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Apical domain polarization localizes actin–myosin activity to drive ratchet-like apical constriction

Nature Cell Biology volume 15, pages 926–936 (2013)Cite this article

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Abstract

Apical constriction promotes epithelia folding, which changes tissue architecture. During Drosophila gastrulation, mesoderm cells exhibit repeated contractile pulses that are stabilized such that cells apically constrict like a ratchet. The transcription factor Twist is required to stabilize cell shape. However, it is unknown how Twist spatially coordinates downstream signals to prevent cell relaxation. We find that during constriction, Rho-associated kinase (Rok) is polarized to the middle of the apical domain (medioapical cortex), separate from adherens junctions. Rok recruits or stabilizes medioapical myosin II (Myo-II), which contracts dynamic medioapical actin cables. The formin Diaphanous mediates apical actin assembly to suppress medioapical E-cadherin localization and form stable connections between the medioapical contractile network and adherens junctions. Twist is not required for apical Rok recruitment, but instead polarizes Rok medioapically. Therefore, Twist establishes radial cell polarity of Rok/Myo-II and E-cadherin and promotes medioapical actin assembly in mesoderm cells to stabilize cell shape fluctuations.

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Figure 1: Rok and E-cadherin exhibit radial cell polarity (RCP) in ventral furrow cells.
Figure 2: Rho1 pathway components exhibit distinct localizations across the apical surface.
Figure 3: Polarized Rok and Myo-II condenses dynamic medioapical F-actin cables.
Figure 4: F-actin polymerization is required to couple the contractile network to the junctions.
Figure 5: Dia restricts E-cadherin to the junctional domain, facilitating contractile network coupling to cell–cell adherens junctions.
Figure 6: Twist mediates medioapical F-actin cable assembly.
Figure 7: Twist is required for medioapical radial cell polarity of Rok and Myo-II.
Figure 8: RCP coordinates Myo-II stabilization, F-actin assembly and E-cadherin localization to facilitate apical constriction.

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Acknowledgements

We thank J. Zallen, S. Simões, R. Fernandez-Gonzalez (Sloan-Kettering Institute, USA), T. Lecuit (Aix-Marseille Universite, France), M. Peifer (University of North Carolina, Chapel Hill, USA), S. Wasserman (University of California, San Diego, USA) and the Bloomington Stock Center for providing fly stocks or antibodies used in this study. In addition, we thank members of the Martin laboratory and A. Sokac, V. Hatini and T. Orr-Weaver for their comments on versions of this manuscript. This work was supported by grant R00GM089826 to A.C.M. from the National Institute of General Medical Sciences. A. C. Martin is a Thomas D. and Virginia W. Cabot Career Development Assistant Professor of Biology.

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  1. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA

    Frank M. Mason, Michael Tworoger & Adam C. Martin

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F.M.M. designed and performed experiments, analysed data and wrote the manuscript. M.T. designed and performed experiments. A.C.M. designed and performed experiments, analysed data, supervised the project and wrote the manuscript.

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Correspondence to Adam C. Martin.

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Integrated supplementary information

Supplementary Figure 1 Venus::Rok(K116A) localizes to medioapical foci during constriction.

During apical constriction, Venus::Rok(K116A) accumulates in medioapical foci in ventral furrow cells, similar to Venus::Rok(WT). Images are from a live embryo expressing Gap43::ChFP (subapical slice) and Venus::Rok(K116A) (apical projection). Apical constriction appears to be unaffected by expression of Rok(K116A). Scale bar is 5 μm.

Supplementary Figure 2 Apical F-actin is dynamic in ventral furrow cells.

(a) Utrophin::GFP (Utr::GFP) labels F-actin throughout the cell, including medioapical, junctional, and subapical structures. Images are from fixed Utr::GFP embryo and stained with phalloidin to label F-actin. Scale bar is 5 m. (b) Orthogonal view/cross-section of same embryo from a. Scale bar is 10 m. (c) Total F-actin levels decrease throughout apical constriction. Quantifications are averages from embryos (n = 4 embryos, at least 35 cells per embryo) expressing Utr::GFP and Myo::ChFP. Area was quantified from sub-apical F-actin images. Total apical F-actin and Myo-II levels were quantified from apical z-projections. Values for individual cells were averaged within an embryo and then averaged across multiple embryos. (d) Quantification of total F-actin levels and area in individual cells from a live embryo expressing Utr::GFP and Gap43::ChFP. F-actin levels during contractile pulses (highlights) are heterogeneous, and can increase (yellow), stabilize (blue), or decrease (magenta). F-actin levels most often increase or remain stable during a contractile pulse. (e) Graph of average behavior of area and F-actin during contractile pulses (n = 44 pulses) from embryo in d. Values are normalized to highest and lowest values during individual pulses. Error bars are standard deviation. On average, F-actin levels are briefly stabilized during contractile pulses. (f) Schematic illustrating the calculation of the time resolved cross-correlation function. Cross-correlation is calculated between constriction rate and myosin rate for various temporal shifts (ΔT). A peak cross-correlation at ΔT∼0 indicates a significant correlation with the value of ΔT indicating the time lag between the two signals.

Supplementary Figure 3 E-Cadherin localizes across the medioapical surface in diaM mutants.

(a,b) Serial z-sections of confocal images from two different fixed diaM embryos expressing Myo::GFP and stained for E-Cadherin. Medioapical E-Cadherin appears to be associated with the cortex and is localized in the same plane as Myo-II (arrows). Medioapical E-Cadherin in diaM mutants can be distinguished from subapical E-Cadherin puncta that are likely to represent endocytic vesicles. Distances indicated are positions relative to the apical z-slice (0 m). Scale bars are 5 μm.

Supplementary Figure 4 Snail is required for dorsal-ventral polarity of apical Rok localization.

Control (twist/ +) and twist mutants have apical GFP::Rok(K116A) (green arrowhead) enrichment in the ventral furrow (green bracket) prior to accumulation in germband cells (green arrowheads). White arrowhead marks the vitelline membrane. snail mutants accumulate GFP::Rok(K116A) in the ventral furrow and in germband cells at the same time. Images are projections of cross-sections from live control, twist, or snail embryos expressing GFP::Rok(K116A). Scale bars are 20 μm.

Supplementary information

Supplementary Information

Supplementary Information (PDF 698 kb)

F-actin and Myo-II structures co-localize as apical Myo-II accumulates.

Live imaging of embryo expressing the F-actin marker Utr–GFP (left, green) and Myo–ChFP (middle, magenta). Images are apical surface projections and were acquired at 6.16 s intervals. The video is displayed at 10 frames per second (fps). Scale bar is 5 μm, which is about the diameter of a cell. (MOV 1935 kb)

F-actin cables form a dynamic medioapical meshwork in ventral furrow cells.

Live imaging of control buffer (water)-injected Utr–GFP embryo. Images are single z-slice and were acquired at 0.78 s intervals. The video is displayed at 30 fps. Scale bar is 5 μm. (MOV 12095 kb)

Rok inhibition prevents medioapical F-actin condensation, but not F-actin cable assembly.

Apical projection of F-actin (Utr–GFP) in Rok inhibitor (Y-27632)-injected embryo (several cells shown). F-actin cables exhibit assembly and disassembly across the apical surface, both medioapically and at junctions. F-actin cables fail to condense after Rok inhibition. Images were acquired at 4 s intervals. The video is displayed at 15 fps. Scale bar is 5 μm. (MOV 3505 kb)

Myo-II assembly and ventral furrow formation in control embryo.

Apical projection of Myo-II–GFP (green) shows accumulation over time, and Myo-II induces pulsatile contraction of cell membranes (Gap43–ChFP, magenta, single subapical z-slice) in solvent (control)-injected embryo. Image was acquired at 5.6 s intervals. The video is displayed at 20 fps. Scale bar is 5 μm. (MOV 17446 kb)

CyoD disrupts Myo-II medioapical network connectivity.

Whereas Myo-II (green, apical projection) can accumulate and cells constrict, injection of CytoD (0.125 mg ml1) disrupts the attachment of medioapical Myo-II to the junctional domain and a failure of cells to maintain cell-cell connectivity. Medioapical Myo-II networks can be seen repeatedly losing and regaining connections. Images are from CytoD-injected Myo–GFP, Gap43–ChFP embryo (magenta, single subapical z-slice). Images were acquired at 5.6 s intervals and every fourth frame is shown. The video is displayed at 7 fps. Scale bar is 20 μm. (MOV 14428 kb)

CytoD destabilizes the connection between the medioapical Myo-II network and junctions.

Medioapical Myo-II (green, apical projection) networks lose and regain connections when F-actin assembly is decreased by CytoD (0.125 mg ml−1). Magnified view of embryo expressing Myo–GFP and Gap43–ChFP (magenta, single z-slice) from Video 4. Images acquired at 5.6 s intervals. The video is displayed at 20 fps. Scale bar is 5 μm. (MOV 7329 kb)

CytoD injection results in gaps in the apical F-actin meshwork.

CytoD injection causes gaps in the F-actin meshwork followed by medioapical and juctional F-actin networks repeatedly losing and regaining connections, similar to Myo-II. CytoD (0.25 mg ml−1) was injected into Utr–GFP embryo and images are apical projection of F-actin (green) and subapical projection marking cell boundaries (magenta). Images were acquired at 7.2 s intervals. The video is displayed at 15 fps. Scale bar is 5 μm. (MOV 10224 kb)

DiaM phenotype resembles CytoD injection.

Myo-II (apical projection) forms dense meshwork across ventral furrow cells in control (dia/+) embryos (top embryo). In diaM embryos (bottom embryo), Myo-II (apical projection) accumulates, but networks lose and regain connectivity (left side of embryo), similar to embryos injected with CytoD. Both control and diaM embryos express Myo–GFP. Control embryo images were acquired at 8.8 s intervals. DiaM embryo images were acquired at 9.3 s intervals. The video is displayed at 20 fps. Scale bars are 20 μm. (MOV 13564 kb)

Snail and Twist are required for a medioapical meshwork of F-actin cables.

Control (twist/+, left), snail (centre) and twist (right) embryos expressing Utr–GFP demonstrate that both Snail and Twist are required for medioapical F-actin cable assembly during ratchet-like constriction. Control embryos have a dense medioapical F-actin network that is dynamic as cells constrict and tissue invaginates. Snail mutants have F-actin puncta that fail to form proper meshwork of F-actin cables. Twist mutants have junctional F-actin cables and condense medioapical F-actin structures, but F-actin cables fail to form/persist. Images were acquired at 9.3 s intervals for control, 7.4 s intervals for snail and 6 s intervals for twist. The video is displayed at 15 fps. Scale bars are 5 μm. (MOV 6023 kb)

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Mason, F., Tworoger, M. & Martin, A. Apical domain polarization localizes actin–myosin activity to drive ratchet-like apical constriction. Nat Cell Biol 15, 926–936 (2013). https://doi.org/10.1038/ncb2796

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