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. 2009 Jan 22;457(7228):495-9.
doi: 10.1038/nature07522. Epub 2008 Nov 23.

Pulsed contractions of an actin-myosin network drive apical constriction

Adam C Martin  1 , Matthias Kaschube, Eric F Wieschaus
Affiliations

Pulsed contractions of an actin-myosin network drive apical constriction

Adam C Martin et al. Nature. .

Abstract

Apical constriction facilitates epithelial sheet bending and invagination during morphogenesis. Apical constriction is conventionally thought to be driven by the continuous purse-string-like contraction of a circumferential actin and non-muscle myosin-II (myosin) belt underlying adherens junctions. However, it is unclear whether other force-generating mechanisms can drive this process. Here we show, with the use of real-time imaging and quantitative image analysis of Drosophila gastrulation, that the apical constriction of ventral furrow cells is pulsed. Repeated constrictions, which are asynchronous between neighbouring cells, are interrupted by pauses in which the constricted state of the cell apex is maintained. In contrast to the purse-string model, constriction pulses are powered by actin-myosin network contractions that occur at the medial apical cortex and pull discrete adherens junction sites inwards. The transcription factors Twist and Snail differentially regulate pulsed constriction. Expression of snail initiates actin-myosin network contractions, whereas expression of twist stabilizes the constricted state of the cell apex. Our results suggest a new model for apical constriction in which a cortical actin-myosin cytoskeleton functions as a developmentally controlled subcellular ratchet to reduce apical area incrementally.

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Figures

Figure 1
Figure 1. Apical constriction of ventral furrow cells is pulsed
a, Schematic of the imaging approach used to visualize ventral furrow cell apical constriction. We selected tangential z-slices 2 μm below the apical surface (red slices) to visualize cell outlines. b, Z-slices (top) and YZ cross-sections (bottom) of cell membranes visualized with Spider-GFP. Scale bar = 10 μm. Apical areas (c) and constriction rates (d) for individual cells of a representative embryo. Each row represents data (see colorbars) for an individual cell. e, Mean apical area (red) and furrow depth (black). Dotted line indicates when tissue invagination initiates. Error bars, s.d. (n = 41 cells). Quantification (f) and time-lapse images (g) of the constriction of an individual cell. The red arrows (c, d) and red dots (g) mark the cell that is quantified in f. C, contraction. S, stabilization. Scale bar = 4 μm. (h) Pulsed constriction is asynchronous in neighboring cells. Constriction rate is colorcoded (see colorbar) and mapped onto the corresponding cells in images at different timepoints.
Figure 2
Figure 2. Constriction pulses are correlated with myosin coalescence
a, Merged images of Myosin-mCherry (Z-projection, 5 μm depth, green) and Spider-GFP (individual z-slice 2 μm below the apical cortex, red). YZ cross-sections at lower magnification to illustrate furrow progression are shown at bottom. b, Mean apical area and myosin intensity (left) and myosin intensity for individual cells (right) for a representative embryo. Error bars, s.d. (n = 37 cells). c, Single channel and merged timelapse images of Myosin-mCherry (green) and Spider-GFP (red). Red arrows indicate spots that will coalesce. Blue arrow indicates myosin fiber that appears between contractions. d, Apical area and myosin intensity vs. time (top) and constriction rate and rate in change of myosin intensity vs. time (bottom) for an individual cell. e, Constriction rate is most highly correlated with medial myosin rather than junctional myosin. The diagram (top) illustrates the purse-string model for contraction in which we expect actin and myosin to become concentrated in the junctional region upon constriction. Data points represent correlation coefficients (r-values) for individual cells and the black bar indicates the mean (n = 37 cells). *, difference between the means is statistically significant (p<0.0001). Scale bars = 4 μm.
Figure 3
Figure 3. Pulsed myosin coalescence and adherens junction bending require an actin-myosin network
a, Cortical myosin (green), cortical F-actin (red), and F-actin 2μm below the apical cortex (white, to illustrate cell shape) were visualized in fixed embryos. b, Timelapse images of Myosin-GFP in control injected (DMSO) and cytochalasin D (CytoD) injected embryos. Arrows indicate individual myosin spots. Note that myosin spots move, but do not coalesce in CytoD treated embryos. c, Single channel and merged timelapse images of Myosin-mCherry (green) and E-Cadherin-GFP (red). Red arrows indicate myosin coalescence. Blue arrows indicate the site where adherens junctions bend inward beneath a myosin spot. Scale bars = 4 μm.
Figure 4
Figure 4. Snail and Twist function at distinct phases of pulsed constriction
a, Timelapse images of Myosin-GFP Z-projections. Blue arrows indicate myosin spots that do not efficiently coalesce in snail mutants. Red arrows indicate myosin coalescence in twist mutants that appears to pull cell junctions. At least one coalescence event that pulled cell junctions occurred over a 6 minute period for 53 % of cells in the twist mutant compared to 4 % of cells in snail and snail twist mutants (n = 60 cells, 3 embryos per mutant). Scale bar = 4 μm. b, Timelapse images of Spider-GFP in snailRNAi or twistRNAi embryos. P[sna] indicates twist independent snail expression. Scale bar = 4 μm. c, Quantification of apical area (red) and constriction rate (blue) for individual cells in snailRNAi or twistRNAi embryos. d, Ratchet model of apical constriction. Myosin (green) contracts an apical actin network (red) that is coupled to adherens junctions (blue) driving constriction. Contractions are pulsed, interrupted by a phase in which the constricted state of the cell is stabilized.
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