Close

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

Read preprint (No Ratings Yet)

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.

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the cell biology category:

The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites

Joelle Lo, Katherine F. Vaeth, Gurprit Bhardwaj, et al.

Selected by 24 September 2024

Felipe Del Valle Batalla

Neuroscience

Pharyngeal neuronal mechanisms governing sour taste perception in Drosophila melanogaster

Bhanu Shrestha, Jiun Sang, Suman Rimal, et al.

Selected by 23 September 2024

Matthew Davies

Cell Biology

Feedback regulation by the RhoA-specific GEF ARHGEF17 regulates actomyosin network disassembly

Vasundhara Rao, Benjamin Grädel, Lucien Hinderling, et al.

Selected by 18 September 2024

Vibha SINGH

Cell Biology

preLists in the cell biology category:

BSCB-Biochemical Society 2024 Cell Migration meeting

This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.

 



List by Reinier Prosee

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.

 



List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.

 



List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage
Close