Article activity feed

  1. eLife assessment

    The manuscript provides valuable insights into the regulatory role and mechanisms of the spectrin cytoskeleton in mechanotransduction in Drosophila. The data are compelling in establishing the role of spectrins, but questions remain regarding some of the precise mechanisms involved. The work will be of interest to cell and developmental biologists, particularly those who focus on mechanotransduction and the cytoskeleton.

  2. Reviewer #1 (Public Review):

    Ibar and colleagues investigate the function of spectrin in Drosophila wing imaginal discs and its effect on the Hippo pathway and myosin activity. The authors find that both βH-Spec and its canonical binding partner α-Spec reduce junctional localization of the protein Jub and thereby restrict Jub's inhibitory effect on Hippo signaling resulting in activation of the Hippo effector Yorkie regulating tissue shape and organ size. From genetic epistasis analysis and analysis of protein localization, the authors conclude that βH-Spec and α-Spec act independently in this regulation. The major point of this study is that the apical localization of βH-Spec and myosin is mutually exclusive and that the proteins antagonize each other's activity in wing discs. In vitro co-sedimentation assays and in silico structural modeling suggest that this antagonization is due to a competition of βH-Spec and myosin for F-actin binding.

    The study's strengths are the genetic perturbation that is the basis for the epistasis analysis which includes specific knockdowns of the genes of interest as well as an elegant CRISPR-based overexpression system with great tissue specificity. The choice of the model for such an in-depth analysis of pathway dependencies in a well-characterized tissue makes it possible to identify and characterize quantitative differences between closely entangled and mutually dependent components. The method of quantifying protein localization and abundance is common for multiple figures which makes it easy to assess differences across experiments.

    A weakness in the methodology is the link to tissue tension and conclusions about tissue mechanics. Methods that directly affect tissue tension and a more thorough and systematic application of laser ablation experiments would be needed to profoundly investigate mechanosensation and consequential effects on tissue tension by the various genetic perturbations. While the in-silico analysis of competing for F-actin binding sites for βH-Spec and myosin appears logical and supports the authors' claims, no point mutation or truncations were used to test these results in vivo. In its current structure the manuscript's strength, the genetic perturbations, is compromised by missing clear assessments of knockdown efficiencies early in the manuscript and other controls such as the actual effect on myosin by ROCK overactivation.

    The flow of experiments is logical and in general, the author's conclusions are supported by the presented data. The findings are very well embedded into the context of relevant literature and both confronting and confirming literature are discussed.

    The study shows how components of the cytoskeleton are directly involved in the regulation of the mechanosensitive Hippo pathway in vivo and thus ultimately regulate organ size supporting previous data in other contexts. The molecular mechanism regulating myosin activity by out-competing it for F-actin binding has been observed for small actin-binding proteins such as cofilin but is a new mode for such a big, membrane-associated actin-binding protein. This may inspire future experiments in different morphogenetic contexts for the investigation of similar mechanisms. For example, the antagonistic activity of βH-Spec and myosin in this tissue context might help explain phenomena in other systems such as spectrin-dependent ratcheting of apical constriction during mesoderm invagination (as the authors discuss). Against the classical view, the work shows that βH-Spec can act independently of α-Spec. Together the results will be of interest to the cell biology community with a focus on the cytoskeleton and mechanotransduction.

  3. Reviewer #2 (Public Review):

    Ibar and colleagues address the role of the spectrin cytoskeleton in the regulation of tissue growth and Hippo signaling in an attempt to elucidate the underlying molecular mechanism(s) and reconcile existing data. Previous reports in the field have suggested three distinct mechanisms by which the Spectrin cytoskeleton regulates Hippo signaling and this is, at least in part, due to the fact that different groups have mainly focused on different spectrins (alpha, beta, or beta-heavy) in previous reports.

    The authors start their investigation by trying to reconcile their previous data on the role of Ajuba in the regulation of Hippo signaling via mechanotransduction and previous observations suggesting that Spectrins affect Hippo signaling independently of any effect on myosin levels or Ajuba localization. Contrary to previous reports, the authors reveal that, indeed, depletion of alpha- and beta-heavy-spectrin leads to an increase in myosin levels at the apical membrane. Moreover, the authors also reveal that the depletion of spectrins leads to an increase in Ajuba levels.

    The authors suggest that Ajuba is required for the effect of beta-heavy spectrin. However, it is still formally possible that this could be a parallel pathway that is being masked by the strong phenotype of Ajuba RNAi flies.

    One of the major points of the manuscript is the observation that alpha- and beta-heavy-spectrin are potentially working independently and not as part of a spectrin tetramer. This is mostly dependent on the observation that alpha- and beta-heavy-spectrin appear to have non-overlapping localizations at the membrane and the fact that alpha- and beta-heavy-spectrin localize at the membrane seemingly independently. It is not entirely obvious that a potential lack of colocalization and the fact that protein localization at the membrane is not affected when the other partner is absent is sufficient to argue that alpha- and beta-heavy-spectrin do not form a complex. Moreover, it is possible that the spectrin complexes are only formed in specific conditions (e.g. by modulating tissue tension).

    If indeed spectrins function independently, would it not be expected to see additive effects when both spectrins are depleted?

    Related to the two previous points, the fact that the authors suggest that both alpha- and beta-heavy-spectrin regulate Hippo signaling via Ajuba would be consistent with the necessity of an alpha- and beta-heavy-spectrin complex being formed. How would the authors explain that both spectrins require Ajuba function but work independently?

    Another major point of the manuscript is the potential competition between beta-heavy-spectrin and myosin for F-actin binding. The authors suggest that there is a mutual antagonism between the two proteins regarding apical F-actin. However, this has not been formally assessed. Moreover, despite the arguments put forward in the discussion, it seems hard to justify a competition for F-actin when beta-heavy-spectrin seems to be unable to compete with myosin. Myosin can displace beta-heavy-spectrin from F-actin but the reciprocal effect seems unlikely given the in vitro data.