authors on more than one occasion misquote the Novak et al. article as saying the spindle has
 right-handed chirality. This is not true, as the article clearly states that the spindle has left-handed
 chirality. in the last section of Discussion, the authors discuss how the interplay of Eg5 and Ncd
 might produce such chirality of the spindle. Taking into account that the spindle is left-handed and
 not right-handed, the authors should adjust their arguments and rewrite the discussion
 accordingly. Statement that Eg5 and Ncd with their movements of opposite handiness and
 direction result in torques that add up would benefit from a graphical depiction to make it more
 understandable. This statement in my opinion is rather important even though it is part of
 discussion, since it tries to explain the possible interplay of motors with different directionalities
 and torques present in vivo.
 Probably my biggest concern with this work is that In Figure 2A the pitches vary considerably with
 the same concentration at 4nM Ncd. Is seems to me they vary much more within this one
 concentration, than in the Figure 4D where the concentration of the Ncd was changed? Also in
 Figure 4D there seems to be no difference between 2.4 nM and 6 nM Ncd and no pitch is close to 3
 um, observable at 4nM in Figure 2A. How do authors explain these inconsistencies apart from the
 fact that in the latter experiment they are using a 2D motility assay and not the suspended
 microtubule assay?
 In a situation with parallel transport microtubules related to Figure 2 and Supplementary Figure 2,
 the comparison between suspended template (Figure 2C) and surface-immobilized template
 (Supplementary Figure 2C) is confusing. The distribution on the surface-immobilized template in
 my opinion does not show a” minor bias to the right” as stated by authors but shows a quite big
 bias to the right. In any case, for me this difference to the bimodal distribution seen in suspended
 template seems unexpected, why the authors think this is happening?
 Also, a clearer way of quantifying this bias would help to avoid imprecise wording.
 The authors attribute to autofluorescence the inability to measure the concentration of Ncd-GFP.
 Would it be possible to measure it with another fluorescent tag? Would a time-gated imaging
 approach be helpful to eliminate autofluorescence in this setup?
 In supplementary figure 4C caption text, I am unconvinced by the hard claim that “At 0.6nM Ncd
 the microtubule did not show any clear rotational movement”. Looking at the example trajectory
 shown below the kymographs it is clearly visible that at 0.6 nM there is rotational movement. To
 me it looks quite similar to what is observed after the switch of concentration considering its much
 shorter duration. Morover, the authors quantify this rotational movement at 0.6 nM Ncd as having
 the rotational frequency similar to 6 nM Ncd (supplementary figure 4B right panel). Also, in Figure
 4 at 0.6 nM Ncd, the rotational pitch is close to 2 whereas at 6 nM Ncd it is close to 0.8. In the
 main text however the authors are more prudent with their wording and talk about “few transport
 microtubules” that rotate at 0.6 nM.
 I noticed in these experiments the very low N for 0.6 nM Ncd experiments (N=7) compared to
 N=29 for 6 nM Ncd. Why is this the case? On the same note both Figure 4 and Supplementary
 Figure 4 lack p-values.
 The mention of chirality observed in yeast spindles in my opinion also merits a mention in the
 discussion, specifically Winey et al. J. Cell Biol 1995; Ding et al. J Cell Biol. 1993.
Supplementary Movie 3 is not referenced in the main text.
Supplementary Figure 4E is referenced in the text, yet there is no Supplementary Figure 4E.