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. 2015 Jul 14;112(28):8620-5.
doi: 10.1073/pnas.1504762112. Epub 2015 Jun 15.

Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes

Edouard Hannezo  1 , Bo Dong  2 , Pierre Recho  3 , Jean-François Joanny  4 , Shigeo Hayashi  5
Affiliations

Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes

Edouard Hannezo et al. Proc Natl Acad Sci U S A. .

Abstract

An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, our model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring our model prediction of a transition from steady to chaotic actin patterns at low cortical friction. Our results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting.

Keywords: Drosophila; actomyosin; biological tubes; biophysics; pattern formation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Periodic supracellular actin rings in tracheal tubular system. (A) Live imaging of actin dynamics during the formation of the pattern, using Lifeact-mEGFP. Actin intensity increases by 70% at the onset of ring formation. (Bottom Right) Enlargement of actin ring structure (phalloidin) at embryonic stage 16. (B) Actin (phalloidin) and myosin II heavy chain (Zip) in the tracheal tubular system at stage 16. (C) At third-instar larval stage, the actin rings (in green, fixed sample) are very regularly spaced, as can be seen in the intensity profile and in the Fourier spectrum, with a period of 1.2 μm. (D) Theoretical model of an actomyosin cortex as a viscous and contractile gel, undergoing turnover and in frictional contact with an ECM. An instability into periodic patterns in actin concentration is very generically expected due to myosin contractility. (Scale bars: 10 μm.)
Fig. 2.
Fig. 2.
Theoretical predictions and validation of the model’s key assumptions. (A) Fluorescence recovery after photobleaching (FRAP) experiments on the apical cortex (marked by Actin-GFP) just before ring formation. We measure both the turnover time and the diffusion coefficient of the actin gel from the recovery curves. (B) Phase diagram of the instability. A cortex is predicted to organize into periodic patterns as soon as the rescaled contractility χ is above a critical value, which increases with the rescaled turnover ϕ. (C) Actin patterns at embryonic stage 16 disappear with contractility inhibition (through incubation of 250 μM Y27632), as predicted by our model. (D) Theoretical profiles of actin concentration for different values of our parameters in 1D. (E) In 2D, if all parameters are isotropic, labyrinth patterns are formed (Left), but circumferential rings are formed for small anisotropies (Right, where the friction coefficient ζθ is increased by 20% in the orthoradial direction). (Scale bars: 10 μm.)
Fig. 3.
Fig. 3.
Model predictions tested through pharmacological perturbation and fly genetics. (A) The period of the ring pattern increased by 70% in Src42 mutants, and 10-fold by genetic removal of the extracellular matrix inside the tube (kkv mutant). One of our model’s core predictions is that decreasing friction should increase the wavelength of the pattern. (B) Wavelength increases in Src42 and kkv mutant (P<10−10), compared with wild type. (C) Local constrictions are observed in kkv mutants lacking an ECM substrate and colocalize with actin (phalloidin staining), and high actin concentration correlates with small tube radius, showing that the rings are contractile. (Bottom Right) Cadherin (green) and actin (purple) stating, showing that only one actin ring is present per cell on average. (D) Actomyosin constrictions in kkv mutant disappeared by lowering contractility through Y-27632 treatment. (Scale bars: 10 μm.)
Fig. 4.
Fig. 4.
(A) Live imaging of rings in control tracheas using moesin-GFP. (A′) Live imaging of rings in kkv mutants using moesin-GFP. (B) Moesin-GFP in kkv mutants. We show three superimposed time steps: t = 0 (green), t = 90 s (red), and t = 180 s (blue), showing that, in kkv mutants, the constrictions are not stationary. (C) Kymograph of patterns in the high- and low-friction regimes, in the case of a nonlinear turnover. A transition toward chaos is observed. (D and E) Heterozygous and homozygous γ-COP mutants. (D′ and E′) Longitudinal section of tracheal tube in heterozygous (D′) and homozygous (E′) γ-COP mutants. (F) COP mutants show a decreased tube diameter, compared with wild type, as well as a decreased cortical actin level. Both phenotypes are stronger for homozygous mutants. (G) Fixed samples of actin (phalloidin) rings in wild type. One can observe that rings form both in the main trunk (G) and in side branches (G′), at the same wavelength. (H) Summary of the proposed model. (Scale bars: 10 μm.)
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