Peer review of: Talin dissociates from RIAM and associates to vinculin sequentially in response to the (2020)

Reviewers' comments:
Reviewer #1 (Remarks to the Author):
This technically very elegant study analyzes the force-dependent interaction of the two adhesion
proteins RIAM and vinculin to talin, a crucial integrin activator and force transducer. By further
developing a previously established in vitro reconstitution assay, the authors demonstrate the
expected actomyosin-dependent binding of vinculin to talin’s R1–R3 domain but also provide direct
evidence for a force-dependent interaction of vinculin with talin R11. The authors then show that
actomyosin forces induce an unbinding of RIAM from talin R1–R3 and R11, while the interaction of
RIAM with talin R7–R8 seems insensitive to actomyosin forces. Combining protein fragments from talin,
vinculin and RIAM in one assay reveals a concomitant RIAM dissociation from and vinculin association
with talin R1–R3 and talin R11 upon actomyosin contraction. Increasing concentrations of RIAM inhibit
vinculin binding to R1–R3, but not to R11 suggesting a differential regulation of force-dependent
vinculin binding to these domains.
Overall, this is a well-written manuscript, the data are clear and statistically sound, and the results
provide intriguing insights into the regulation of talin by mechanical force. Previous work already
indicated that RIAM and vinculin undergo mutual exclusive interaction with R1–R3 (Goult et al. JBC,
2013), which is nicely confirmed in this paper. In addition, the manuscript shows how the additional
RIAM/vinculin binding sites in R7–R8 and in R11 are regulated by actomyosin force. It would be nice
to see whether increasing concentrations of vinculin (i.e. higher concentrations than used in Fig. 4E, G)
can displace RIAM from talin, as this has been suggested before (Li e al, MBoC, 2013). However, the
current data sets are convincing and do not require, from my perspective, any major adjustments.
The only minor drawback of the study is the absence of any attempts to confirm the observations in
an independent fashion. It would certainly increase the impact of the study if some of the predictions
were being tested in cell culture experiments. I would not consider such cell culture experiments
mandatory, because the data are conclusive and deserve to be published.
Reviewer #2 (Remarks to the Author):
In this manuscript, the authors use an elegant in vitro reconstitution system with time-lapse imaging
to investigate mechanoresponses of talin in RIAM and vinculin bindings. They clearly show that RIAM
dissociates from and vinculin binds to talin domains in response to assembly of the actomyosin
cytoskeleton. They observe that different talin domains show distinct responses in actomyosin-
dependent RIAM dissociation and vinculin association. With kinetics analyses, they show the sequential
actions of talin in which actomyosin accumulation induces RIAM dissociation that is followed by
vinculin binding. Furthermore, they reveal that RIAM inhibits actomyosin-dependent binding of vinculin
to talin.
This is an interesting and well-written paper that would contribute to our understanding of molecular
basis for mechanoresponses of cell adhesion complexes. However, the reviewer has a few concerns
that should be addressed before publication.
1) The authors assume that “force” is responsible for actomyosin-dependent RAIM dissociation from
and vinculin association with talin. However, they applied actin and myosin II at a time and did not
distinguish individual actions of these components, e.g., actin polymerization, F-actin bundling by
myosin II, and force generation by actin-myosin II sliding. Therefore, it is currently unclear whether
force is a true stimulus that induces the RAIM dissociation and the vinculin association in the system
of this study. Several additional controls should be tested. For example, addition of 1) actin alone, and
2) actin together with myosin II that can crosslink F-actin but has no motor activity (e.g., N-
 ethylmaleimide-treated myosin II; Smith et al. 2007 Biophys J) would provide good controls.
 Furthermore, effects of inhibition of myosin II ATPase activity need to be tested. Even though
 conventional blebbistatin may not be compatible with time-lapse fluorescence imaging, a photostable
 and non-fluorescent derivative of blebbistatin is now commercially available.
 2) Even in the presence of actomyosin, talin R7-R8 shows no apparent vinculin binding and only
 moderate RIAM dissociation. The authors discuss that the poor response of R7-R8 in vinculin binding
 and RIAM dissociation may arise from high mechanical stability of R7-R8. However, considering the
 fact that R7-R8 directly binds to F-actin (Atherton et al. 2015 Nat Commun), a simple possibility is
 that F-actin binding to R7-R8 may block RIAM dissociation from and vinculin binding to R7-R8.
 Alternatively, simultaneous pulling at R13 and at R7-R8 by actomyosin through F-actin binding to R13
 and R7-R8 may result in no apparent tension development between R13 and R7-R8, leading to little
 RIAM dissociation/vinculin association.
 3) In this in vitro study, the authors use the vinculin head domain as a vinculin model and show its
 actomyosin-dependent binding to talin domains. On the other hand, studies using cells have reported
 that in contrast to full-length vinculin, the head domain only of vinculin constitutively binds to talin at
 focal adhesions independently of actomyosin activity (Humphries et al. 2007 J Cell Biol; Carisey et al.
 2013 Curr Biol; Hirata et al. 2014 Am J Physiol Cell Physiol). Please discuss why the vinculin head
 domain behaves differently in talin binding between in vitro and in cells and how relevant the results
 of vinculin head-talin binding obtained in this study to vinculin-talin binding at focal adhesions in cells.