Peer review of: Tug-of-war between actomyosin-driven antagonistic forces determines the positioning (2020)

Reports from round 1:
Rev#1
The authors have done an excellent job addressing the reviewer concerns, including addition of significant new experiments, all of which support their model. All critiques have been addressed, this is a lovely paper.
Rev#2
The authors have sufficiently addressed referee concerns. The manuscript is improved and appropriate for publication in Nature Communications
Rev#3
 The authors went a long way to answer my previous and other reviewer comments. Overall the
 manuscript has improved with the new experiments and the qualitative understanding of the
 process seems to be supported by the data. I only have two concerns left.
One minor is that the authors provide new analysis to distinguish between bulk contraction vs ring
contraction. For that they refer to the new supplementary figure 4 that shows that the intensity of
the ring goes up as it contracts whereas the bulk intensity remain constant. This would indeed
suggest that the contraction is happening in the ring. However, this seems to contradict their exact
same measurements in supplementary figure 2 :”(f) Fluorescence intensity along the each broken
line in e, which also corresponds to the intensity at the arrows with the same color in d. Both the
F-actin densities on the rim and inside of the wave increased during the contraction. The thickness
of the spacer was 100 μm. Scale bars, 1 mm. “ Could the authors clarify the discrepancy? Does it
depend on day to day or example taken?
My main concern is still related to the theory:
For their theory the authors state: “We have proposed that the off-centering is mediated by the
percolated actomyosin bridge”, and that the bridge emerges from “low number of remained F-actin
and newly assembled F-actin stochastically form bridges between the cluster and the boundary.”
The authors are assuming the bridge is pre-formed and just calculate the probability that cross
linkers connect the filaments that form the bridge. But, why don’t we see the bridge than initially
after the wave? From their images it looks that actin is actually polymerized as the bridge is form
(See figure 4c. After laser ablation a new bridge is formed but no wave has formed before it).
Since the authors only consider the top 5% of actin lengths, shouldn’t this have a dramatic effect
on time scales too? Meaning that it may be very difficult to actually have a bridge made of actin
filaments longer than the 5% population as opposed of having a bridge made of short actin
filaments. Of course that time scale to percolate a bridge of shorter filaments is longer, but it may
be compensated by the time scales (or probability required to create enough long (~6 um)
filaments. Also, how can the authors really justify to use 6 um as length based on their measured
length distributions? These distributions clearly show an average of ~1um, and almost no
filaments larger than 5 um.I think it is necessary that the authors clarify this point, comment on
the origin of actin in these bridges, what would be the time scales associated to having only the
5% population of actin, and recalculate their predictions based on the average actin length they
measure.
The answer the authors give to use ~6 um is flawed. They argue “In the proposed model, the
probability that a bridge is formed by the length L of F-actin in a droplet with radius R is written as
P=(1/2)R/L. If we compare the probability with different actin length, L=10 μm and L=5 μm, in a
droplet with radius R=50 μm, we can estimate P=(1/2)5 and P=(1/2)10 respectively. Thus, only 2
times change in F-actin length decreases the bridge formation probability ~30 times.”. The authors
are missing the probability of actually getting actin filaments of that particular length. With their
measured length distribution, this can also change by a decade from ~1 to ~5 um so it could
counteract their estimated probability, so the authors need to take this into account in their
argument.
The authors distinguish gelation vs percolation to explain the difference of timescales, which they
 argue leads to a faster gelation time. However, my understanding is that gelation is a connectivity
 transition, and that previous research has used percolation as a theory to explain the gelation
 process. In both cases, they are network-connectivity transitions. Gelation further implies the
 emergence of material properties such as a finite young modulus, whereas percolation theory
 makes no statement (or can’t explain) about it and it is just restricted to the emergence of a
 system size cluster. Thus, I don’t really understand what the authors imply by “Since percolation is
 possible even without gelation, it is natural to assume that the bridge formation does not have to
 rely on the gelation process.”, and why would we expect that the percolation would take longer?
 Could the authors explain what the explicit differences between percolation and gelation they are
 impling?