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

ART I C L E S

Apical domain polarization localizes actin–myosinactivity to drive ratchet-like apical constrictionFrank M. Mason1, Michael Tworoger1 and Adam C. Martin1,2

Apical constriction promotes epithelia folding, which changes tissue architecture. During Drosophila gastrulation, mesoderm cellsexhibit repeated contractile pulses that are stabilized such that cells apically constrict like a ratchet. The transcription factor Twistis required to stabilize cell shape. However, it is unknown how Twist spatially coordinates downstream signals to prevent cellrelaxation. 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 contractsdynamic medioapical actin cables. The formin Diaphanous mediates apical actin assembly to suppress medioapical E-cadherinlocalization and form stable connections between the medioapical contractile network and adherens junctions. Twist is notrequired for apical Rok recruitment, but instead polarizes Rok medioapically. Therefore, Twist establishes radial cell polarity ofRok/Myo-II and E-cadherin and promotes medioapical actin assembly in mesoderm cells to stabilize cell shape fluctuations.

Apical constriction is an epithelial cell shape change that promotestissue bending during developmental processes such as gastrulationand neural tube closure1–3. Apical constriction bends epithelia bytransforming columnar cells to a wedge shape4. During Drosophilagastrulation, the apical constriction of presumptive mesoderm cellsalong the ventral midline results in ventral furrow formation and tissueinvagination5,6. Apical constriction and ventral furrow formation areinduced by the expression of two transcription factors, Twist andSnail5,7,8. A major question in the field has been how Twist and Snailpromote force generation and apical constriction at themolecular level.Forces that drive apical constriction are generated by the contraction

of an actin filament (F-actin) network by the molecular motornon-muscle myosin II (Myo-II; refs 9–12). In ventral furrow cells, andmany other contractile systems, Myo-II contractions and cell shapechanges occur in a pulsed or ratchet-likemanner13–24. Contractile pulsesin ventral furrow cells occur in the F-actin/Myo-II network spanningthe apical domain (medioapical cortex), which pull peripheral adherensjunctions inward20 (Fig. 1a). After a contraction pulse, the constrictedstate of the cell is stabilized to incrementally decrease apical area, similarto the mechanism of a ratchet20. Twist and Snail regulate distinctsteps of this ratchet-like constriction20. Snail is required to initiatecontractile pulses, but the mechanism is unclear. Twist is required tostabilize cell shape between pulses. Two Twist transcriptional targets,Folded gastrulation (Fog) and T48, could function in parallel toactivate the Rho1 GTPase apically25,26 (Fig. 1b). It is thought that apicalsecretion of Fog activates Rho1 signalling and Myo-II recruitment

1Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.2Correspondence should be addressed to A.C.M. (e-mail: [email protected])

Received 4 February 2013; accepted 29 May 2013; published online 7 July 2013; DOI: 10.1038/ncb2796

across the apical surface of ventral furrow cells27–29. HowRho1 stabilizescell shape is not known and could depend on tension generatedby medioapical or junctional cytoskeletal networks13,22,30. Therefore,elucidating the ratchet mechanism requires determining how Rho1and its effectors regulate medioapical and junctional actin–myosinnetworks in response to Twist and Snail.Contractile forces must be coupled to the adherens junctions

to generate cell and tissue shape changes28,30–34. Adherens junctionremodelling accompanies ventral furrow cell apical constriction, withsubapical adherens junctions being disassembled, which requires Snail,and spot junctions assembling at the apical cell–cell interfaces, whichseems to require Twist26,28,30 (Fig. 1c). It is not known how signals thatactivate Myo-II are coordinated with adherens junction remodelling tocouple contraction to adherens junctions.Here we visualize how the dynamics of the Myo-II, F-actin and

adherens junctions are coordinated with the Rho1 GTPase pathway todissect themechanism of ratchet-like apical constriction.

RESULTSVentral furrow cells exhibit radial cell polarity of Rok/Myo-IIand E-cadherinThe Rho1 effector Rok phosphorylates and activates Myo-II and isrequired for ventral furrow cell apical constriction, suggesting thatapical Fog-dependent activation of Rok and Myo-II triggers apicalconstriction28,35. The importance of Rok in polarizing contractionis supported by the fact that planar polarized Rok localizes Myo-II

926 NATURE CELL BIOLOGY VOLUME 15 | NUMBER 8 | AUGUST 2013

© 2013 Macmillan Publishers Limited. All rights reserved.

mailto:[email protected]

http://www.nature.com/doifinder/10.1038/ncb2796

ART I C L E S

JunctionalMedioapical

Twist

DRhoGEF2

T48Fog

Cta

Snail

Contraction Stabilization

Twist

a

c

e

b d

g

f h

rok2

muta

nt;

V

enus::R

ok (W

T)

cro

ss-s

ectio

n

4.3 min0 min 2.3 min

6.3 min 8.4 min 10.6 min

Rok

Rok E-cad Merge

Surf

ace

Cro

ss-s

ectio

n

Rok

Apical

Basal

Subapical

Myosin F-actin Adherens junctions Rho1

Rok

Myo-II activation

Dia (F-actin assembly)

11 min

6.7 min

2.3 min

18.1 minSubapical junctions Apical junctions Apical constriction

rok2

muta

nt;

V

enus::R

ok (W

T)

surf

ace v

iew

Myosin MergeMembranes Merge

Figure 1 Rok and E-cadherin exhibit radial cell polarity (RCP) in ventralfurrow cells. (a) Diagram of ventral furrow cell and model of apicalconstriction. Snail is required to initiate contraction, and Twist stabilizes thecontracted cell shape. During the stabilization, F-actin and Myo-II are oftenremodelled from foci into fibres. (b) Schematic of the Twist pathway. Twistactivates the expression of Fog and T48, leading to Rho1 activation, whichis proposed to activate Myo-II through Rok and stimulate F-actin assemblythrough Dia. (c) Model of adherens junction remodelling in ventral furrowcells. Adherens junctions initially assemble subapically and then moveapically into spot junctions when contraction initiates. (d) Rok is apicallyenriched in ventral furrow cells (arrowhead) before more lateral germbandcells (arrow). Images are projections of cross-sections from a time-lapse

movie of a rok2 germline mutant embryo expressing Venus::Rok(WT). Thedotted yellow line marks the vitelline membrane. (e) Rok localizes tomedioapical foci and patches. Surface view of the same embryo as in d.(f) Rok is polarized to a medioapical focus in individual cells. Images arefrom a live embryo expressing Venus::Rok(K116A) (apical surface projection)and Gap43::ChFP (plasma membranes, subapical section). (g) Rok localizesto the medioapical surface between adherens junctions. Images are from afixed embryo expressing GFP::Rok(K116A) (apical projection) and stainedfor E-cadherin (subapical section). Images are surface view (top) andcross-sections (bottom). (h) Rok co-localizes with apical Myo-II. Images arefrom a live embryo expressing Myo::ChFP and GFP::Rok(K116A). Scale bars,5 µm.

contraction to anterior–posterior cell interfaces during convergentextension of the Drosophila germband cells36–40. In addition, planarpolarized Rok excludes Bazooka/Par-3 from the cortex, establishingcomplementary domains of Rok/MyoII and adherens junctionproteins40. To test the role of Rok in ventral furrow cells, weexamined Rok localization dynamics using either a kinase-deadRok allele, Venus(or GFP)::Rok(K116A), or a Venus::Rok(WT)transgene expressed in rok2 mutant germline clones40. Venus(orGFP)::Rok(K116A) and Venus::Rok(WT) localization patterns wereindistinguishable, and Venus::Rok(WT) rescued the ventral furrowinvagination defect of rok2 mutants28, suggesting that both Venus::Roktransgenes indicate the localization of endogenous Rok (Fig. 1d,e,fand Supplementary Fig. S1). Rok exhibited apical accumulation inventral furrow cells before Rok apical localization in the germband(Fig. 1d). Apical domain proteins normally localize directly aboveadherens junctions or uniformly across the apical surface of epithelialcells41,42. However, Rok exhibited an unexpected apical organization,initially exhibiting dispersed staining and then accumulating inmedioapical foci as cells constricted (Fig. 1e,f and SupplementaryFig. S1). In contrast to germband cells, where Rok is present atcell–cell interfaces40, Rok intensity seemed lowest at the junctions

(Fig. 1f,g). Medioapical Rok foci co-localized with Myo-II, consistentwith Rok recruiting or stabilizing Myo-II (Fig. 1h). Thus, similarto germband cells, Rok/Myo-II and E-cadherin are enriched indifferent domains. However, in ventral furrow cells, Rok/Myo-IIis concentrated in medioapical foci and adherens junctions arepresent around the cell circumference, a pattern that we termradial cell polarity (RCP).

The Rho1 GTPase and its effectors exhibit distinctlocalization patternsTo determine whether other Twist pathway components are polarizedto medioapical foci, we examined the localization of Rho1 and aRho1 effector that mediates unbranched actin polymerization andF-actin cable formation, the formin Diaphanous27,29,43 (Dia). Rho1localized to medioapical foci that partially overlapped Rok foci,but also localized to the junctional domain (Fig. 2a). Dia partiallyco-localized with medioapical Rho1/Rok foci and junctional Rho1(Fig. 2b,c). However, Dia exhibited more diffuse staining across theapical domain, which largely co-localized with the F-actin/Myo-IImeshwork (Fig. 2d). Therefore, Rho1, Rok and Dia are differentiallylocalized in the apical domain of ventral furrow cells, which suggests

NATURE CELL BIOLOGY VOLUME 15 | NUMBER 8 | AUGUST 2013 927


ART I C L E S

Rho1Rok Merge

DiaRho1 Merge

DiaRok Merge

Subapical F-actin

Apical projection Merge

Dia

Myo

sin

F-a

ctin

d

a

c

b

Figure 2 Rho1 pathway components exhibit distinct localizations acrossthe apical surface. (a) Rho1 localizes to medioapical foci and thejunctional domain. Apical surface projection of a fixed embryo expressingGFP::Rok(K116A) and stained for Rho1. (b) Diaphanous (Dia) co-localizeswith Rho1 in both the medioapical and junctional domains. Images areapical surface projections from a fixed embryo expressing GFP::Rho1 andstained for Dia. (c) Co-localization of Rok and Dia. Images are apical surfaceprojections from a fixed embryo expressing GFP::Rok(K116A) and stainedfor Dia. (d) Dia localizes to the medioapical F-actin and Myo-II meshwork.Images are apical surface projections from the same fixed Myo::GFP embryostained for Dia and F-actin (phalloidin). Scale bars, 5 µm.

that Twist and Rho1 spatially regulate F-actin and Myo-II activityduring ratchet-like constriction.

Rok is required for medioapical F-actin network condensationWe reasoned that the RCP of Rok might spatially control Myo-IIactivation or stabilization to regulate contractility or the organizationof the apical F-actin network, both of which are important for apicalconstriction44. To determine how medioapical Rho1–Rok foci regulateF-actin network contraction in ventral furrow cells, we visualizedF-actin and Myo-II dynamics by expressing the F-actin bindingdomain of Utrophin fused to GFP (Utr::GFP) and the regulatorylight chain of Myo-II (spaghetti squash, sqh in Drosophila) fused tomCherry fluorescent protein (Myo::ChFP; refs 20,22). Utr::GFP labelsmedioapical, junctional and basolateral networks, similar to phalloidin

staining in fixed embryos, demonstrating that Utr::GFP labels F-actinnetworks in an unbiased manner (Supplementary Fig. S2a,b). Similarto Rok localization, apical Myo-II recruitment occurs in ventral furrowcells before germband cells and was associated with structural changesin the F-actin network (Fig. 3a, and Supplementary Videos S1 andS2). To analyse F-actin dynamics, we first used custom software tosegment individual cells and quantify total apical F-actin levels duringapical constriction45. In contrast to the increase in apical Myo-IIintensity, we found that average total apical F-actin levels decreaseover the course of apical constriction, suggesting that F-actin networkdepolymerization accompanies constriction (Supplementary Fig. S2c).However, during contraction pulses we often observed brief increases orstabilization of F-actin intensity (Fig. 3b and Supplementary Fig. S2d,e).The increase or stabilization of F-actin intensity was associatedwith F-actin condensation into medioapical foci (Fig. 3c). FollowingF-actin condensation, F-actin foci were subsequently remodelled,often into cables, and F-actin intensity seemed to decrease as areastabilized (Fig. 3c, and Supplementary Fig. S2d). The mechanism forthis F-actin remodelling is unclear, but F-actin depolymerizationis possibly required to regenerate actin monomers that are thenassembled into new F-actin cables. Medioapical F-actin condensationand dissolution occurred simultaneously with Myo-II (Fig. 3d–f),suggesting that F-actin condensation represents contraction of themedioapical F-actin cortex. To determine whether medioapical F-actincondensation requires Rok, we injected embryos with the Rok inhibitorY-27632 (ref. 46). Y-27632-injected embryos failed to accumulatecortical Myo-II, consistent with previous observations47, and cells didnot constrict (Fig. 3g). However, medioapical F-actin was prominentin ventral furrow cells after Y-27632 injection. F-actin filamentsor cables continuously assembled and disassembled (Fig. 3h,i, andSupplementary Video S3). F-actin cables originated medioapicallyand junctionally, consistent with Rho1 and Dia localization at bothlocations (Fig. 3i). Despite robust medioapical F-actin assembly,F-actin cables failed to condense into medioapical foci in Y-27632-injected embryos (Supplementary Videos S2 and S3). Thus, RCP ofRok is dispensable for medioapical F-actin assembly, but is requiredto condense medioapical F-actin cables that span the apical domain,presumably by targetingMyo-II to themedioapical cortex.

F-actin polymerization is required to couple medioapicalcontraction to cell–cell junctionsOur visualization of F-actin cable assembly at both the medioapicalcortex and junctions of Y-27632-injected ventral furrow cells suggestedthat Rho1 could activate Dia to stimulate F-actin assembly across theapical domain. Furthermore, Y-27632-injected germband cells haveless pronounced medioapical F-actin cables than in ventral furrowcells (Fig. 3h), supporting a previous study that suggested that signalsin ventral furrow cells enhance medioapical F-actin assembly relativeto germband cells48. To test the function of F-actin assembly duringapical constriction, we injected embryos with cytochalasin D (CytoD),which binds the barbed end of F-actin and inhibits polymerization49.High CytoD concentrations blocked apical constriction and disruptedthe F-actin/Myo-II network, resulting in apical Myo-II spots thatfail to coalesce, as previously reported20 (Fig. 4a,b). We titrated theamount of injected CytoD to identify processes that were most sensitiveto reduced F-actin assembly. Lower CytoD concentrations allowed



ART I C L E S

Apical F-actin + Y-27632

0 s 20 s 40 s

60 s 80 s 100 s

Ju

nctio

nal p

oly

merizatio

n4 s

Med

ioap

ical 0 s 8 s

0 100 200F-a

ctin a

nd

Myo

(a.u

.)

Time (min)

Quantification of total intensity for individual pulse

r

Merge

0 5 10

Time (min)

Control

+Y-27632

0 1 2 3 4 5 6 7

F-a

ctin (a.u

.)

Quantification of total apical

intensity for individual cell

Myo

sin

F-a

ctin

(a

pic

al)

F-a

ctin

(s

ub

ap

ical)

90 s

F-a

ctin

Myo

-II

Merg

e

50

40

30

20

10

0Ap

ical are

a (

µm2)

No

rm.

Myo

(a.u

. µm

–2)

600

400

200

0

800

No

rm.

Myo

(a.u

. µm

–2)

600

400

200

0

800

Time (min)

0 5 10

Ap

ical are

a (

µm2)

Ap

ical are

a (

µm2)

50

40

30

20

10

0

0 53–53

0

–0.2

0.2

0.4

0.6

0

500

1,000

1,500

0

20

40

60

4.6

min

5.2

min

5.7

min

Co

ntr

ol

+Y

-27

63

2+

Y-2

76

32

Apical F-actin

Subapical F-actin

Ven

tral fu

rro

wG

erm

ban

d

250

200

150

100

50

0

Time (s) Time offset (s)

0 min 5 min 10 min 0 s 18 s 36 s 54 s 72 sF-actin Membranes Merge dc

e f

h i

b

a

g

Figure 3 Polarized Rok and Myo-II condenses dynamic medioapical F-actincables. (a) F-actin meshwork organization during ventral furrow formation.Time-lapse images are from live embryos expressing Myo::ChFP (Myosin) andUtr::GFP (F-actin). Apical Myo-II and F-actin are apical z projections andsubapical F-actin is one subapical section. (b) F-actin levels slightly increaseor remain stable during contraction pulses. Quantification of total apicalF-actin levels (green) and area (black) in an individual cell from a live embryoexpressing Utr::GFP and Gap43::ChFP. Contractile pulses are highlighted(yellow) and the arrowheads indicate a slight increase in F-actin intensitythat corresponds to F-actin condensation in c. (c) F-actin is dynamicallyremodelled during and after contraction pulses. Time-lapse images representdifferent phases of the contraction cycle. Contractile pulses and increasesin F-actin levels (yellow highlights) are associated with the condensation ofmedioapical F-actin into foci (arrowheads). During stabilization phases (grey),F-actin foci are remodelled, often into cables (arrow). Images of cell andarrowheads corresponds to pulses in b. (d) F-actin condensation occurs withMyo-II accumulation. Time-lapse images are from a live embryo expressing

Myo::ChFP and Utr::GFP. (e) Graph of a representative contraction pulsedemonstrates that total F-actin and Myo-II intensities within a subcellularregion increase and decrease at the same time. (f) Myo-II and F-actincoalesce simultaneously. Graph of mean time-resolved cross-correlationanalysis of F-actin and Myo-II intensities within pulses (n = 25 pulses)demonstrates that correlation peaks at zero seconds. (g) Rok is requiredfor Myo-II accumulation and apical constriction. Curves represent averageapical area (black) and apical Myo-II intensity (magenta) in control (n=77cells) and Y-27632-injected (n = 82 cells) embryos. (h) Condensation ofmedioapical F-actin cables requires Rok activity. Representative images fromembryos expressing Utr::GFP and injected with solvent (control) or Y-27632(Rok inhibitor). Note the presence of F-actin cables in Y-27632-injectedventral furrow cells, which are not prominent in germband cells. (i) Rok isnot required for medioapical F-actin assembly. Representative time-lapseimages are from a Utr::GFP-expressing embryo that is injected with Y-27632.Arrowheads indicate F-actin cable appearance and elongation. Scale bars,5 µm (a,c,h,i) and 3 µm (d).

ventral furrow cells to assemble an apical F-actin/Myo-II network andinitiate constriction (Fig. 4a,b). Normally, ventral furrow cells undergocontractile pulses, resulting in a significant positive cross-correlationbetween calculated constriction rate and Myo-II accumulation ratedue to in-phase contraction pulses20,30 (Supplementary Fig. S2f).Despite undergoing apical constriction and Myo-II accumulation, wefailed to observe obvious Myo-II pulses in embryos injected with

low CytoD concentrations, which was supported by the lack of across-correlation between constriction rate and myosin rate (Fig. 4c,d).Also, low CytoD concentrations destabilized the connection betweenthe medioapical actin–myosin network and cell–cell junctions, withnetworks transiently losing and then regaining connections to eachother across the tissue (Fig. 4a, and Supplementary Videos S4–S6).Low CytoD concentrations resulted in gaps in the medioapical F-actin



ART I C L E S

0

–1

1r

Time offset (min)

Cell

ind

ex

Cell

ind

ex

Cell

ind

ex

DMSO

Time offset (min)

Low CytoD

r

DMSOLow

High

d

a

c e

b

∗∗∗∗

Co

ntr

ol

Lo

w C

yto

DH

igh

Cyto

D

Lo

w C

yto

DC

on

tro

lIn

t. C

yto

DHigh CytoD

0 1 2 3 4 5 6 7 8

Time (min)

Control

High CytoD

Low CytoD

MembranesMyosin Merge

Apical F-actin

Subapical F-actin Merge0

25

50

0 1–1–2 2

0

20

40

600 1–1

Time offset (min)

0

20

40

60

0 1–1

Time offset (min)

0 1–1–2 2

0

0.2

0.4

–0.2

50

40

30

20

10

0

Ap

ical are

a (

μm2)

Myo

sin

in

ten

sity (a.u

.)

0 1 2 3 4 5 6 7 8

Time (min)

50

40

30

20

10

0

Ap

ical are

a (

μm2)

Myo

sin

in

ten

sity (a.u

.)

0 1 2 3 4 5 6

Time (min)

50

40

30

20

10

0

Ap

ical are

a (

μm2)

Myo

sin

in

ten

sity (a.u

.)

Figure 4 F-actin polymerization is required to couple the contractile networkto the junctions. (a) Titration of actin dynamics with CytoD identifiesdistinct functions for F-actin polymerization. Images are from live embryosexpressing Myo::GFP and Gap43::ChFP. Embryos were injected withsolvent (control), 0.125mgml−1 CytoD (low CytoD) or 5mgml−1 CytoD(high CytoD). Note low CytoD destabilizes the connection between themedioapical Myo-II network and cell–cell junctions, leading to tears in thesupracellular Myo-II network (arrowheads). Cellular and tissue-wide Myo-IInetworks are disrupted with high CytoD. (b) Mean apical area and Myo-IIintensity for different CytoD doses. Data are from representative embryosinjected with solvent (control, n =56 cells), low CytoD (n =86 cells) andhigh CytoD (n =64 cells). Note that low-CytoD embryos initially constrictbefore losing adhesion (arrowhead). (c) CytoD disrupts contractile pulses.The colour bar represents cross-correlation between constriction rate and

the rate of change in Myo-II intensity. Different cells are plotted (Cellindex) for different temporal offsets. Note the significant cross-correlationpeak around 0 offset in the solvent control. (d) Mean cross-correlationbetween constriction rate and rate in change of Myo-II intensity (P value<0.0001, using an unpaired, two-tailed test for control compared withlow or high CytoD at time 0). Data are from representative embryosinjected with solvent (n = 56 cells), low CytoD (n = 86 cells) and highCytoD (n =64 cells). (e) CytoD injection causes gaps in the medioapicalF-actin meshwork before its dissociation from junctions. Image from a liveUtr::GFP embryo, injected with low CytoD, demonstrates that the F-actinnetwork stretches before cell–cell separation (arrowhead). Image from alive Utr::GFP embryo, injected with 0.25mgml−1 CytoD (intermediate, Int.CytoD) shows a fragmented medioapical F-actin network that is separatedfrom junctions. Scale bars, 20 µm (a) and 5 µm (e).

cortex that preceded junction detachments, demonstrating that defectsin medioapical F-actin network continuity precipitate separationof the medioapical network from cell–cell junctions (Fig. 4e, andSupplementary Videos S7). Overall, these results demonstrate thatrobust apical F-actin assembly is critical for medioapical contractionpulses and to form a stable connection between the contractilemedioapical domain and adherens junctions.

Diaphanous couples the contractile network to junctions,possibly by polarizing adherens junction localizationDia was previously shown to be important for ventral furrowformation50. To determine whether Dia mediates F-actin assembly

and junction attachment, we generated germline clones using thepartial loss-of-function dia5 allele (diaM)50. We were unable to analyseventral furrow formation in diaM embryos with strong phenotypes,because these had severe defects during cellularization, which precedesgastrulation51–53. However, diaM embryos with mild cellularizationphenotypes had apical Myo-II accumulation and exhibited transientlosses in the connections between medioapical the F-actin/Myo-IInetwork and junctions, similar to embryos injected with low CytoDconcentrations (Fig. 5a,b, and Supplementary Video S8). Together withthe CytoD injections, our results suggest that Dia-dependent F-actinassembly in part mediates medioapical and junctional F-actin assemblyto link themedioapical cortex to adherens junctions.



ART I C L E S

Adherens junction depolarization

Co

ntr

ol

Cyto

Dd

ia/+

0 min 0.6 min 1.3 min 1.8 min

Lo

w C

yto

Dd

iaM



Myosin

Loss of F-actin assembly

Network dissolutionMyosin

F-actin

Adherens junctions

Diffuse adherens junctions

dia

/+d

iaM

Subapical E-cad MergeMyosin

dia

/+d

iaM

Apical E-cadSubapical

E-cad Merge

Apical E-cadSubapical

E-cad Merge

a

d e

cb

Figure 5 Dia restricts E-cadherin to the junctional domain, facilitatingcontractile network coupling to cell–cell adherens junctions. (a) Dia isrequired to couple the medioapical contractile network to junctions. IndiaM embryos, Myo-II puncta (arrowheads) lose adhesion and separatebut can reattach by forming new connections (arrow). Control embryos,dia/+, exhibit new connections forming (arrow) and contraction of Myo-IIpuncta. The diaM embryos have similar phenotypes to embryos injectedwith low CytoD (0.125mgml−1). Images are apical surface projectionsfrom live embryos expressing Myo::GFP. (b) In dia/+ controls, Myo-IIforms a supracellular meshwork that spans the medioapical surface andacross junctions, whereas diaM embryos have fragmented medioapical

meshworks that fail to maintain connections to junctions. Images arefrom fixed embryos expressing Myo::GFP (apical surface projection) andstained for E-cadherin (subapical section). (c) Apical E-cadherin localizesacross the apical surface and loses RCP in diaM embryos. Images areapical surface projections or subapical section of E-cadherin from thesame embryos as in b. (d) Inhibition of F-actin assembly by CytoDcauses accumulation of E-cadherin across the apical surface, similarto the phenotype in diaM. Images are from live embryos expressingE-cad::GFP, injected with solvent (control) or CytoD (0.25mgml−1).(e) Models for loss of contractile network coupling in dia mutants. Scalebars, 5 µm.

Dia-mediated F-actin assembly could facilitate apical constriction bygenerating medioapical F-actin cables to facilitate Myo-II contraction(Figs 3c and 4e), but could also regulate the organization of adherensjunctions39,50,54,55. As Twist has been proposed to be required for apicaladherens junction formation26, we examined E-cadherin localizationin diaM mutants. We found that E-cadherin localized apically in diaM

mutants. However, E-cadherin was not restricted to the junctionaldomain, but spread across the entire apical surface, which is differentfrom E-cadherin localization in diaM mutants for other cell types50

(Fig. 5c). Although it is possible that some of this apical E-cadherinis present in endocytic vesicles and not cortical50,56 (SupplementaryFig. S3), it is clear that there is a disruption in the localizationand polarity of E-cadherin in diaM mutants. E-cadherin polaritywas also lost after CytoD injection (Fig. 5d). Interestingly, defectsin Dia-mediated F-actin assembly result in a similar loss of corticaldomains or compartments during the process of cellularization,potentially from misregulation of endocytic activity53,57,58. Thus,Dia-mediated F-actin assembly is required in ventral furrow cells forthe RCP of apical E-cadherin, restricting E-cadherin to the junctionaldomain. The cell–cell attachment defect is unlikely to result fromlower E-cadherin levels at the junctional domain because medioapicalnetworks can regain attachments (Fig. 5a), whereas adherens junctionmutants result in permanent dissociations between the medioapicalnetwork and the junctions30. We propose that Dia-mediated F-actinassembly could facilitate the attachment between the medioapical

contractile network and junctions by spatially segregating contractileand junctional components into distinct domains, providing definitiveanchor points at the cell periphery (Fig. 5e, adherens junctiondepolarization). In addition, Dia could be required to assemblemedioapical and junctional F-actin cables to link the two domains(Fig. 5e, Network dissolution).

Twist is required for a continuous medioapical F-actin meshworkOur results demonstrate that the spatial regulation of Myo-IIrecruitment, F-actin assembly and adherens junction organizationcooperate to achieve ratchet-like apical constriction. Thus, Twistexpression could stabilize pulsed contractions by controlling F-actin,Myo-II or adherens junctions in specific cellular domains. Recently,cell shape fluctuations resulting from medioapical contractions wereproposed to be stabilized by junctional actin–myosin networks13,22. AsRho1 and Dia localize both medioapically and junctionally in ventralfurrow cells, we investigatedwhether Twist is required for either of theseF-actin networks. In contrast to wild-type ventral furrow cells, whichexhibit a continuous meshwork of medioapical and junctional F-actincables, visualization of Utr::GFP in twist mutants demonstrated thatthe twist mutant results in dynamicmedioapical F-actin puncta that failto form cables across the apical domain (Fig. 6a, and SupplementaryVideo S9). Importantly, twist mutants have junctional F-actin cables(Fig. 6a,b), which exhibit a similar intensity to junctional F-actinin lateral germband cells (furrow/germband ratio = 0.96, n = 20



ART I C L E S

0 3 6 9 12

snail twistControl

500

1,000

750

Control

NS

twist pulse

Med

ial/ju

nctio

nal

ap

ical F

-actin

ratio

Control snail twist

∗∗∗∗NS

snai

l

Apical F-actinSubapical

F-actin Merge

Co

ntr

ol

Control pulse

∗∗∗∗

Time (min)

0 1 2 3 4 5

twisttwis

t

0 s 17 s 25 s 58 s 66 s

cApical F-actin

Subapical F-actin Merge

Ap

ical F

-actin

Su

bap

ical F

-actin

Merg

e

Su

bap

ical an

d a

pic

al

F-a

ctin

in

ten

sity (a.u

.)

120

0

60

0 2.5 5.0 7.5

Distance (μm) Distance (μm)

120

0

60

0 3 6 9 12

120

0

60

0

0.5

1.0

1.5

0

0.5

1.0

–30 300–30 300

No

rmaliz

ed

are

a

an

d F

-actin (a.u

.)

0

0.5

1.0

500

900

700

40

50

60

Ap

ical are

a (

μm2)

Time (min)

0 1 2 3 4 65

F-a

ctin

(a.u

.)F

-actin

(a.u

.)

0

25

50

Ap

ical are

a (

μm2)

a

d

Time (s)Distance (μm) Time (s)

b e

Figure 6 Twist mediates medioapical F-actin cable assembly. (a) Twist andSnail are required for medioapical F-actin cable formation. Representativeimages are from time-lapse movies of control embryos (twist/+) and snailor twist mutants expressing Utr::GFP. Control embryos have more obviousF-actin cables (arrowheads). snail mutants have F-actin puncta that fail toform a fibrous meshwork. Twist mutant constricting cells (arrowhead) havemedioapical F-actin condensation during the pulse, but lack visible F-actincables before and after pulse. Non-pulsing cells (arrow) have junctionalF-actin, but lack medioapical F-actin cables. Scale bars, 5 µm. (b) Twist isrequired for the medioapical F-actin meshwork, but not the circumferentialF-actin belt. Graphs represent averages of mean fluorescence intensity ofapical and subapical F-actin from line scans across cells (n = 6) fromembryos in a. In control cells, F-actin is present in the medioapical cortexand accumulates at junctions. snail mutants equally lack both medioapicaland junctional F-actin. Error bars are standard deviation. (c) Twist mutantshave a 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 andtwist is significant (P <0.0001, two-tailed, Mann–Whitney test) but snailis not significant (NS). Error bars represent standard deviation. (d) Twist isrequired to stabilize F-actin levels during contractile pulses. Quantificationof apical area and F-actin intensity in individual control and twist mutantcells from the embryos in a. The control cell contracts (yellow highlight)and F-actin and cell shape is stabilized during contraction. Twist cellshave pulsed contraction (yellow highlight) but do not maintain F-actinstabilization. (e) Graphs of average behaviour of area and F-actin duringcontractile pulses from embryos in a (left, control n = 51 pulses, right,twist n = 45 pulses). Values are normalized to highest and lowest valuesin individual pulses. Error bars are standard deviation. Yellow highlightsindicate time when F-actin levels are significantly increased from apicalarea in control (P < 0.0001, two-tailed, Mann–Whitney test) but not intwist mutants (NS).

cells). Twist mutants exhibited a lower medioapical/junctional F-actinratio, suggesting that twist mutants are defective in the formationof the medioapical F-actin network relative to junctional F-actin(Fig. 6c). This contrasts with snail mutants, which lack medioapicaland apical junctional F-actin cables (Fig. 6a–c and SupplementaryVideo S9). As Twist is required for cell shape stabilization afterpulses, we examined F-actin dynamics over the course of individualpulsatile events. In wild-type cells, F-actin levels usually increase orremain stable during the contraction and persist as F-actin cables(Fig. 3b). However, in twist mutants, the F-actin network is alwayspunctate and F-actin levels decrease during contraction (Fig. 6a,d,e).Overall, these results suggest that Twist facilitates medioapical F-actinassembly. Importantly, twist mutants did not result in E-cadherin

depolarization, suggesting that the medioapical meshwork of F-actincables contributes to cell shape stabilization independently fromregulating E-cadherin RCP (Fig. 7e).

Twist mutants result in the inversion of Rok and Myo-II RCPWe previously demonstrated that twist is required for apical Myo-IIpersistence following pulsed contractions30. The RCP of Rok suggeststhat medioapical Myo-II phosphorylation could be coordinatedwith F-actin assembly to stabilize medioapical actin–myosin cablesthat prevent cell relaxation. Therefore, we examined whether Twistregulates the RCP of Rok. Previous work suggested that the Twistpathway is required for the apical localization of the Rho1 activatorDRhoGEF2 (Fig. 1b; ref. 26). Unexpectedly, we found apical Rok



ART I C L E S

twist

snail

Myosin

twis

t

RokVM

Co

ntr

ol

snai

l

twist

snail

Control cross-section

Control cross-section

ApicalMyosin

dcb

Rok Membranes Merge

twist cross-sectionsnail cross-sectionControl cross-section

Co

ntr

ol

snai

ltw

ist

Myosin

E-cad

Merge

a

e

Figure 7 Twist is required for medioapical radial cell polarity of Rok andMyo-II. (a) twist and snail mutants result in an inversion of Rok RCP.Images are apical surface projections from live control, snail or twist mutantembryos expressing GFP::Rok(K116A) and Gap43::ChFP. Rok abnormallylocalizes to junctions at three-way vertices in snail and twist mutants.(b) Rok localizes subapically in snail mutants, similar to the position ofthe junctions, whereas Rok is present apically in control (twist/+) or twistmutants. Cross-section views of embryos from a. Dotted lines indicate thevitelline membrane (VM, white arrowhead). (c) Myo-II localization in snailand twist mutants mirrors Rok localization. Images are from live, controland snail or twist mutant embryos expressing Myo::GFP. Control embryos

(twist/+) possess dense medioapical Myo-II meshwork, whereas Myo-IIlocalizes to cell vertices in both snail and twist mutants (arrowheads).Myo-II transiently appears in medioapical foci in twist mutants duringconstriction pulses (arrow). (d) The apical–basal position of Myo-II mirrorsthe position of Rok. Images are cross-section views of embryos from c.Myo-II remains subapical in snail mutants, but localizes apically in controland twist mutants. (e) The apical–basal position of Myo-II corresponds tothe position of adherens junctions. Myo-II localizes to subapical adherensjunctions in snail mutants and to apical junctions in twist embryos. Imagesare cross-sections of fixed embryos expressing Myo::GFP and stained forE-cadherin. Scale bars, 5 µm.

enrichment in twist mutant ventral furrow cells (SupplementaryFig. S4). However, the normal RCP of Rok was inverted in twistmutants, with Rok exhibiting junctional localization at three-wayvertices, rather than localizing medioapically (Fig. 7a). snail mutantsalso exhibited a defect in medioapical Rok recruitment (Fig. 7a).However, Rok localizes to a subapical position, consistent with theposition of adherens junctions in snail mutants26 (Fig. 7b). SubapicalRok localization did not exhibit a clear dorsal–ventral polarity in snailmutants, demonstrating that Twist requires Snail to recruit apicalRok (Supplementary Fig. S4). Consistent with Rok locally stabilizingMyo-II, changes in Myo-II localization mirrored Rok, with Myo-IIbecoming enriched in apical or subapical vertices in twist and snailmutants, respectively (Fig. 7c,d). In twist and snail mutants, Myo-II,and presumably Rok, co-localized with apical and subapical adherensjunctions, respectively (Fig. 7e). Thus, Twist and Snail are requiredto polarize Rok to the medioapical domain and away from adherensjunctions. Twist mutants continued to exhibit transient medioapicalMyo-II pulses between Myo-II foci at vertices, but medioapical Myo-IIwas not stabilized (Fig. 7c). Thus,medioapical Rok foci seem to stabilizeMyo-II in the medioapical cortex of ventral furrow cells. We proposethat the function of Twist is not simply to activate apical Rho1 andRok, but that Twist polarizes Rho1 signalling in the plane of theapical surface, leading to the polarization of Rok and Myo-II to themedioapical domain.

DISCUSSIONWe have demonstrated a spatial organization to Twist pathwaycomponents, which polarizes Rok/Myo-II and E-cadherin/adherensjunctions to medioapical and junctional domains, respectively. Theenrichment of Rok/Myo-II and E-cadherin/adherens junctions toseparate domains is similar to the process of cell–cell intercalationduring Drosophila germband extension36–40. However, Rok/Myo-IIand adherens junction components exhibit planar cell polarity ingermband cells, whereas they exhibit RCP in ventral furrow cells(Fig. 8a). Dia-mediated F-actin polymerization seems to maintainE-cadherin polarity and link the medioapical and junctionaldomains in ventral furrow cells, allowing contractile forces tobe stably transmitted across adherens junctions. Thus, Myo-IIstabilization, adherens junction organization and F-actin assemblyare temporally and spatially coordinated to drive ratchet-like apicalconstriction (Fig. 8b).During ventral furrow cell apical constriction, Rok becomes

concentrated as medioapical foci and is absent or present at lowlevels at adherens junctions. The RCP of Rok seems to target Myo-IIaccumulation to the medioapical domain. The importance of Roklocalization is strongly supported by the fact that the RCPs of bothRok and Myo-II are inverted in twist and snail mutants, with Rok andMyo-II accumulating inappropriately at adherens junctions. AlthoughRok is improperly localized in both mutants, twist mutants still



ART I C L E S

Myo-II

F-actin

Adherens junctions

Stabilized Myo-II

PCPE-cadherin and Par3/Baz Rok and Myo-II

Rho1 localization

E-cadherin Rok

E-cadherin Rok

E-cadherin

Myo-II stabilizationF-actin assembly

Ventral furrow/apical constriction

Germband/cell intercalation

RCP

twist RCP inversion

diaM

RCP loss

Twist Fog/T48

Contraction Stabilization

Rok localizationDia localization

a b

Figure 8 RCP coordinates Myo-II stabilization, F-actin assembly andE-cadherin localization to facilitate apical constriction. (a) Comparison ofcell polarity in germband cells (planar cell polarity, PCP) and ventral furrowcells (radial cell polarity, RCP). Changes in RCP are shown for twist and diamutants. Note that we have not analysed Rok localization in dia mutants.

(b) Model for the Twist-mediated actin–myosin ratchet. Periodic medioapicalMyo-II pulses transiently contract the cell (Contraction). Myo-II stabilizationand F-actin cable assembly are coordinated to form medioapical contractilefibres that are anchored at adherens junctions and stabilize cell shapefluctuations elicited by pulsing.

exhibit contractile pulses, suggesting that the apical Rok at adherensjunctions or low levels of Rok in the medioapical domain are sufficientto promote Myo-II pulses. We propose that the localization ofRok increases Myo-II phosphorylation when Myo-II reaches themedioapical domain either by diffusion or contractile flow, leadingto increased minifilament assembly and stabilization of Myo-IIstructures59,60 (Fig. 8b). The inappropriate localization of Rok in twistmutants suggests that Myo-II assembly in the medioapical domain,rather than junctions, is critical to stabilize cell shape between pulsesfor cells to constrict like a ratchet.Medioapical contractility and epithelial tension requires that the

contractile cortex be coupled to the junctional domain30,33. Rho1 andDia localize to both the medioapical and junctional domains, and weobserve F-actin cables originating from both locations in the absenceof Rok and Myo-II activity. Reducing F-actin assembly using CytoDor diaM mutants destabilizes the connection between the medioapicalcontractile network and the junctional domain. Interestingly, CytoDand diaM mutants cause E-cadherin to accumulate across the entireapical surface rather than being restricted to the junctional domain.Dia-mediated F-actin assembly could polarize E-cadherin throughclathrin-mediated endocytosis, as was described in germband cells39,thus removing medioapical E-cadherin. F-actin-dependent regulationof endocytosis has also been proposed to establish cortical domainsduring Drosophila cellularization53,57,58. Thus, Dia could function toseparate E-cadherin from peak Rok activity, creating discrete anchorpoints at cell–cell interfaces that can be pulled inwards by Myo-II. Inaddition, F-actin assembly is required to form a continuous meshworkof F-actin cables that connects the medioapical and junctional domains.twist mutants maintain E-cadherin RCP but lack a continuousmeshwork of medioapical F-actin cables, suggesting that F-actin

assembly contributes to the actin–myosin ratchet independently fromregulating adherens junction polarity.Our data suggest that Myo-II localization and F-actin assembly are

coordinated to prevent cell relaxation. First, disruption of F-actinturnover and meshwork formation using F-actin inhibitors, such asCytoD, does not disrupt apical Myo-II recruitment20. Second, F-actincable assembly occurs continuously in Rok-inhibitor-injected embryosthat lack apical Myo-II. Therefore, we propose that the combination oflocalizing Myo-II stabilization to the medioapical cortex while morebroadly activating medioapical and junctional F-actin assembly resultsin the formation of contractile cables that span the apical domainof ventral furrow cells and become anchored at discrete adherensjunction sites (Fig. 8b). These actin–myosin cables could function likestress-fibres to prevent cell relaxation after contractile pulses and forma supracellular meshwork30.Planar polarized actomyosin and adherens junction localization

results in cell–cell intercalation whereas radially polarized actomyosinand adherens junction localization causes apical constriction36,37,40,48

(Fig. 8a). This is illustrated by the fact that Rok and Dia localizemedioapically in ventral furrow cells to drive apical constriction,whereas Dia activation in the germband induces Myo-II accumulationat vertical junctions that contain Rok48. Therefore, developmental cues,such as Twist and Snail, must properly orient Myo-II, F-actin assemblyand adherens junction organization to elicit the appropriate cell shapechange48. Importantly, we show that the function of Twist is not simplyto activate, but to polarize apical signals such as Rok to spatially regulateMyo-II stabilization and F-actin assembly. Determining themechanismthat establishes RCP will be critical to determine how the commonset of signalling and cytoskeletal proteins are regulated to generatedifferent cell shape changes during morphogenesis61,62. �



ART I C L E S

METHODSMethods and any associated references are available in the onlineversion of the paper.

Note: Supplementary Information is available in the online version of the paper

ACKNOWLEDGEMENTSWe thank J. Zallen, S. Simões, R. Fernandez-Gonzalez (Sloan-Kettering Institute,USA), T. Lecuit (Aix-Marseille Universite, France), M. Peifer (University of NorthCarolina, Chapel Hill, USA), S. Wasserman (University of California, San Diego,USA) and the Bloomington Stock Center for providing fly stocks or antibodies usedin 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 NationalInstitute of General Medical Sciences. A. C. Martin is a Thomas D. and Virginia W.Cabot Career Development Assistant Professor of Biology.

AUTHOR CONTRIBUTIONSF.M.M. designed and performed experiments, analysed data and wrote themanuscript. M.T. designed and performed experiments. A.C.M. designed andperformed experiments, analysed data, supervised the project and wrote themanuscript.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Published online at www.nature.com/doifinder/10.1038/ncb2796Reprints and permissions information is available online at www.nature.com/reprints

1. Leptin, M. Gastrulation movements: the logic and the nuts and bolts. Dev. Cell 8,305–320 (2005).

2. Martin, A. C. Pulsation and stabilization: Contractile forces that underliemorphogenesis. Dev. Biol. 341, 114–125 (2010).

3. Sawyer, J. M. et al. Apical constriction: a cell shape change that can drivemorphogenesis. Dev. Biol. 341, 5–19 (2010).

4. Odell, G. M., Oster, G., Alberch, P. & Burnside, B. The mechanical basisof morphogenesis. I. Epithelial folding and invagination. Dev. Biol. 85,446–462 (1981).

5. Leptin, M. & Grunewald, B. Cell shape changes during gastrulation in Drosophila.Development 110, 73–84 (1990).

6. Sweeton, D., Parks, S., Costa, M. & Wieschaus, E. Gastrulation in Drosophila: theformation of the ventral furrow and posterior midgut invaginations. Development112, 775–789 (1991).

7. Leptin, M. twist and snail as positive and negative regulators during Drosophilamesoderm development. Genes Dev. 5, 1568–1576 (1991).

8. Nusslein-Volhard, C., Wieschaus, E. & Kluding, H. Mutations affecting the patternof the larval cuticle in Drosophila melanogaster : I. Zygotic loci on the secondchromosome. Roux’s Arch. Dev. Biol. 193, 267–282 (1984).

9. Hildebrand, J. D. Shroom regulates epithelial cell shape via the apical positioningof an actomyosin network. J. Cell Sci. 118, 5191–5203 (2005).

10. Kasza, K. E. & Zallen, J. A. Dynamics and regulation of contractile actin-myosinnetworks in morphogenesis. Curr. Opin. Cell Biol. 23, 30–38 (2011).

11. Lecuit, T., Lenne, P. F. & Munro, E. Force generation, transmission, andintegration during cell and tissue morphogenesis. Annu. Rev. Cell Dev. Biol. 27,157–184 (2011).

12. Young, P. E., Pesacreta, T. C. & Kiehart, D. P. Dynamic changes in the distributionof cytoplasmic myosin during Drosophila embryogenesis. Development 111,1–14 (1991).

13. Blanchard, G. B., Murugesu, S., Adams, R. J., Martinez-Arias, A. & Gorfinkiel, N.Cytoskeletal dynamics and supracellular organisation of cell shape fluctuationsduring dorsal closure. Development 137, 2743–2752 (2010).

14. Burnette, D. T. et al. A role for actin arcs in the leading-edge advance of migratingcells. Nat. Cell Biol. 13, 371–381 (2011).

15. David, D. J., Tishkina, A. & Harris, T. J. The PAR complex regulates pulsedactomyosin contractions during amnioserosa apical constriction in Drosophila.Development 137, 1645–1655 (2010).

16. Fernandez-Gonzalez, R. & Zallen, J. A. Oscillatory behaviors and hierarchical assem-bly of contractile structures in intercalating cells. Phys. Biol. 8, 045005 (2011).

17. Giannone, G. et al. Periodic lamellipodial contractions correlate with rearward actinwaves. Cell 116, 431–443 (2004).

18. He, L., Wang, X., Tang, H. L. & Montell, D. J. Tissue elongation requires oscillatingcontractions of a basal actomyosin network. Nat. Cell Biol. 12, 1133–1142 (2010).

19. Kim, H. Y. & Davidson, L. A. Punctuated actin contractions during convergentextension and their permissive regulation by the non-canonical Wnt-signalingpathway. J. Cell Sci. 124, 635–646 (2011).

20. Martin, A. C., Kaschube, M. & Wieschaus, E. F. Pulsed contractions of an actin-myosin network drive apical constriction. Nature 457, 495–499 (2009).

21. Munro, E., Nance, J. & Priess, J. R. Cortical flows powered by asymmetricalcontraction transport PAR proteins to establish and maintain anterior–posteriorpolarity in the early C. elegans embryo. Dev. Cell 7, 413–424 (2004).

22. Rauzi, M., Lenne, P. F. & Lecuit, T. Planar polarized actomyosin contractile flowscontrol epithelial junction remodelling. Nature 468, 1110–1114 (2010).

23. Sawyer, J. K. et al. A contractile actomyosin network linked to adherensjunctions by Canoe/afadin helps drive convergent extension. Mol. Biol. Cell 22,2491–2508 (2011).

24. Solon, J., Kaya-Copur, A., Colombelli, J. & Brunner, D. Pulsed forces timed by aratchet-like mechanism drive directed tissue movement during dorsal closure. Cell137, 1331–1342 (2009).

25. Costa, M., Wilson, E. T. & Wieschaus, E. A putative cell signal encoded by the foldedgastrulation gene coordinates cell shape changes during Drosophila gastrulation.Cell 76, 1075–1089 (1994).

26. Kölsch, V., Seher, T., Fernandez-Ballester, G. J., Serrano, L. & Leptin, M. Control ofDrosophila gastrulation by apical localization of adherens junctions and RhoGEF2.Science 315, 384–386 (2007).

27. Barrett, K., Leptin, M. & Settleman, J. The Rho GTPase and a putative RhoGEFmediate a signaling pathway for the cell shape changes in Drosophila gastrulation.Cell 91, 905–915 (1997).

28. Dawes-Hoang, R. E. et al. folded gastrulation, cell shape change and the control ofmyosin localization. Development 132, 4165–4178 (2005).

29. Hacker, U. & Perrimon, N. DRhoGEF2 encodes a member of the Dbl family ofoncogenes and controls cell shape changes during gastrulation in Drosophila. GenesDev. 12, 274–284 (1998).

30. Martin, A. C., Gelbart, M., Fernandez-Gonzalez, R., Kaschube, M. & Wieschaus,E. F. Integration of contractile forces during tissue invagination. J. Cell Biol. 188,735–749 (2010).

31. Cox, R. T., Kirkpatrick, C. & Peifer, M. Armadillo is required for adherens junctionassembly, cell polarity, and morphogenesis during Drosophila embryogenesis. J. CellBiol. 134, 133–148 (1996).

32. Gorfinkiel, N. & Arias, A. M. Requirements for adherens junction components in theinteraction between epithelial tissues during dorsal closure in Drosophila. J. Cell Sci.120, 3289–3298 (2007).

33. Roh-Johnson, M. et al. Triggering a cell shape change by exploiting preexistingactomyosin contractions. Science 335, 1232–1235 (2012).

34. Sawyer, J. K., Harris, N. J., Slep, K. C., Gaul, U. & Peifer, M. The Drosophila afadinhomologue Canoe regulates linkage of the actin cytoskeleton to adherens junctionsduring apical constriction. J. Cell Biol. 186, 57–73 (2009).

35. Bresnick, A. R. Molecular mechanisms of nonmuscle myosin-II regulation. Curr.Opin. Cell Biol. 11, 26–33 (1999).

36. Bertet, C., Sulak, L. & Lecuit, T. Myosin-dependent junction remodelling controlsplanar cell intercalation and axis elongation. Nature 429, 667–671 (2004).

37. Blankenship, J. T., Backovic, S. T., Sanny, J. S., Weitz, O. & Zallen, J. A.Multicellular rosette formation links planar cell polarity to tissue morphogenesis. Dev.Cell 11, 459–470 (2006).

38. Fernandez-Gonzalez, R., Simoes Sde, M., Roper, J. C., Eaton, S. & Zallen, J. A.Myosin II dynamics are regulated by tension in intercalating cells. Dev. Cell 17,736–743 (2009).

39. Levayer, R., Pelissier-Monier, A. & Lecuit, T. Spatial regulation of Dia andMyosin-II by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelialmorphogenesis. Nature Cell Biol. 13, 529–540 (2011).

40. Simoes Sde, M. et al. Rho-kinase directs Bazooka/Par-3 planar polarity duringDrosophila axis elongation. Dev. Cell 19, 377–388 (2010).

41. Fletcher, G. C., Lucas, E. P., Brain, R., Tournier, A. & Thompson, B. J. Positivefeedback and mutual antagonism combine to polarize Crumbs in the Drosophilafollicle cell epithelium. Curr. Biol.: CB 22, 1116–1122 (2012).

42. Harris, T. J. & Peifer, M. The positioning and segregation of apical cuesduring epithelial polarity establishment in Drosophila. J. Cell Biol. 170,813–823 (2005).

43. Goode, B. L. & Eck, M. J. Mechanism and function of formins in the control of actinassembly. Annu. Rev. Biochem. 76, 593–627 (2007).

44. Fox, D. T. & Peifer, M. Abelson kinase (Abl) and RhoGEF2 regulate actinorganization during cell constriction in Drosophila. Development 134,567–578 (2007).

45. Gelbart, M. A. et al. Volume conservation principle involved in cell lengthening andnucleus movement during tissue morphogenesis. Proc. Natl Acad. Sci. USA 109,19298–19303 (2012).

46. Uehata, M. et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389, 990–994 (1997).

47. Royou, A. Cortical recruitment of nonmuscle myosin II in early syncytial Drosophilaembryos: its role in nuclear axial expansion and its regulation by Cdc2 activity. J.Cell Biol. 158, 127–137 (2002).

48. Bertet, C., Rauzi, M. & Lecuit, T. Repression of Wasp by JAK/STAT signalling inhibitsmedial actomyosin network assembly and apical cell constriction in intercalatingepithelial cells. Development 136, 4199–4212 (2009).

49. Cooper, J. A. Effects of cytochalasin and phalloidin on actin. J. Cell Biol. 105,1473–1478 (1987).







http://www.nature.com/reprints

ART I C L E S

50. Homem, C. C. & Peifer, M. Diaphanous regulates myosin and adherens junctionsto control cell contractility and protrusive behavior during morphogenesis.Development 135, 1005–1018 (2008).

51. Afshar, K., Stuart, B. & Wasserman, S. A. Functional analysis of the Drosophiladiaphanous FH protein in early embryonic development. Development 127,1887–1897 (2000).

52. Grosshans, J. et al. RhoGEF2 and the formin Dia control the formation of the furrowcanal by directed actin assembly during Drosophila cellularisation. Development132, 1009–1020 (2005).

53. Yan, S. et al. The F-BAR protein Cip4/Toca-1 antagonizes the forminDiaphanous in membrane stabilization and compartmentalization. J. Cell Sci. 126,1796–1805 (2013).

54. Carramusa, L., Ballestrem, C., Zilberman, Y. & Bershadsky, A. D. Mammaliandiaphanous-related formin Dia1 controls the organization of E-cadherin-mediatedcell–cell junctions. J. Cell Sci. 120, 3870–3882 (2007).

55. Sahai, E. & Marshall, C. J. ROCK and Dia have opposing effects on adherensjunctions downstream of Rho. Nat. Cell Biol. 4, 408–415 (2002).

56. Roeth, J. F., Sawyer, J. K., Wilner, D. A. & Peifer, M. Rab11 helps maintain apicalcrumbs and adherens junctions in the Drosophila embryonic ectoderm. PLoS One4, e7634 (2009).

57. Sokac, A. M. & Wieschaus, E. Zygotically controlled F-actin establishes corticalcompartments to stabilize furrows during Drosophila cellularization. J. Cell Sci. 121,1815–1824 (2008).

58. Sokac, A. M. & Wieschaus, E. Local actin-dependent endocytosis is zygoticallycontrolled to initiate Drosophila cellularization. Dev. Cell 14, 775–786 (2008).

59. Watanabe, T., Hosoya, H. & Yonemura, S. Regulation of myosin II dynamics byphosphorylation and dephosphorylation of its light chain in epithelial cells.Mol. Biol.Cell 18, 605–616 (2007).

60. Uehara, R. et al. Determinants of myosin II cortical localization during cytokinesis.Curr. Biol. 20, 1080–1085 (2010).

61. Mason, F. M. & Martin, A. C. Tuning cell shape change with contractile ratchets. Curr.Opin. Genet. Dev. 21, 671–679 (2011).

62. Montell, D. J. Morphogenetic cell movements: diversity from modular mechanicalproperties. Science 322, 1502–1505 (2008).



DOI: 10.1038/ncb2796 METHODS

METHODSFly stocks and genetics. The following fluorescent-protein fusion stockswere used: Utr::GFP, Myo::mCherry (sqhAX3; sqhp–sqh::mCherry(ChFP) sqhp–Utr::GFP/CyO; ref. 22), Myo::mCherry (sqhp–sqh::mCherryA11; ref. 20),sqhp–Utr::GFP/CyO; (ref. 22), rho1p–GFP::Rho1 (ref. 63), ubi–ECad2::GFP(ref. 64), UASp–Venus::Rok(K116A) (ref. 40), UASp–Venus::Rok (WT), sqhp–GFP::Rok(K116A) (gifts from J. Zallen, S. Simões, and R. Fernandez-Gonzalez),halo snailIIG05/CyO, sqhp–sqh::GFP and halo twistey53/CyO, sqhp–sqh::GFP, wheresqhp is the sqh promoter and rho1p is the Rho1 promoter. The CyO, sqhp-sqh::GFP is a CyO balancer chromosome with a Myo::GFP transgene insertedinto the chromosome. The halo mutant was used to identify homozygoustwist or snail mutants during cellularization65. To visualize membranes andMyo-II, Gap43::mCherry/CyO; sqhp–sqh::GFP (ref. 30) was crossed to Myo::GFP(sqhAX3; sqhp–sqh::GFP(II)) to remove potential phenotypes resulting from theCyO balancer47. UASp–Venus::Rok(K116A) (ref. 40) females were crossed tomales containing the maternal GAL4 driver mat15:GAL4. Resultant females(UASp–Venus::Rok(K116A)/+; mat15/+) were mated to OregonR males and theirembryos were imaged. Germline clones for dia were generated by heat-shockinghsFLP; dia5 FRT40A/ovoDFRT40A; sqhp–sqh::GFP/+ larvae for two hours at37 ◦C for three to four days. Controls for the germline clones were hsFLP; dia5

FRT40A/Sco; sqhp–sqh::GFP/+ embryos. To generate twist mutant embryos thatexpress Utr::GFP, halo twistey53/CyO, sqh::GFP were crossed to Utr::GFP/CyO.halo twistey53/Utr::GFP females were then mated to halo twistey53/CyO, sqh::GFPmales and their embryos were imaged. As halo can be separated from twist byrecombination, twist homozygous mutant embryos were selected on the basis oftheir failure to undergo ventral furrow formation. The same strategy was applied tohalo snailIIG05/CyO, sqh::GFP mutants. To generate twist mutant embryos that ex-pressGAP43::ChFP andGFP::Rok(K116A), halo twistey53/CyO, sqh::GFP was crossedto Gap43::ChFP/CyO; sqhp-GFP::Rok(K116A) flies. halo twistey53/GAP43::ChFP;GFP::Rok(K116A) females were crossed to halo twistey53/CyO, sqh::GFP malesand twist homozygous mutants were selected on the basis of their failure toundergo ventral furrow formation. The same strategy applied for snailIIG05 andGap43::ChFP ; sqhp–GFP::Rok(K116A). Controls for twist or snail experi-ments were halo twistey53/halo or halo snailIIG05/halo expressing the appropriatetransgene. For Rok germline clones and rescue, rok2 FRT19A/ovoDFRT19A;hsFLP UAS–Venus::Rok(WT)/+; mat15/+ larvae were heat-shocked at 37 ◦C fortwo hours on two days.

Live and fixed imaging. For live imaging, embryos were dechorionated with 50%bleach then washed with water and mounted, ventral side up, onto a slide coatedwith glue (double-sided tape soaked in heptane). Spacer coverslips (No. 1.5) wereattached using glue and a coverslip was attached to create a chamber. Halocarbon27 oil was added to the chamber. Embryos were not compressed. All imaging wasperformed at room temperature (∼23 ◦C).

For fixed imaging, embryos were dechorionated with bleach then fixed in4% paraformaldehyde in 0.1M phosphate buffer at pH 7.4 with 50% heptanefor 20–30min and manually devitellinized. To visualize F-actin, embryos werethen incubated with AlexaFluor568 phalloidin (Invitrogen) diluted in PBS+0.1%Triton X-100 (PBST) overnight. Embryos were then blocked with 10% BSA in PBST.Primary and secondary antibodies were diluted in 5% BSA in PBST. Embryos werethen mounted using AquaPolymount (Polysciences). Diaphanous was recognizedusing antibody (a gift from S. Wasserman) diluted at 1:5,000, E-cadherin (DCad2,from Developmental Studies Hybridoma Bank) at 1:50, and Rho1 (p1D9, fromDevelopmental Studies Hybridoma Bank) at 1:50. Secondary antibodies used wereAlexaFluor488, 568 or 647 (Invitrogen; catalogue numbers A11034, A11036, A21245,A11031, A21235 and A21247), diluted at 1:500.

All images were acquired on a Zeiss LSM 710 confocal microscope, with a40×/1.2 Apochromat water objective (Zeiss), an argon ion, 561 nm diode, 594 nmHeNe, and 633 nm HeNe lasers. Pinhole settings ranged from 1–2 airy units. Fortwo-colour, live imaging, simultaneous excitation was used with band-pass filtersset at ∼499–561 nm for GFP and ∼599–696 nm for ChFP. All images shown arerepresentative of at least four embryos unless otherwise stated.

Drug injections. Dechorionated embryos weremounted ventral side up for furrowinjection or lateral side up for germband injections then desiccated for 5-10minusing Drierite (Drierite Company). A 3:1 mixture of halocarbon 700/halocarbon27 oils was added over the embryo for injection. Embryos were injected laterallyduring mid-to-late cellularization (furrow canals at base of nuclei) for ventralfurrow cells or injected dorsally after initiation of pole cell movement for germbandcells. Embryos were injected with a drop size of around 40 µm, estimating a

1:200–300 dilution of the drug. Y-27632 (Enzo Life Sciences) was resuspendedand injected at 50mM in water. Sixteen Y-27632-injected embryos were imagedand quantifications in Fig. 3g are from a representative embryo. CytoD (Enzo LifeSciences) was resuspended at 5mgml−1 in DMSO, diluted in DMSO and injectedat 0.125mgml−1 (low), 0.25mgml−1 (intermediate) and 5mgml−1 (high). ForCytoD experiments, 6 control (DMSO solvent), 7 low-dose CytoD and 5 high-doseCytoD GAP43::ChFP, Myo::GFP embryos were imaged and quantifications in Fig. 4are from representative embryos from each condition. Images from E-cad::GFP-injected embryos with intermediate CytoD are representative from three individualembryos. Embryos were imaged 3–5min after injection.

Image processing and analysis. Images were processed using MATLAB (Math-Works), Fiji (http://fiji.sc/wiki/index.php/Fiji) and Photoshop CS5.1 (Adobe Sys-tems). A Gaussian filter (kernel = 1 pixel) was applied to images. Apical images aremaximum intensity projections of multiple z sections (∼2–3 µm). Subapical imagesare one z slice,∼1–2 µm below the apical sections.

Image segmentation for quantification of area and F-actin intensities wasperformed using custom MATLAB software titled EDGE (Embryo DevelopmentGeometry Explorer)45. Raw images were processed through background subtractionto remove cytoplasmic Utr::GFP and/or Myo::ChFP by clipping cytoplasmicintensity values two standard deviations from the mean. In addition, a rotationallysymmetric Gaussian smoothing filter (kernel = 3 pixels, σ = 0.5) was applied.We made maximum intensity projections, summing the top two highest-intensityvalues. Images were imported into EDGE, and cell membranes were automaticallydetected and manually corrected. Cell area and integrated fluorescence intensityof Myo-II and Utr were quantified (Figs 3, 4 and 6 and Supplementary Fig. S2).For pulse intensities (Fig. 3d–f), maximum intensity projections of apical 2–3 µmwere generated in FIJI. We generated kymographs of pulses and intensity valueswere obtained by drawing lines through pulses in the kymograph. Pulse dataare from one representative embryo of four individual embryos where pulseswere quantified. Temporal cross-correlation was determined using the ‘crosscoef’function in MATLAB. To calculate cross-correlation between constriction rate andmyosin rate in CytoD- and control-injected embryos (Fig. 4c,d), we first smoothedapical area and myosin intensity curves by averaging values at a given time pointwith immediately neighbouring time points (3 time points total).We then calculateda rate for each time point by subtracting the value following a time point by thepreceding time point and dividing by the time interval. Pearson correlations werecalculated using 50 time points for each cell. Quantifications of F-actin (Utr::GFP)for Fig. 6 are from a representative embryo of 9 twist, 10 snail or 3 control (twist/+)embryos imaged. To determine average pulsing behaviour in control (twist/+) andtwist mutants (Fig. 6e), individual constriction pulses (n=51 for control, n=45 fortwist ) were identified by thresholding area constriction rate and were then alignedat their peak constriction rate (time zero). Values for each pulse were normalizedto initial and final areas and F-actin levels then averaged. Line scans of Utr::GFPin cells were generated using FIJI and a linewidth of 25 (Fig. 6b). More than 30cells were analysed for each genotype, and six representative cells of similar sizewere averaged. Ratios of medioapical to junctional F-actin were obtained usingFIJI, by manually drawing a line (width= 4) around cell junctions to determineaverage intensity (Fig. 6c). Circles or ellipseswere drawnover themedioapical regionto obtain average intensity. For each cell, junctional intensities were divided bymedioapical intensity and then averaged for each genotype.

Statistical analysis Statistical analyses were performed in Prism (Graphpad).Data sets were subjected to Komolgorov–Smirnov tests to determine distributions.Non-normal distributions resulted in non-parametric analyses using a two-tailed,Mann–Whitney test for average pulse behaviour (Fig. 6e) and medial-to-junctionalF-actin ratio (Fig. 6c). An unpaired, two-tailed test was used to determinesignificance of mean cross-correlation for CytoD pulsing data (Fig. 4d). All errorbars are standard deviation and results of tests are represented as P values. Nostatistical method was used to predetermine sample size. The experiments were notrandomized. The investigators were not blinded to allocation during experimentsand outcome assessment.

63. Rosales-Nieves, A. E. et al. Coordination of microtubule and microfilament dynamicsby Drosophila Rho1, Spire and Cappuccino. Nat. Cell Biol. 8, 367–376 (2006).

64. Oda, H. & Tsukita, S. Real-time imaging of cell–cell adherens junctions reveals thatDrosophila mesoderm invagination begins with two phases of apical constriction ofcells. J. Cell Sci. 114, 493–501 (2001).

65. Gross, S. P., Guo, Y., Martinez, J. E. & Welte, M. A. A determinant for directionalityof organelle transport in Drosophila embryos. Curr. Biol. 13, 1660–1668 (2003).

NATURE CELL BIOLOGY


http://fiji.sc/wiki/index.php/Fiji

S U P P L E M E N TA RY I N F O R M AT I O N

WWW.NATURE.COM/NATURECELLBIOLOGY 1

DOI: 10.1038/ncb2796

Rok Membranes Merge

0 m

in1.

8 m

in3.

5 m

in5.

1 m

in6.

8 m

in8.

3 m

in

Supplemental Figure 1

32

4

1

32

4

1

5

5

32

4

1

5

3 24

1

5

Figure S1 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 5mm.


http://www.nature.com/doifinder/10.1038/ncbxxxx


2 WWW.NATURE.COM/NATURECELLBIOLOGY

Utr::GFP Phalloidin MergeA

pica

l F-a

ctin

z-pr

ojec

tion

Sub

apic

al F

-act

in

Orth

ogon

al V

iew

Pha

lloid

in

Apical

Basal

Medioapical Junctionala b

0

20

40

60

80

1000120014001600180020002200

0 1 2 3 4 5 6 7Time (min)

Api

cal a

rea

(μm

2 )

F-actin intensity (a.u.)

Cell 6

c

Orth

ogon

al V

iew

Utr:

:GFP

d

e

Api

cal a

rea

(μm

2 ) 50403020

10

00 5 10

800

600

400

200

0

Time (min)

F-actin and Myo

intensity (a.u.)

f

0.0

0.2

0.4

0.6

0.8

1.0

-30 0 30Time (sec)

Nor

mal

ized

are

aan

d F-

actin

(a.u

.)

30

40

50

60

70

800

1000

1200

1400

1600

1800

0 1 2 3 4 5 6Time (min)

Api

cal a

rea

(μm

2 )


Cell 4

0

20

40

60

0

500

1000

1500

0 1 2 3 4 5 6 7Time (min)

Api

cal a

rea

(μm

2 )


Cell 13

Examples where F-actin levels during pulses are increasing, stabilizing or decreasing

0

20

40

60

600

800

1000

1200

1400

1600

Time (min)

Api

cal a

rea

(μm

2 )


Cell 2

0 1 2 3 4 5 6 7

0 1 2 3 4−40

−20

0

20

40

Time (min)

Con

stric

tion

Rat

e M

yosi

n R

ate

ΔT

Time offset

Cro

ss-C

orre

latio

n

ΔT

0


Figure S2 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 5mm. (b) Orthogonal view/cross-section of same embryo from a. Scale bar is 10mm. (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 (DT). A peak cross-correlation at DT~0 indicates a significant correlation with the value of DT indicating the time lag between the two signals.




Myosin E-Cadherin MergeApical surfaceOptical section

a

Myosin E-Cadherin Merge

b

Apical surfaceOptical section

0μm

-0.0

8μm

-0.5

4μm

-0.1

5μm

-0.2

3μm

-0.3

1μm

-0.3

9μm

-0.4

6μm

0μm

-0.0

8μm

-0.1

5μm

-0.2

3μm

-0.3

1μm


Figure S3 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 (0mm). Scale bars are 5mm.



4 WWW.NATURE.COM/NATURECELLBIOLOGY

0 m

in5

min

10 m

in

twist; GFP::Rok(K116A)

0 m

in5

min

10 m

in

snail; GFP::Rok(K116A)

0 m

in5

min

Control; GFP::Rok(K116A)


Figure S4 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 20mm.




Supplemental Video Legends

Supplemental Video 1: F-actin and Myo-II structures colocalize 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 6.16s intervals. The video is displayed at 10 frames per second (fps). Scale bar is 5mm, which is about the diameter of a cell.

Supplemental Video 2: 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 0.78s intervals. The video is displayed at 30 fps. Scale bar is 5mm.

Supplemental Video 3: 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 4s intervals. The video is displayed at 15 fps. Scale bar is 5mm.

Supplemental Video 4: 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 5.6s intervals. The video is displayed at 20 fps. Scale bar is 20mm.

Supplemental Video 5: CyoD disrupts Myo-II medioapical network connectivity. While Myo-II (green, apical projection) can accumulate and cells constrict, injection of CytoD (0.125mg/mL) 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 5.6s intervals and every fourth frame is shown. The video is displayed at 7 fps. Scale bar is 20mm.

Supplemental Video 6: 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 (0.125mg/mL). Magnified view of embryo expressing Myo::GFP and Gap43::ChFP (magenta, single z-slice) from Video 4. Image acquired at 5.6s intervals. The video is displayed at 20 fps. Scale bar is 5mm.

Supplemental Video 7: 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 (0.25mg/mL) 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 7.2s intervals. The video is displayed at 15 fps. Scale bar is 5mm.

Supplemental Video 8: 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 8.8s intervals. diaM embryo images were acquired at 9.3s intervals. The video is displayed at 20 fps. Scale bars are 20mm.

Supplemental Video 9: Snail and Twist are required for a medioapical meshwork of F-actin cables. Control (twist/+, left), snail (center), 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 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 9.3s intervals for control, 7.4s intervals for snail, and 6s intervals for twist. The video is displayed at 15 fps. Scale bars are 5mm.


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

Documents