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. 2013 Aug;15(8):926-36.
doi: 10.1038/ncb2796. Epub 2013 Jul 7.

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

Frank M Mason  1 , Michael Tworoger, Adam C Martin
Affiliations

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

Frank M Mason et al. Nat Cell Biol. 2013 Aug.

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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Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Rok and E-Cadherin exhibit “radial” cell polarity (RCP) in ventral furrow cells. (a) Diagram of ventral furrow cell and model of apical constriction. Snail is required to initiate contraction, and Twist stabilizes the contracted cell shape. During the stabilization, F-actin and Myo-II are often remodeled from foci into fibers. (b) Schematic of the Twist pathway. Twist activates the expression of Fog and T48, leading to Rho1 activation, which is proposed to activate Myo-II via Rok and stimulate F-actin assembly through Dia. (c) Model of AJ remodeling in ventral furrow cells. AJ initially assemble subapically then move apically into spot junctions when contraction initiates. (d) Rok is apically enriched in ventral furrow cells (arrowhead) prior to more lateral germband cells (arrow). Images are projections of cross-sections from a time-lapse movie of a rok2 germline mutant embryo expressing Venus::Rok(WT). Dotted yellow line marks the vitelline membrane. (e) Rok localizes to medioapical foci and patches. Surface view of same embryo in d. (f) Rok is polarized to a medioapical focus in individual cells. Images are from live embryo expressing Venus::Rok(K116A) (apical surface projection) and Gap43::ChFP (plasma membranes, subapical section). (g) Rok localizes to the medioapical surface between AJs. Images are from fixed embryo expressing GFP::Rok(K116A) (apical projection) and stained for E-Cadherin (subapical section). Images are surface view (top) and cross-sections (bottom). (h) Rok colocalizes with apical Myo-II. Images are from live embryo expressing Myo::ChFP and GFP::Rok(K116A). Scale bars are 5μm.
Figure 2
Figure 2
Rho1 pathway components exhibit distinct localizations across the apical surface. (a) Rho1 localizes to medioapical foci and the junctional domain. Apical surface projection of a fixed embryo expressing GFP::Rok(K116A) and stained for Rho1. (b) Diaphanous (Dia) colocalizes with Rho1 in both the medioapical and junctional domains. Images are apical surface projections from a fixed embryo expressing GFP::Rho1 and stained for Dia. (c) Colocalization of Rok and Dia. Images are apical surface projections from a fixed embryo expressing GFP::Rok(K116A) and stained for Dia. (d) Dia localizes to the medioapical F-actin and Myo-II meshwork. Images are apical surface projections from the same fixed Myo::GFP embryo stained for Dia and F-actin (phalloidin). All scale bars are 5μm.
Figure 3
Figure 3
Polarized Rok and Myo-II condenses dynamic medioapical F-actin cables. (a) F-actin meshwork organization during ventral furrow formation. Time-lapse images are from live embryos expressing Myo::ChFP (Myosin) and Utr::GFP (F-actin). Apical Myo-II and F-actin are apical z-projections and sub-apical F-actin is one subapical section. Scale bar is 5μm. (b) F-actin levels slightly increase or remain stable during contraction pulses. Quantification of total apical F-actin levels (green) and area (black) in an individual cell from a live embryo expressing Utr::GFP and Gap43::ChFP. Contractile pulses are highlighted (yellow) and the arrowheads indicate a slight increase in F-actin intensity that corresponds to F-actin condensation in c. (c) F-actin is dynamically remodeled during and after contraction pulses. Time-lapse images represent different phases of the contraction cycle. Contractile pulses and increases in F-actin levels (yellow highlights) are associated with the condensation of medioapical F-actin into foci (arrowheads). During stabilization phases (grey), F-actin foci are remodeled, often into cables (arrow). Images of cell and arrowheads corresponds to pulses in b. Scale bar is 5μm. (d) F-actin condensation occurs with Myo-II accumulation. Time-lapse images are from live embryo expressing Myo::ChFP and Utr::GFP. Scale bar is 3μm. (e) Graph of a representative contraction pulse demonstrates that total F-actin and Myo-II intensities within a subcellular region increase and decrease at the same time. (f) Myo-II and F-actin coalesce simultaneously. Graph of mean time-resolved cross-correlation analysis of F-actin and Myo-II intensities within pulses (n=25 pulses) demonstrates that correlation peaks at zero seconds. (g) Rok is required for Myo-II accumulation and apical constriction. Curves represent average apical area (black) and apical Myo-II intensity (magenta) in control (n=77 cells) and Y-27632 injected (n=82 cells) embryos. (h) Condensation of medioapical F-actin cables requires Rok activity. Representative images from embryos expressing Utr::GFP and injected with solvent (control) or Y-27632 (Rok inhibitor). Note the presence of F-actin cables in Y-27632 injected ventral furrow cells, which are not prominent in germband cells. Scale bar is 5μm. (i) Rok is not required for medioapical F-actin assembly. Representative time-lapse images are from a Utr::GFP expressing embryo that is injected with Y-27632. Arrowheads indicate F-actin cable appearance and elongation. Scale bar is 5μm.
Figure 4
Figure 4
F-actin polymerization is required to couple the contractile network to the junctions. (a) Titration of actin dynamics with CytoD identifies distinct functions for F-actin polymerization. Images are from live embryos expressing Myo::GFP and Gap43::ChFP. Embryos were injected with solvent (control), 0.125 mg/mL CytoD (low CytoD), or 5 mg/mL CytoD (high CytoD). Note low CytoD destabilizes the connection between the medioapical Myo-II network and cell-cell junctions, leading to tears in the supracellular Myo-II network (arrowheads). Cellular and tissue-wide Myo-II networks are disrupted with high CytoD. Scale bars are 20μm. (b) Mean apical area and Myo-II intensity for different CytoD doses. Data are from representative embryos injected with solvent (control, n=56 cells), low CytoD (n=86 cells), and high CytoD (n=64 cells). Note that low CytoD embryos initially constrict before losing adhesion (arrowhead). (c) CytoD disrupts contractile pulses. Colorbar represents cross-correlation between constriction rate and the rate of change in Myo-II intensity. Different cells are plotted (Cell index) for different temporal offsets. Note the significant cross-correlation peak around 0 offset in the solvent control. (d) Mean cross-correlation between constriction rate and rate in change of Myo-II intensity (p-value <0.0001, using unpaired, two-tailed test for control compared to low or high CytoD at time 0). Data are from representative embryos injected with solvent (n=56 cells), low CytoD (n=86 cells), and high CytoD (n=64 cells). (e) CytoD injection causes gaps in the medioapical F-actin meshwork prior to its dissociation from junctions. Image from live Utr::GFP embryo, injected with Low CytoD, demonstrates that F-actin network stretches prior to cell-cell separation (arrowhead). Image from live Utr::GFP embryo, injected with 0.25mg/mL CytoD (intermediate, Int. CytoD), shows fragmented medioapical F-actin network that is separated from junctions. Scale bars are 5μm.
Figure 5
Figure 5
Dia restricts E-Cadherin to the junctional domain, facilitating contractile network coupling to cell-cell AJs. (a) Dia is required to couple the medioapical contractile network to junctions. In diaM embryos, Myo-II puncta (arrowheads) lose adhesion and separate but can reattach by forming new connections (arrow). Control embryos, dia/+, exhibit new connections forming (arrow) and contraction of Myo-II puncta. The diaM embryos have similar phenotypes to embryos injected with low CytoD (0.125 mg/mL). Images are apical surface projections from live embryos expressing Myo::GFP. (b) In dia/+ controls, Myo-II forms supracellular meshwork that spans the medioapical surface and across junctions, whereas diaM embryos have fragmented medioapical meshworks that fail to maintain connections to junctions. Images are from fixed embryos expressing Myo::GFP (apical surface projection) and stained for E-Cadherin (subapical section). (c) Apical E-Cadherin localizes across the apical surface and loses RCP in diaM embryos. Images are apical surface projections or subapical section of E-Cadherin from same embryos in b. (d) Inhibition of F-actin assembly by CytoD causes accumulation of E-Cadherin across the apical surface, similar to phenotype in diaM. Images are from live embryos expressing E-Cad::GFP, injected with solvent (control) or CytoD (0.25 mg/ml). (e) Models for loss of contractile network coupling in dia mutants. Scale bars are 5μm.
Figure 6
Figure 6
Twist mediates medioapical F-actin cable assembly. (a) Twist and Snail are required for medioapical F-actin cable formation. Representative images are from time-lapse movies of control embryos (twist/+) and snail or twist mutants expressing Utr::GFP. Control embryos have more obvious F-actin cables (arrowheads). snail mutants have F-actin puncta that fail to form a fibrous meshwork. twist mutant constricting cell (arrowhead) has medioapical F-actin condensation during the pulse, but lacks visible F-actin cables before and after pulse. Non-pulsing cells (arrow) have junctional F-actin, but lack medioapical F-actin cables. Scale bars are 5μm. (b) Twist is required for the medioapical F-actin meshwork, but not the circumferential F-actin belt. Graphs represent averages of mean fluorescent intensity of apical and subapical F-actin from linescans across cells (n=6) from embryos in a. In control cells, F-actin is present in the medioapical cortex and accumulates at junctions. snail mutants equally lack both medioapical and junctional F-actin. Error bars are standard deviation. (c) twist mutants have lower ratio of medioapical:junctional F-actin. Quantifications of average ratio of medioapical to junctional apical F-actin for control (n=25), snail (n=25), and twist (n=20) cells. Difference between control and twist is significant (p<0.0001, two-tailed, Mann-Whitney test) but snail is not significant (n.s.). Error bars represent standard deviation. Scale bars are 5μm. (d) Twist is required to stabilize F-actin levels during contractile pulses. Quantification of apical area and F-actin intensity in individual control and twist mutant cell from embryo in a. Control cell contracts (yellow highlight) and F-actin and cell shape is stabilized during contraction. twist cells have pulsed contraction (yellow highlight) but do not maintain F-actin stabilization. (e) Graphs of average behavior of area and F-actin during contractile pulses from embryos in a (above, control n=51 pulses, below, twist n=45). Values are normalized to highest and lowest values in individual pulses. Error bars are standard deviation. Yellow highlights indicate time when F-actin levels are significantly increased from apical area in control (p<0.0001, two-tailed, Mann-Whitney test) but not in twist mutants (n.s.).
Figure 7
Figure 7
Twist is required for medioapical radial cell polarity of Rok and Myo-II. (a) twist and snail mutants result in an inversion of Rok RCP. Images are apical surface projections from live control, snail, or twist mutant embryos expressing GFP::Rok(K116A) and Gap43::ChFP. Rok abnormally localizes to junctions at three-way vertices in snail and twist mutants. (b) Rok localizes subapically in snail mutants, similar to position of the junctions, while Rok is present apically in control (twist/+) or twist mutants. Cross-section views of embryos from a. Dotted lines indicate the vitelline membrane (VM, white arrowhead). (c) Myo-II localization in snail and twist mutants mirrors Rok localization. Images are from live, control and snail or twist mutant embryos expressing Myo::GFP. Control embryos (twist/+) possess dense medioapical Myo-II meshwork, whereas Myo-II localizes to cell vertices in both snail and twist mutants (arrowheads). Myo-II transiently appears in medioapical foci in twist mutants during constriction pulses (arrow). (d) The apical-basal position of Myo-II mirrors the position of Rok. Images are cross-section views of embryos from c. Myo-II remains subapical in snail mutants, but localizes apically in control and twist mutants. (e) Apical-basal position of Myo-II corresponds to position of adherens junctions. Myo-II localizes to subapical adherens junctions in snail mutants and to apical junctions in twist embryos. Images are cross-sections of fixed embryos expressing Myo::GFP and stained for E-Cadherin. Scale bars are 5μm.
Figure 8
Figure 8
Radial cell polarity (RCP) coordinates Myo-II stabilization, F-actin assembly, and E-Cadherin localization to facilitate apical constriction. (a) Comparison of cell polarity in germband cells (planar cell polarity, PCP) and ventral furrow cells (radial cell polarity, RCP). Changes in RCP are shown for twist and dia mutants. Note that we have not analyzed Rok localization in dia mutants. (b) Model for the Twist-mediated actin-myosin ratchet. Periodic medioapical Myo-II pulses transiently contract the cell (Contraction). Myo-II stabilization and F-actin cable assembly are coordinated to form medioapical contractile fibers that are anchored at AJs and stabilize cell shape fluctuations elicited by pulsing.
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