Complementary Cytoskeletal Feedback Loops Control Signal Transduction Excitability and Cell Polarity
Posted on: 7 March 2024 , updated on: 8 October 2025
Preprint posted on 13 February 2024
Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-025-62799-3
The cytoskeleton talks back and has a bi-directional influence on cell polarity.
Selected by Vibha SINGH
Categories: cell biology
Updated 8 October 2025 with a postLight by Vibha SINGH, PhD
This study was recently published in Nature Communications (https://www.nature.com/articles/s41467-025-62799-3) and includes interesting updates and revision experiments. The reviewers mainly questioned how the authors’ molecular observations are related to whole-cell behavior. While the core findings remain the same, the authors effectively responded to the reviewers’ questions, eventually resulting in the paper’s acceptance. I have summarized the key differences between the published Nature communication article and preprint version.
- Experimental additions to validate biosensor data.
The most substantial changes were made to Fig.1 and S1 to address concerns about the reliability of the Ras biosensor (RBD) data – particularly regarding varying expression levels and the absence of a membrane marker. Authors performed a direct quantification of RBD expression between populations (Fig 1D), showing control cells (AX3) had slightly higher expression than the ABN-null cells, disproving the possibility that the increased signal was an expression artifact. Moreover, the authors present a panel showing membrane staining in AX3 and ABN null cells and observed no differences in membrane morphology, suggesting the observed Ras localization changes were not artifacts.
- Polished dose-response data (Fig. 2).
The authors have updated preprint figures 2I-J with new experiments with more diverse cAMP concentrations and improved signal-to-noise. To address concerns that Latrunculin (pan-actin inhibitor) might cause non-specific effects (e.g., on adhesion or endocytosis), the authors conducted control experiments. The new results demonstrate that Latrunculin alone did not increase Ras activation in un-pretreated cells (if anything, it slightly decreased it), supporting the conclusion that the inhibitory effect was specific to the type of actin network left behind after CK666 treatment.
- Better clarity and originality.
The authors made numerous textual revisions in response to the reviewers’ feedback improving clarity and distinguishing novel findings from established literature. They now describe which conclusions were novel and unpredicted (such as actomyosin inhibition acting acutely on the basal activity of signaling networks and Arp2/3 inhibition blocking STEN activity despite no overall F-actin reduction). They also modified the discussion section, clarifying how their new model differs from previous models, including their own, emphasizing the explicit inclusion of a back-promoting loop from actomyosin. New data where the myosin-null phenotype was rescued by overexpressing GFP-Myosin, supports the claim that Myosin II plays a central role in regulating the persistence of PIP3 patches and supports originality.
Overall, this remains a comprehensive and important study. Thanks to peer review, substantial textual and experimental revisions were added to the final version. These changes further boost the central model discussed in the earlier preprint version.
Background.
Cell migration mechanisms, signaling networks, and cytoskeletal dynamics are tightly interlinked and play pivotal roles in various physiological and pathological cellular processes. This preprint presents a comprehensive investigation of the multi-step regulation of front-back polarity, membrane protrusion, and the contribution of signaling molecules (specifically, Ras GTPase and phosphoinositide) in the regulation of actin dynamics. Through both experimental and computational approaches, the authors successfully uncovered feedback loops between branched actin networks and the actomyosin cortex, governing the activation of the Signal Transduction Excitable Network (STEN), and the Cytoskeletal Excitable Network (CEN). The findings in this study describe complicated, yet deeply regulated molecular mechanisms which help cells navigate through complex environments. The outcomes also have implications for potential therapeutic approaches in diseases characterized by aberrant cell migration processes.
Key findings.
The authors of this preprint have managed to successfully demonstrate several pivotal points that enhance our understanding of the interplay between signaling networks and cytoskeletal dynamics in coordinating cell polarity and migration. One unique finding that stood out for me is the discovery of bidirectional feedback loops between the cytoskeleton and signaling networks, which challenges the conventional views of one-way regulation in cellular migration.
I’ve listed some of the key findings from this article below:
(a) Positive feedback loop from actin network to regulate STEN activity. Actin polymerization was boosted at the cell-front by utilizing mutated actobindins (Lampert et al. 2017) which led to increased Ras activation. This result uncovers a positive regulatory function of actin involving elevated Arp2/3 activity and the formation of branched actin which regulates STEN activity.
(b) Negative feedback loop from Myosin II to STEN. Myosin heavy chain kinase C (MHCKC) was exploited to disintegrate the actomyosin cortex at the cell-rear, which resulted in elevated STEN and Ras activity. In contrast, myosin activity increased the STEN activation threshold, thereby hindering front state and protrusion formation.
(c) Actin disruption inhibits STEN activity. CK666 was used to inhibit the nucleation of branched actin Arp2/3, which diminished STEN activity, as could be observed by decreased PIP3 production, Ras activation, and STEN waves. Furthermore, inhibition of branched actin boosted cortical actin formation and actin cross-linkers, which in turn further suppressed STEN activity.
(d) RacE Actin disruption inhibits STEN activity. RacE (member of Rac/Rho subfamily) regulates actin nucleation, myosin contraction, and cell mechanics (Zhou et al. 2010; Gerald et al. 1998; Luo et al. 2013). Stimulation of RacE resulted in enhanced actin polymerization which was independent of Arp2/3, and led to the inhibition of STEN activity and Ras activation. Moreover, using optogenetics to induce the local recruitment of RacE led to protrusion formation and localized modification in STEN activity, emphasizing its crucial role in the regulation of cell polarity.
This elaborate set of observations describes some multifaceted and complementary feedback loops between the cytoskeleton and signaling networks and, thereby, offers valuable insights that expand our understanding of molecular mechanisms governing cell polarity and migration.
Significance and Implications.
The results presented in this article have significant implications for numerous research fields, ranging from basic cell biology to therapeutic development. Previous studies have often focused on conventional one-directional signaling pathways while studying the molecular mechanism regulating cell polarity/migration. This article, however, uncovers how the cytoskeleton can actively participate in and influence upstream signaling events. These results suggest that due to these feedback loops, targeting just one signaling molecule in a therapeutic setting may be insufficient and that there need to be potential combination targeting strategies involving both the cytoskeleton and signaling networks.
The identification of front- and back-promoting mechanisms, coupled with evidence of how they impact cellular behavior and respond to external stimuli, contributes much to our understanding of cellular regulation. Another highly important discovery reported in this article is that cells have intricate ways to talk back to the control center governing the operation. In other words, cells can send signals/indications to tell central command (the upstream governing machinery) how to act.
In summary, the results from this preprint article contribute to our understanding of how cells function, and how we might target and treat diseases in the future.
Open questions.
(a) Is there a possibility of microtubules being involved in these complementary loops between actin/actomyosin networks and signaling networks?
(b) Do cells in different mechanical microenvironments (say higher mechanical stress) still have functional complementary loops? Actin could depolymerize under higher stress (a possible scenario in some cancerous conditions). Could there possibly be a shift in the balance of these complementary loops in such a cell?
doi: https://doi.org/10.1242/prelights.36742
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