Assembly of a persistent apical actin network by the formin Frl/Fmnl tunes epithelial cell deformability

Benoit Dehapiot, Raphaël Clément, Gabriella Gazsó-Gerhát, Jean-Marc Philippe, Thomas Lecuit

Preprint posted on 23 June 2019 https://www.biorxiv.org/content/10.1101/680033v1

Article now published in Nature Cell Biology at https://www.nature.com/articles/s41556-020-0524-x

Formin’ the right shape: The formin Frl/Fmn1 modulates a persistent actin network that tunes epithelial cell deformations and enables the propagation of contractile forces to direct tissue-scale morphogenesis.

Selected by Melanie White

Categories: cell biology, developmental biology

Background

How small changes in cell shape are generated and transmitted across tissues to cause large-scale rearrangements of tissue architecture is a fundamental question in developmental biology. During various morphogenetic processes, actomyosin foci form at the apical cell cortex and undergo periodic contractions, or pulses. These actomyosin pulses pull on actin filaments connected to adherens junctions to drive changes in cell shape.

The mechanisms regulating actomyosin pulsation have been well studied and many of the underlying signalling molecules have been described^1,2. Yet, it remained unclear how the myosin pulses interact with the cortical actin network to both effect changes in shape within the cell, and propagate forces across cell boundaries throughout a tissue.

In this preprint by Dehapiot et al, the authors identify two subpopulations of cortical actin filaments in Drosophila embryos: a pulsatile network and a persistent network. By combining genetic manipulations, live imaging and computational modelling, they show how the pulsatile network promotes cell shape changes and the persistent network supports the transmission of contractile forces through the tissue.

Key findings

There are two differentially regulated subpopulations of actin at the medioapical cortex of Drosophila ectodermal cells. In addition to pulsatile F-actin polymerisation occurring in synchrony with the well-known Myosin II pulses, there is also a persistent homogeneous network of actin filaments.

Unlike the pulsatile F-actin polymerisation, assembly of the persistent actin network does not require RhoA activity.

The formin Frl/Fmn1 regulates the density of the persistent actin network and counteracts the medial actomyosin pulsatility by modulating Rho activity.

The pulsatile actomyosin drives cell deformations and the persistent actin network supports the transmission of these contractile forces to cell junctions, enabling their propagation throughout the tissue.

Disrupting the persistent actin network reduces the propagation range of contractile forces and results in tissue-scale defects in germband extension and dorsal closure.

Why I chose this preprint & how it moves the field forward

The molecular and mechanical details of actomyosin networks can sometimes be heavy reading for the non-specialist but this paper is beautifully written. The authors proceed logically through the experiments explaining the rationale behind each one and allowing the reader to follow their thinking. The experiments themselves are often relatively simple but the imaging is excellent and the analysis is elegant. The use of nicely designed schematics also helps to explain the findings at a glance.

Importantly, the authors’ approach allows them to look beyond the pulsatile myosin contractility that has been well studied during morphogenesis, and begin to tease apart the effects of the organisation of the cortical actin network. This is interesting because it provides new insights into an additional level of actin-mediated regulation of epithelial cell dynamics that is likely to be relevant to many other developmental contexts. By differentially modulating the subpopulations of actin filaments, cells may be able to fine tune the degree of cellular deformation and the spatial propagation of forces at different times during development and within different tissues.

More broadly, I think this paper is a wonderful example of how variations in the expression level of a single protein can cause molecular alterations within the cell that alter mechanical properties and have far-reaching consequences for morphogenesis.

Future directions and questions for the authors.

This paper identified a new role for formins distinct from their well-known roles in lamellipodia/filopodia formation. Future work will likely reveal how formin activity is regulated in this context and which specific formins are involved.

The authors showed that the persistent actin network acts to inhibit Rho activity but it remains to be shown how this happens.

Actomyosin pulsing also occurs during development in C.elegans³, Xenopus⁴ and the preimplantation mouse embryo⁵. It will be interesting to see whether similar subpopulations of cortical actin filaments exist in these systems and if they also act to tune cell deformation and the propagation of forces across the tissue.

Q1: What motivated you to look for a subpopulation of actin filaments distinct from the contractile network?

Q2: Previous research on apical constriction proposed the existence of a “molecular clutch” that modulates the strength of the connection between adherens junctions and the apical actomyosin network⁶. Although this is usually thought of in terms of adhesion complex regulation, could Frl/Fmn1 also be thought of as a clutch mechanism by controlling the density of the persistent actin network, and therefore the strength of the connection between actin and the junctions?

Q3: Formins also regulate the microtubule cytoskeleton and microtubules have recently been shown to promote connections between medioapical actomyosin and adherens junctions in Drosophila epithelia⁷. Did you consider any potential effects of the formin manipulations on the microtubule network?

References

1 Coravos, J. S., Mason, F. M. & Martin, A. C. Actomyosin Pulsing in Tissue Integrity Maintenance during Morphogenesis. Trends Cell Biol 27, 276-283 (2017).

2 Blanchard, G. B., Etienne, J. & Gorfinkiel, N. From pulsatile apicomedial contractility to effective epithelial mechanics. Current opinion in genetics & development 51, 78-87 (2018).

3 Munro, E., Nance, J. & Priess, J. R. Cortical flows powered by asymmetrical contraction transport PAR proteins to establish and maintain anterior-posterior polarity in the early C. elegans embryo. Developmental cell 7, 413-424 (2004).

4 Skoglund, P., Rolo, A., Chen, X., Gumbiner, B. M. & Keller, R. Convergence and extension at gastrulation require a myosin IIB-dependent cortical actin network. Development 135, 2435-2444, (2008).

5 Maitre, J. L., Niwayama, R., Turlier, H., Nedelec, F. & Hiiragi, T. Pulsatile cell-autonomous contractility drives compaction in the mouse embryo. Nature cell biology 17 (2015).

6 Roh-Johnson, M. et al. Triggering a cell shape change by exploiting preexisting actomyosin contractions. Science 335 (2012).

7 Ko, C. S., Tserunyan, V. & Martin, A. C. Microtubules promote intercellular contractile force transmission during tissue folding. The Journal of cell biology (2019).

Tags: actin, cell biology, developmental biology, drosophila, flies, formin, morphogenesis

Posted on: 24 July 2019 , updated on: 3 June 2020

doi: https://doi.org/10.1242/prelights.12373

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Author's response

Benoit Dehapiot shared

Q1 : What motivated you to look for a subpopulation of actin filaments distinct from the contractile network?

We first sought to understand how the medio-apical F-Actin behaves upon Rho1 pathway inhibitions (e.g. C3-trans and RhoGEF2^-/-). We soon realized that a persistent F-Actin network was maintained under these inhibitory conditions and we next identified Frl as critical nucleator promoting its assembly. We decided to focus our attention on this persistent subpopulation since it had never been described before and this offered us the opportunity to better understand how the pulsatile contractile forces propagate in cells and tissues. However, it will be also very interesting to better characterize the pulsatile medial pool of F-Actin to see how it influences the spatio-temporal dynamics of MyoII pulses.

Q2: Previous research on apical constriction proposed the existence of a “molecular clutch” that modulates the strength of the connection between adherens junctions and the apical actomyosin network. Although this is usually thought of in terms of adhesion complex regulation, could Frl/Fmn1 also be thought of as a clutch mechanism by controlling the density of the persistent actin network, and therefore the strength of the connection between actin and the junctions?

Our findings demonstrate that the persistent medial F-Actin network, assembled by Frl, influences the level of connectivity between actomyosin pulses and adherens junctions. However, our observations revealed that the persistent network is not required for cells to undergo apical contractility. Indeed, we found that cells still deform upon pulsatile contractility in the absence of the persistent network (frl^59/59 null mutant). This can be explained by the fact that actomyosin pulses can autonomously connect to the adherens junctions at short distances. The persistent network instead promotes longer-range connectivity and the uniform distribution of contractile forces to the cell periphery. Therefore, if the persistent network is involved in a “molecular clutch”, as described by Roh-Johnson and colleagues, this would be in an undirect manner by modulating, for example, the viscoelastic properties of the cellular cortex.

Q3: Formins also regulate the microtubule cytoskeleton and microtubules have recently been shown to promote connections between medio-apical actomyosin and adherens junctions in Drosophila epithelia. Did you consider any potential effects of the formin manipulations on the microtubule network?

Unfortunately, we did not have time to study the influence of Frl on microtubules. However, in light of recent findings, we are convinced that it would certainly be worth it.

1 comment

4 years

Benoit Dehapiot

What were the most important things you improved in your study as a result of peer review?

Since the release of this prelight our work have been published in Nature Cell Biology (https://www.nature.com/articles/s41556-020-0524-x). One of the questions posed by the reviewers opened new perspectives for future research. Indeed, we have been asked: why the increased cell intercalation observed in the frl59/59 mutants does not accelerate the extension of the germband?

This is in short what we answered to the reviewers:

Ectodermal cell intercalation is one of the key events occurring during germband extension. However, it has been shown that intercalation alone cannot account for tissue extension. Indeed, inhibiting cell intercalation, using eve, runt or Toll-2,6,8 mutants, only reduces extension by 40% (Irvine and Wieschaus, 1994 ; Paré et al, 2014). Other forces are required in this process such as those produced by the mesoderm and the posterior midgut (PMG) invagination (Butler et al, 2009 ; Collinet et al, 2015 ; Lye et al, 2015 ; Bailles et al, 2019). Cell intercalation contributes internal stress to extend the ectoderm, but also provide a means to dissipate elastic energy that accumulates due to PMG pulling on the ectoderm (Collinet et al 2015).
Interestingly, the increased cell intercalation observed in the frl59/59 mutant does not seem to affect the overall tissue extension. In the context detailed above, we would argue that the rate of PMG invagination is somewhat dominant over cell intercalation in defining the pace of germband extension once intercalation is taking place. Intercalation is fast enough to dissipate internal stress due to PMG pulling on the ectoderm.
While many mutants are known to inhibit cell intercalation (see above), the frl59/59 mutant is one of the few known experimental conditions in which the opposite can be observed. The latter could prove to be a very useful tool to further documents the importance of internal and external stresses in tuning the rate of tissue extension.

Irvine, K. D. & Wieschaus, E. Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes. Development 120, 827–841 (1994).
Paré, A. C. et al. A positional Toll receptor code directs convergent extension in Drosophila. Nature 515, 523–527 (2014).
Butler LC, Blanchard GB, Kabla AJ, et al. Cell shape changes indicate a role for extrinsic tensile forces in Drosophila germ-band extension. Nat Cell Biol. 2009;11(7):859‐864.
Collinet, C., Rauzi, M., Lenne, P.-F. & Lecuit, T. Local and tissue-scale forces drive oriented junction growth during tissue extension. Nat. Cell Biol. 17, 1247–1258 (2015).
Lye, C. M. et al. Mechanical Coupling between Endoderm Invagination and Axis Extension in Drosophila. PLOS Biol. 13, e1002292 (2015).
Bailles A, Collinet C, Philippe JM, Lenne PF, Munro E, Lecuit T. Genetic induction and mechanochemical propagation of a morphogenetic wave. Nature. 2019;572(7770):467‐473.

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Assembly of a persistent apical actin network by the formin Frl/Fmnl tunes epithelial cell deformability

Background

Key findings

Why I chose this preprint & how it moves the field forward

Future directions and questions for the authors.

References

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