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Excitable Rho dynamics drive cell contractions by sequentially inducing ERM protein-mediated actin-membrane attachment and actomyosin contractility

Seph Marshall-Burghardt, Rodrigo A. Migueles-Ramírez, Qiyao Lin, Nada El Baba, Rayan Saada, Mustakim Umar, Arnold Hayer

Posted on: 12 February 2024

Preprint posted on 20 December 2023

"Unraveling cellular dance”: Rho directs edge retractions, ERMs lead the rhythm.

Selected by Vibha SINGH

Categories: cell biology

Background.

The role of the small GTPases Rac1 and Cdc42 in driving cell protrusion via actin polymerization in various contexts and cell types is well acknowledged. Rho activity, despite being a well-known regulator of cell contractility via Rho-associated kinase (ROCK), and non-muscle myosin II (NMII), has not been fully characterized, as Rho activity has also been reported in cell protrusions (possibly via downstream effector formin, mDia1). This study aimed to clarify the roles of Rho effectors ROCK/NMII and SLK/LOK/ERM in cell morphology and Rho-driven cell contractions.

Overall, the authors manage to highlight the crucial roles of ROCK and SLK/LOK in Rho-mediated cell contractions and emphasize the significance of SLK/LOK-dependent ERM activation in safeguarding cell integrity.

Key findings.

(a) Excitability of Rho Activity and its dynamics: The spatiotemporal analysis of a FRET-based RhoB sensor revealed consistently elevated Rho activity during cell edge retractions, but not during protrusions, indicative of a distinct role in mediating cell contraction via ROCK-dependent mechanisms. Rac and Cdc42 reporters displayed the expected responses to GEF translocations, with reciprocal activation upon Rho activation/inactivation.

(b) Role of MLC and Ezrin: NMII accumulation at retracting cell edges occurred with a delay relative to Rho activity, while ERM activation showed strong co-localization without a detectable delay. This indicates that ERMs act as immediate responders to Rho activity, enhancing force transmission during retractions. Rho was further shown to activate ERMs, facilitating membrane-cortex attachment during retraction initiation, while NMII exerts contractile forces to further drive edge retraction.

(c) Feedback Mechanisms: Potential positive and negative feedback mechanisms – involving Rho, ERMs, and focal adhesion dynamics, contributing to the regulation of cell edge dynamics during, and after retraction onset, indicative of an excitable Rho signaling network has been highlighted. Positive feedback loops enhance Rho activation, resulting in pulsatile behavior and propagating waves. Besides the RhoB sensor, DORA-RhoA and RhoA2G probes also exhibit pulsatile activation patterns, supporting the existence of an excitable Rho signaling network during retractions.

(d) SLK/LOK inhibition effects: Inhibition of SLK/LOK kinase resulted in morphological phenotypes similar to ROCK inhibition, underlining the importance of Rho-dependent ERM activation for cellular integrity and contractility. A SLK/LOK inhibitor (Cpd31) caused a rapid and almost complete loss of pERM signal, suggesting a high turnover rate of ERM phosphorylation. Thrombin and nocodazole, known Rho activators, induced simultaneous increases in Rho activity and pERM signals, which were suppressed by Cpd31 but not by ROCK inhibitor (Y27632). This highlights the crucial role of SLK/LOK kinases in mediating rapid ERM activation downstream of Rho, underlining the important role of the Rho-SLK/LOK-ERM signaling axis in regulating actin-membrane attachment and force transmission.

Significance and Implications:

The authors of this study have uncovered intricate spatiotemporal dynamics of Rho activity during endothelial cell edge retractions, validating pulsatile excitability and feedback mechanisms. This delineation not only improves our understanding of cellular motility, but also uncovers the roles of Ezrin-Radixin-Moesin (ERMs) and non-muscle myosin II (NMII) in governing cell shape modifications (Figure 1). The conclusions from this study have broad implications for the field of cell migration, morphogenesis, and the regulatory networks underlying dynamic cellular responses. Furthermore, the identification of Rho excitability and ERMs as early effectors unlock avenues for exploring novel therapeutic targets and allows more detailed investigations into cellular behavior across diverse contexts.

Figure 1. (Reproduced from figure 7j). Model showing how excitable Rho activity drives cell contractions through both ERM activation via SLK/LOK and ROCK-mediated actomyosin contractility.

Open questions.

(1) Since RhoA activity has been reported during cell edge protrusions, could there be a spatiotemporal correlation between edge-proximal RhoA activity and ERMs in contexts where the Rho-SLK/LOK-ERM module exists?

(2) Could activated ERM also interact/activate NMII, further accelerating cell retraction?

 

 

doi: https://doi.org/10.1242/prelights.36479

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Author's response

Arnold Hayer shared

Author’s response.

Q1: Since RhoA activity has been reported during cell edge protrusions, could there be a spatiotemporal correlation between edge-proximal RhoA activity and ERMs in contexts where the Rho-SLK/LOK-ERM module exists?

While several studies have reported elevated Rho in cell edge protrusions, it has more recently been shown that protrusion initiation requires decreased actin-membrane attachment and depleted ERM activation1,2. If Rho activated ERMs in protrusions, we expect this would stall protrusions. Since we have not observed elevated Rho in protrusions, we were not able to explore this further. We found it interesting that while activated NMII is found both near the front and in the rear of migrating cells, NMII in the front or in protrusions is preferentially activated MLCK rather than by Rho/ROCK3,4. Our results argue that Rho in the front would immediately activate ERMs, which would enhance actin membrane attachment and therefore slow down or stop protrusions, explaining why MLCK, and not Rho/ROCK, activates NMII in protrusions.

Q2: Could activated ERMs also interact/activate NMII, further accelerating cell retraction?

Both positive and negative feedback mechanisms from ERMs to Rho have been described5-7. However, given the tight spatiotemporal coupling between Rho activity and ERM activation that we observed, positive feedback from ERMs to Rho is more likely than negative feedback, in the context of cell contractions in HUVEC. We would expect positive feedback between ERMs and Rho to result in increasing NMII activation. This question deserves further exploration in future research. One striking observation was that acutely inhibiting SLK/LOK kinases in blebbing cells counterintuitively stopped blebbing and resulted in cells to attach and spread. This was unexpected, since ERMs are known to be involved in bleb retraction and suggests that ERM activation is required to maintain cells in a contractile, blebbing state.

References:

  1. Anjali Bisaria et al. Membrane-proximal F-actin restricts local membrane protrusions and directs cell migration. Science 368,1205-1210 (2020).
  2. Erik S. Welf et al. Actin-Membrane Release Initiates Cell Protrusions. Developmental Cell. 55, 723-736 (2020).
  3. Go Totsukawa et al. Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts. J Cell Biol 2 2004; 164 (3): 427–439.
  4. Beach, J., Bruun, K, Shao, L. et al. Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments. Nat Cell Biol 19, 85–93 (2017).
  5. Riasat Zaman et al. Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain. J Cell Biol 7 June 2021; 220.
  6. Speck, O., Hughes, S., Noren, N. et al. Moesin functions antagonistically to the Rho pathway to maintain epithelial integrity. Nature 421, 83–87 (2003).
  7. Jiao M, Wu D, Wei Q. Myosin II-interacting guanine nucleotide exchange factor promotes bleb retraction via stimulating cortex reassembly at the bleb membrane. Mol Biol Cell. 2018, 29(5):643-656.

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