Integer topological defects organize stresses driving tissue morphogenesis

Pau Guillamat, Carles Blanch-Mercader, Karsten Kruse, Aurélien Roux

Posted on: 27 August 2020

Preprint posted on 2 June 2020

Article now published in Nature Materials at http://dx.doi.org/10.1038/s41563-022-01194-5

A peek into the dynamic patterns of myoblasts around integer defects.

Selected by Yamini Ravichandran, Giulia Paci

Categories: biophysics, cell biology, developmental biology

Background

Long-range alignment and collective motion can be observed in biological systems across scales, from cytoskeletal filaments to bacteria, cells and even entire organisms like fish schools. These systems can be described as nematic liquid crystals [1], formed by elongated elements with a long-range orientational order. Importantly, biological systems are active, meaning that the elements can self-propel through energy-consuming processes, creating stresses. When neighbours have mismatched orientations, topological defects can arise, causing a local disruption of the orientation field. These defects can be classified based on their topological strength s, which indicates the change in the orientation of molecules around the singular points around a 360° rotation.

Half-integer defects (s = ± 1/2) have been observed in several biological systems including cell colonies and monolayers, where they have correlations with changes in cell density and extrusion events [2, 3]. The role of integer defects (s = ±1) in living systems is, on the other hand, still unknown: here, Guillamat et al. investigate their role in modelling tissues.

Approach and observations

A circular confinement below the inter-defect characteristic length is applied to myoblast C2C12 cells by seeding them on disc micropatterns, after which they are let to proliferate and create a monolayer. The cell density within the monolayer determines the fate of the self-organization patterns that occur around integer topological defects.

Two patterns of organization corresponding to s = ±1 defects are observed in the monolayers: spirals and asters (Figure). Upon reaching confluence, the cells self-organize into spiral patterns with persistent rotation that can be stabilized by chemically inhibiting further proliferation. Asters are observed when the cells exhibiting a spiral pattern continue to proliferate, and cease rotating.

After transitioning from spirals to asters further proliferation leads to the formation of cellular mounds in the center. The 3D organization of the mounds reveals that they present aster-to-spiral transitions from the bottom to the top of the mound, capable of generating differentiated myotube-like structures. Contrarily, upon adding C2C12 cells incapable of differentiation cellular mounds turn into cylindrical multicellular protrusions.

To dissect the stress fields present in the monolayer, the authors employed particle image velocimetry (PIV) to measure the velocity and orientation fields and developed a 2D active nematic theory, which is described in two associated preprints [4,5]. Stress patterns in the center of the aster are probed by a micropillar constriction assay, which consists in seeding cells in a circular confinement with micropillars placed strategically in the center. Deformations induced on the base of the pillar by the cells are quantified to measure the forces exerted by the cells in asters.

Figure.1 A,D Cellular spiral and aster configurations respectively. B,E depict average velocity of the cells represented by the colormap and the orientation fields of the cells depicted by the arrowed streamlines.

Key findings

Increased cell density triggers spirals-to-asters transitions in monolayers and the reverse is true for asters-to-spirals transitions in cellular mounds
Formation of these self-organized cellular structures is strictly dependent on the circular confinement, in the absence of which mounds and protrusions collapse.
The stress patterns generated by aster defects can trigger localized differentiation, in this case with the presence of multinucleated myotube-like structures.
Integer topological defects can organize multi-cellular stresses required for tissue morphogenesis

Why this work is important?

This work investigates for the first time the active mechanics of integer topological defects, showing that they can localize myoblast differentiation and the formation of 3D protrusions.

Questions for the authors

What about extrusion events in these cellular mounds? Couldn’t cell extrusion compensate for the stress experienced in the core of cellular mounds?
With regard to active anisotropic cytoskeletal stress gradients would you be interested in studying Cdc42/Rac gradients and how they could influence collective cellular rearrangements in these patterns?
How important is the size of fibronectin-coated discs to obtain self-organized patterns around integer defects? Did you ever observe more than an individual integer defect (e.g. using larger discs)?

References

[1] Doostmohammadi, A., Ignés-Mullol, J., Yeomans, J. M., & Sagués, F. (2018). Active nematics. Nature communications, 9(1), 1-13.
[2] Kawaguchi, K., Kageyama, R., & Sano, M. (2017). Topological defects control collective dynamics in neural progenitor cell cultures. Nature, 545 (7654), 327-331.
[3] Saw, T. B., Doostmohammadi, A., Nier, V., Kocgozlu, L., Thampi, S., Toyama, Y., … & Ladoux, B. (2017). Topological defects in epithelia govern cell death and extrusion. Nature, 544(7649), 212-216.
[4] Blanch-Mercader, C., Guillamat, P., Roux, A., & Kruse, K. (2020). Integer topological defects of cell monolayers–mechanics and flows. arXiv preprint arXiv:2006.01725.
[5] Blanch-Mercader, C., Guillamat, P., Roux, A., & Kruse, K. (2020). Quantifying material properties of cell monolayers by analyzing integer topological defects. arXiv preprint arXiv:2006.01575.

Tags: -1, defects, development, growth, muscle, patterns

doi: https://doi.org/10.1242/prelights.22399

Read preprint

(2 votes)

Author's response

The author team shared

Dear Yamini and Giulia,

We would like to thank you for writing this nice Prelight on our Bioarxiv preprint and for your very interesting questions and suggestions!

Here, our reply to your questions:

1. What about extrusion events in these cellular mounds? Couldn’t cell extrusion
compensate for the stress experienced in the core of cellular mounds?

Indeed, extrusion is a mechanism by which tissues can reorganize localized compressive forces; in our case, this occurs at the core of topological defects. Defect-mediated extrusion has been reported for semi-integer defects [1,2]. In the case of differentiation-competent C2C12 cells, cell fusion leads to the formation of multinucleated myostructures and then mounds stop growing. In the case of the differentiation-incompetent cells, proliferation stabilizes defect arrangements and thus the stress patterns within the cell monolayers. We think this can lead to continuous extrusion, mainly localized at the defect cores, and consequently, promotes the growth of cylindrical cellular protrusions.

2. With regard to active anisotropic cytoskeletal stress gradients would you be
interested in studying Cdc42/Rac gradients and how they could influence collective
cellular rearrangements in these patterns?

This is a very nice suggestion. Although from the flow and the orientational fields one can make quite accurate guesses about the patterns of cell polarization, it is indeed interesting to characterize how cellular topological defects are organized bottom-up from the subcellular processes.

3. How important is the size of fibronectin-coated discs to obtain self-organized
patterns around integer defects? Did you ever observe more than an individual
integer defect (e.g. using larger discs)?

From an energetic point of view, integer topological defects are not favored in passive nematic liquid crystals. Nevertheless, when the system is strongly confined and anchoring at the boundaries is homogeneous, then the system is enforced to form an integer topological defect. Similar effects were reported in biological systems, such as vortices in bacterial discs [3], actin spirals inside confined fibroblasts [4], or microtubule-based active gel swirls [5]. If we used larger discs, indeed, more defects would arise, but they would then feature semi-integer charges as reported in Ref. [6]. Keep in mind that the total charge inside a circular disc with homogeneous anchoring has to be equal to +1!

Thanks again for your interest in our work!

All the best,

Pau, Carles, Karsten & Aurélien

[1] K. Kawaguchi, R. Kageyama, and M. Sano, Nature 545, 327 (2017).
[2] T. B. Saw, A. Doostmohammadi, V. Nier, L. Kocgozlu, S. Thampi, Y. Toyama, P. Marcq, C. T. Lim, J. M. Yeomans, and B. Ladoux, Nature 544, 212 (2017).
[3] H. Wioland, F. G. Woodhouse, J. Dunkel, J. O. Kessler, and R. E. Goldstein, Phys. Rev. Lett. 110, 268102 (2013).
[4] S. Jalal, S. Shi, V. Acharya, R. Y.-J. Huang, V. Viasnoff, A. D. Bershadsky, and Y. H. Tee, J. Cell Sci. 132, jcs220780 (2019).
[5] A. Opathalage, M. M. Norton, M. P. N. Juniper, B. Langeslay, S. A. Aghvami, S. Fraden, and Z. Dogic, Proc. Natl. Acad. Sci. 116, 4788 (2019).
[6] G. Duclos, C. Erlenkamper, J.-F. Joanny, and P. Silberzan, Nat Phys 13, 58 (2017).

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Integer topological defects organize stresses driving tissue morphogenesis

Background

Approach and observations

Key findings

Increased cell density triggers spirals-to-asters transitions in monolayers and the reverse is true for asters-to-spirals transitions in cellular mounds

Formation of these self-organized cellular structures is strictly dependent on the circular confinement, in the absence of which mounds and protrusions collapse.

The stress patterns generated by aster defects can trigger localized differentiation, in this case with the presence of multinucleated myotube-like structures.

Integer topological defects can organize multi-cellular stresses required for tissue morphogenesis

Why this work is important?

This work investigates for the first time the active mechanics of integer topological defects, showing that they can localize myoblast differentiation and the formation of 3D protrusions.

Questions for the authors

What about extrusion events in these cellular mounds? Couldn’t cell extrusion compensate for the stress experienced in the core of cellular mounds?

With regard to active anisotropic cytoskeletal stress gradients would you be interested in studying Cdc42/Rac gradients and how they could influence collective cellular rearrangements in these patterns?

Share this:

Have your say Cancel reply

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

Also in the biophysics category:

Engineered Nanotopographies Induce Transient Openings in the Nuclear Membrane

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

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

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Specialized signaling centers direct cell fate and spatial organization in a limb organoid model

Adult caudal fin shape is imprinted in the embryonic fin fold

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