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Excitable RhoA dynamics drive pulsed contractions in the early C. elegans embryo.

Jonathan B Michaux, Francois B Robin, William M McFadden, Edwin M Munro

Preprint posted on July 04, 2018 https://www.biorxiv.org/content/early/2018/07/04/076356

Single molecule imaging study performed in C. elegans early embryos sheds light on the mechanisms of pulsatility in the actomyosin cortex.

Selected by Sundar Naganathan

Background

The cell cortex is a thin filamentous network of actin and myosin bound to the plasma membrane in most animal cells. Myosin pulls on actin filaments thereby generating contractile tension in the cortex, a key determinant of cell surface tension. The actomyosin cortex has been observed to be pulsatile with periodic accumulation of actin, myosin and associated components, which contracts into a patch, followed by its disassembly. This pulsatile dynamics is critical for several morphogenetic processes including polarization of the C. elegans zygote, tissue invagination, elongation and closure in developing embryos across species as well as wound healing. Even though cortical pulsation is observed in several contexts, the mechanisms by which pulsed contractions are initiated and terminated are poorly understood. Michaux et al. addressed this problem in C. elegans early embryos and uncover that a combination of positive and negative feedback of activation of RhoA, an upstream regulator of actin and myosin, leads to pulsatile oscillations.

Key findings

The authors analyzed individual cortical pulsing events in 2-cell C. elegans embryos by performing single molecule imaging using near-total internal reflection fluorescence microscopy. They first characterized the hierarchy of accumulation and disappearance of actin, myosin and active RhoA during pulsation. Interestingly, they observed that RhoA was the first to accumulate in a broad spatial domain, followed by actin and myosin localization, after which contraction commenced. Furthermore, they observed that an increase in actomyosin density during the contraction phase is largely due to a net imbalance of assembly/disassembly of cortical filaments, with a minimal contribution from contraction itself.

How does RhoA accumulate in the cortex before contraction occurs? The authors first showed that myosin is not required for RhoA pulsation as myosin knockdown failed to abolish RhoA accumulation and disappearance, consistent with previously published data (Nishikawa et al., 2017). Furthermore, depletion of anillin, an actomyosin crosslinking protein, did not stop RhoA pulsation either. The authors then make a critical observation that the rate of active RhoA accumulation intensified with increasing RhoA recruitment to the cortex suggesting a positive feedback loop in operation.

RhoA disappears from the cortex before disassembly of actomyosin filaments. To understand the termination of RhoA activity during each pulse, the authors turned towards Rho GAPs RGA-3/4, which are known to negatively regulate RhoA activity. Through live imaging of RGA-3, the authors show that rapid accumulation of RGA-3 strongly coincides with the disappearance of RhoA from the cortex. Furthermore, this cortical accumulation of RGA-3 was observed to be dependent on F-actin, where depolymerization of F-actin by administering Latrunculin A resulted in rapid disappearance of both actin and RGA-3, while myosin and RhoA remained densely clustered in the cortex. Thus, a delayed negative feedback mediated by RGA-3 terminates RhoA activity during pulsation.

Finally, the authors build a simple theoretical model that corroborates the idea that a fast positive and a delayed negative feedback loop involving RhoA and RGA-3 can account quantitatively for locally pulsatile RhoA dynamics and predicts excitable behavior.

Why I chose this preprint?

  1. The authors clearly show that in a locally contracting patch of actomyosin, an increase in fluorescence is predominantly due to the increased assembly of actomyosin with only a minor contribution from contraction itself. This finding is novel and is certainly a valuable contribution towards an in-depth understanding of actomyosin cortex dynamics.
  2. RhoA and its regulation has been extensively studied in various contexts, however the mechanism by which it displays pulsatile dynamics has been elusive. In this study, the authors uncover associated feedback processes that lead to pulsatile RhoA activity.
  3. The application of near total internal reflection fluorescence microscopy to investigate spatiotemporal cortex dynamics in this study can be extended to give novel insights in other morphogenetic processes.

Open questions

The authors point out that the model with components of fast positive feedback and a delayed negative feedback predicts excitable RhoA activity. However, the extent to which RhoA really exhibits excitable dynamics is not clear. In the resting state, a stochastic increase in active RhoA positively amplifies its own activity, leading to actomyosin contraction, followed by recruitment of RGA-3 that in turn decreases RhoA activity. This cycle continues at regular intervals pointing towards self-sustained oscillations rather than an excitable behavior. Further experiments are required to distinguish between the two possibilities.

Pulsed actomyosin contractility has been observed in several contexts during morphogenesis. For instance, during germband extension in Drosophila embryos (Munjal et al. 2015), inhibiting myosin activity abolishes RhoA pulses. However, in developing C. elegans embryos RhoA oscillations are set up independent of myosin activity. It will be interesting to compare the mechanisms of spatiotemporal RhoA regulation in different model systems and developmental contexts.

As described previously (Nishikawa et al. 2017) and also observed in this study, RhoA pulsation can get affected by increased cortical flow. Therefore, RhoA spatial patterning is certainly influenced by mechanical feedback from the contracting cortex. A mechanochemical model is therefore required to address spatial patterning of RhoA in the cortex.

References

  1. Nishikawa M. et al., Controlling contractile instabilities in the actomyosin cortex, eLife, 2017.
  2. Munjal A et al., A self-organized biomechanical network drives shape changes during tissue morphogenesis, Nature, 2015.

Tags: cortical flow, feedback loops, self-sustained oscillation, spatiotemporal pattern

Posted on: 2nd August 2018

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