Apical domain polarization localizes actin–myosin activity
to drive ratchet-like apical constriction
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Citation
Mason, Frank M., Michael Tworoger, and Adam C. Martin.
“Apical Domain Polarization Localizes Actin–myosin Activity to
Drive Ratchet-Like Apical Constriction.” Nature Cell Biology 15,
no. 8 (July 7, 2013): 926–936.
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http://dx.doi.org/10.1038/ncb2796
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Wed May 25 19:05:47 EDT 2016
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Detailed Terms
Apical domain polarization localizes actin-myosin
activity to drive ratchet-like apical constriction
Frank M. Mason1, Michael Tworoger1, Adam C. Martin1
1
Department of Biology,
Massachusetts Institute of Technology, Cambridge, MA 02142, USA
Word count: 3,513
Corresponding author:
Adam C. Martin
acmartin@mit.edu
31 Ames St.
Cambridge, MA 02142, USA
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 (AJs). 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 AJs. 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.
Introduction
Apical constriction is an epithelial cell shape change that promotes tissue
bending during developmental processes such as gastrulation and neural tube closure13
. Apical constriction bends epithelia by transforming columnar cells to a wedge shape4.
During Drosophila gastrulation, the apical constriction of presumptive mesoderm cells
along the ventral midline results in ventral furrow (VF) formation and tissue
invagination5,6. Apical constriction and VF formation are induced by the expression of
two transcription factors, Twist and Snail5,7,8. A major question in the field has been how
Twist and Snail promote force generation and apical constriction at the molecular level.
Forces that drive apical constriction are generated by the contraction of an actin
filament (F-actin) network by the molecular motor non-muscle myosin II (Myo-II)9-12. In
VF cells, and many other contractile systems, Myo-II contractions and cell shape
changes occur in a pulsed or ratchet-like manner13-24. Contractile pulses in VF cells
occur in the F-actin-Myo-II network spanning the apical domain (medioapical cortex),
which pull peripheral adherens junctions (AJs) inward (Fig. 1a)20. After a contraction
pulse, the constricted state of the cell is stabilized to incrementally decrease apical area,
similar to the mechanism of a ratchet20. Twist and Snail regulate distinct steps of this
ratchet-like constriction20. Snail is required to initiate contractile pulses, but the
mechanism is unclear. Twist is required to stabilize cell shape between pulses. Two
Twist transcriptional targets, Folded gastrulation (Fog) and T48, could function in parallel
to activate the Rho1 GTPase apically (Fig. 1b)25,26. It is thought that apical secretion of
Fog activates Rho1 signaling and Myo-II recruitment across the apical surface of VF
cells27-29. How Rho1 stabilizes cell shape is not known and could depend on tension
generated by medioapical or junctional cytoskeletal networks13,22,30. Therefore,
elucidating the ratchet mechanism requires determining how Rho1 and its effectors
regulate medioapical and junctional actin-myosin networks in response to Twist and
Snail.
Contractile forces must be coupled to the AJs in order to generate cell and tissue
shape changes28,30-34. AJ remodeling accompanies VF cell apical constriction, with
subapical AJs being disassembled, which requires Snail, and spot junctions assembling
at the apical cell-cell interfaces, which appears to require Twist (Fig. 1c)26,28,30. It is not
known how signals that activate Myo-II are coordinated with AJ remodeling to couple
contraction to AJs.
Here we visualize how the dynamics of the Myo-II, F-actin, and AJs are
coordinated with the Rho1 GTPase pathway to dissect the mechanism of ratchet-like
apical constriction.
Results
Ventral furrow cells exhibit radial cell polarity (RCP) of Rok/Myo-II and E-Cadherin
The Rho1 effector Rho-associated kinase (Rok) phosphorylates and activates
Myo-II and is required for VF cell apical constriction, suggesting that apical Fogdependent activation of Rok and Myo-II triggers apical constriction28,35. The importance
of Rok in polarizing contraction is supported by the fact that planar polarized Rok
localizes Myo-II contraction to anterior-posterior cell interfaces during convergent
extension of the Drosophila germband cells36-40. Additionally, planar polarized Rok
excludes Bazooka/Par-3 from the cortex, establishing complementary domains of
Rok/MyoII and AJ proteins40. To test the role of Rok in VF cells, we examined Rok
localization dynamics using either a kinase-dead Rok allele, Venus(or
GFP)::Rok(K116A), or a Venus::Rok(WT) expressed in rok2 mutant germline clones40.
Venus(or GFP)::Rok(K116A) and Venus::Rok(WT) localization patterns were
indistinguishable, and Venus::Rok(WT) rescued the VF invagination defect of rok2
mutants28, suggesting that both Venus::Rok alleles indicate the localization of
endogenous Rok (Fig. 1d,e,f, and Supplemental Fig. 1). Rok exhibited apical
accumulation in VF cells prior to Rok apical localization in the germband (Fig. 1d).
Apical domain proteins normally localize directly above AJs or uniformly across the
apical surface of epithelial cells41,42. However, Rok displayed an unexpected apical
organization, initially exhibiting dispersed staining and then accumulating in medioapical
foci as cells constricted (Fig. 1e,f, and Supplemental Fig. 1). In contrast to germband
cells, where Rok is present at cell-cell interfaces40, Rok intensity appeared lowest at the
junctions (Fig. 1f,g). Medioapical Rok foci colocalized with Myo-II, consistent with Rok
recruiting or stabilizing Myo-II (Fig. 1h). Thus, similar to germband cells, Rok/Myo-II and
E-Cadherin are enriched in different domains. However, in VF cells, Rok/Myo-II is
concentrated in medioapical foci and AJs are present around the cell circumference, a
pattern that we term “radial” cell polarity (RCP).
The Rho1 GTPase and its effectors exhibit distinct localization patterns
To determine if other Twist pathway components are polarized to medioapical
foci, we examined the localization of Rho1 and a Rho1 effector that mediates
unbranched actin polymerization and F-actin cable formation, the formin Diaphanous
(Dia)27,29,43. Rho1 localized to medioapical foci that partially overlapped Rok foci, but
also localized to the junctional domain (Fig. 2a). Dia partially colocalized with
medioapical Rho1/Rok foci and junctional Rho1 (Fig. 2b,c). However, Dia exhibited
more diffuse staining across the apical domain, which largely colocalized with the Factin-Myo-II meshwork (Fig. 2d). Therefore, Rho1, Rok, and Dia are differentially
localized in the apical domain of VF cells, which suggests that Twist and Rho1 spatially
regulate F-actin and Myo-II activity during ratchet-like constriction.
Rok is required for medioapical F-actin network condensation
We hypothesized that the RCP of Rok might spatially control Myo-II activation
or stabilization to regulate contractility or the organization of the apical F-actin network,
both of which are important for apical constriction44. To determine how medioapical
Rho1-Rok foci regulate F-actin network contraction in VF cells, we visualized F-actin and
Myo-II dynamics by expressing the F-actin binding domain of Utrophin fused to GFP
(Utr::GFP) and the regulatory light chain of Myo-II (spaghetti squash, sqh in Drosophila)
fused to mCherry fluorescent protein (Myo::ChFP)20,22. Utr::GFP labels medioapical,
junctional, and basolateral networks, similar to phalloidin staining in fixed embryos,
demonstrating that Utr::GFP labels F-actin networks in an unbiased manner
(Supplemental Fig. 2a,b). Similar to Rok localization, apical Myo-II recruitment occurs in
VF cells before germband cells and was associated with structural changes in the Factin network (Fig. 3a, and Supplemental Videos 1, 2). To analyze F-actin dynamics, we
first used custom software to segment individual cells and quantify total apical F-actin
levels during apical constriction45. In contrast to the increase in apical Myo-II intensity,
we found that average total apical F-actin levels decrease over the course of apical
constriction, suggesting that F-actin network depolymerization accompanies constriction
(Supplemental Fig. 2c). However, during contraction pulses we often observed brief
increases or stabilization of F-actin intensity (Fig. 3b, and Supplemental Fig. 2d,e). The
increase or stabilization of F-actin intensity was associated with F-actin condensation
into medioapical foci (Fig. 3c). Following F-actin condensation, F-actin foci were
subsequently remodeled, often into cables, and F-actin intensity appeared to decrease
as area stabilized (Fig. 3c,d, and Supplemental Fig. 2d). The mechanism for this F-actin
remodeling is unclear, but F-actin depolymerization is possibly required to regenerate
actin monomers that are then assembled into new F-actin cables. Medioapical F-actin
condensation and dissolution occurred simultaneously with Myo-II (Fig. 3d-f), suggesting
that F-actin condensation represents contraction of the medioapical F-actin cortex. To
determine whether medioapical F-actin condensation requires Rok, we injected embryos
with the Rok inhibitor, Y-2763246. Y-27632-injected embryos failed to accumulate
cortical Myo-II, consistent with previous observations47, and cells did not constrict (Fig.
3g). However, medioapical F-actin was prominent in VF cells after Y-27632 injection. Factin filaments or cables continuously assembled and disassembled (Fig. 3h,i, and
Supplemental Video 3). F-actin cables originated from medioapically and junctionally,
consistent with Rho1 and Dia localization at both locations (Fig. 3i). Despite robust
medioapical F-actin assembly, F-actin cables failed to condense into medioapical foci in
Y-27632 injected embryos (Supplemental Videos 2, 3). Thus, RCP of Rok is
dispensable for medioapical F-actin assembly, but is required to condense medioapical
F-actin cables that span the apical domain, presumably by targeting Myo-II to the
medioapical cortex.
F-actin polymerization is required to couple medioapical contraction to cell-cell
junctions
Our visualization of F-actin cable assembly at both the medioapical cortex and
junctions of Y-27632-injected VF cells suggested that Rho1 could activate Dia to
stimulate F-actin assembly across the apical domain. Furthermore, Y-27632-injected
germband cells have less pronounced medioapical F-actin cables than in VF cells (Fig.
3h), supporting a previous study that suggested that signals in VF cells enhance
medioapical F-actin assembly relative to germband cells48. To test the function of Factin assembly during apical 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 disrupted the F-actin-Myo-II
network, resulting in apical Myo-II spots that fail to coalesce, as previously reported (Fig.
4a,b)20. We titrated the amount of injected CytoD to identify processes that were most
sensitive to reduced F-actin assembly. Lower CytoD concentrations allowed VF cells to
assemble an apical F-actin-Myo-II network and initiate constriction (Fig. 4a,b). Normally,
VF cells undergo contractile pulses, resulting in a significant positive cross-correlation
between calculated constriction rate and Myo-II accumulation rate due to in-phase
contraction pulses (Supplemental Fig. 2f)20,30. Despite undergoing apical constriction
and Myo-II accumulation, we failed to observe obvious Myo-II pulses in embryos injected
with low CytoD concentrations, which was supported by the lack of a cross-correlation
between constriction rate and myosin rate (Fig. 4c,d). Also, low CytoD concentrations
destabilized the connection between the medioapical actin-myosin network and cell-cell
junctions, with networks transiently losing and then regaining connections to each other
across the tissue (Fig. 4a, and Supplemental Videos 4-6). Low CytoD concentrations
resulted in gaps in the medioapical F-actin cortex that preceded junction detachments,
demonstrating that defects in medioapical F-actin network continuity precipitate
separation of the medioapical network from cell-cell junctions (Fig. 4e, and Supplemental
Video 7). Overall, these results demonstrate that robust apical F-actin assembly is
critical for medioapical contraction pulses and to form a stable connection between the
contractile medioapical domain and AJs.
Diaphanous couples the contractile network to junctions, possibly by polarizing
AJ localization
Dia was previously shown to be important for VF formation50. To determine
whether Dia mediates F-actin assembly and junction attachment, we generated germline
clones using the partial loss-of-function dia5 allele (diaM)50. We were unable to analyze
VF formation in diaM embryos with strong phenotypes, because these had severe
defects during cellularization, which precedes gastrulation51-53. However, diaM embryos
with mild cellularization phenotypes had apical Myo-II accumulation and exhibited
transient losses in the connections between medioapical F-actin-Myo-II network and
junctions, similar to embryos injected with low CytoD concentrations (Fig. 5a,b, and
Supplemental Video 8). Together with the CytoD injections, our results suggest that Diadependent F-actin assembly in part mediates medioapical and junctional F-actin
assembly to link the medioapical cortex to AJs.
Dia-mediated F-actin assembly could facilitate apical constriction by generating
medioapical F-actin cables to facilitate Myo-II contraction (Fig. 3c and 4e), but could also
regulate the organization of AJs39,50,54,55. Because Twist has been proposed to be
required for apical AJ formation26, we examined E-Cadherin localization in diaM mutants.
We found that E-Cadherin localized apically in diaM mutants. However, E-Cadherin was
not restricted to the junctional domain, but spread across the entire apical surface, which
is different from E-Cadherin localization in diaM mutants for other cell types50 (Fig. 5c).
While it is possible that some of this apical E-Cadherin is present in endocytic vesicles
and not cortical (Supplemental Fig. 3)50,56, it is clear that there is a disruption in the
localization and polarity of E-Cadherin in diaM mutants. E-Cadherin polarity was also
lost after CytoD injection (Fig. 5d). Interestingly, defects in Dia-mediated F-actin
assembly results in a similar loss of cortical domains or “compartments” during the
process of cellularization, potentially from misregulation of endocytic activity53,57,58.
Thus, Dia-mediated F-actin assembly is required in VF cells for the RCP of apical ECadherin, restricting E-Cadherin to the junctional domain. The cell-cell attachment
defect is unlikely to result from lower E-Cadherin levels at the junctional domain because
medioapical networks can regain attachments (Fig. 5a), whereas AJ mutants result in
permanent dissociations between the medioapical network and the junctions30. We
propose that Dia-mediated F-actin assembly could facilitate the attachment between the
medioapical contractile network and junctions by spatially segregating contractile and
junctional components into distinct domains, providing definitive anchor points at the cell
periphery (Fig. 5e, AJ depolarization). In addition, Dia could be required to assemble
medioapical and junctional F-actin cables to link the two domains (Fig. 5e, Network
dissolution).
Twist is required for a continuous medioapical F-actin meshwork
Our results demonstrate that the spatial regulation of Myo-II recruitment, F-actin
assembly, and AJ organization cooperate to achieve ratchet-like apical constriction.
Thus, Twist expression could stabilize pulsed contractions by controlling F-actin, Myo-II,
or AJs in specific cellular domains. Recently, cell shape fluctuations resulting from
medioapical contractions were proposed to be stabilized by junctional actin-myosin
networks13,22. Because Rho1 and Dia localize both medioapically and junctionally in VF
cells, we tested whether Twist is required for either of these F-actin networks. In
contrast to wild-type VF cells, which exhibit a continuous meshwork of medioapical and
junctional F-actin cables, visualization of Utr::GFP in twist mutants demonstrated that the
twist mutant results in dynamic medioapical F-actin puncta that fail to form cables across
the apical domain (Fig. 6a, and Supplemental Video 9). Importantly, twist mutants have
junctional F-actin cables (Fig. 6a,b), which display similar intensity to junctional F-actin in
lateral germband cells (furrow:germband ratio=0.96, n=20 cells). twist mutants exhibited
a lower medioapical:junctional F-actin ratio, suggesting that twist mutants are defective
in the formation of the medioapical F-actin network relative to junctional F-actin (Fig. 6c).
This contrasts with snail mutants, which lack medioapical and apical junctional F-actin
cables (Fig. 6a-c, and Supplemental Video 9). Because Twist is required for cell shape
stabilization after pulses, we examined F-actin dynamics over the course of individual
pulsatile events. In wild-type cells, F-actin levels usually increase or remain stable
during the contraction and persist as F-actin cables (Fig. 3b). However, in twist mutants,
the F-actin network is always punctate and F-actin levels decrease during contraction
(Fig. 6a,d,e). Overall, these results suggest that Twist facilitates medioapical F-actin
assembly. Importantly, twist mutants did not result in E-Cadherin depolarization,
suggesting that the medioapical meshwork of F-actin cables contributes to cell shape
stabilization independently from regulating E-Cadherin RCP (Fig. 7e).
twist mutants result in the inversion of Rok and Myo-II RCP
We previously demonstrated that twist is required for apical Myo-II persistence
following pulsed contractions30. The RCP of Rok suggests that medioapical Myo-II
phosphorylation could be coordinated with F-actin assembly to stabilize medioapical
actin-myosin cables that prevent cell relaxation. Therefore, we examined whether Twist
regulates the RCP of Rok. Previous work suggested that the Twist pathway is required
for the apical localization of the Rho1 activator, DRhoGEF2 (Fig. 1b)26. Unexpectedly,
we found apical Rok enrichment in twist mutants VF cells (Supplemental Fig. 4).
However, the normal RCP of Rok was inverted in twist mutants, with Rok exhibiting
junctional localization at three-way vertices, rather than localizing medioapically (Fig.
7a). snail mutants also exhibited a defect in medioapical Rok recruitment (Fig. 7a).
However, Rok localizes to a subapical position, consistent with the position of AJs in
snail mutants (Fig. 7b)26. Subapical Rok localization did not exhibit a clear dorsal-ventral
polarity in snail mutants, demonstrating that Twist requires Snail to recruit apical Rok
(Supplemental Fig. 4). Consistent with Rok locally stabilizing Myo-II, changes in Myo-II
localization mirrored Rok, with Myo-II becoming enriched in apical or subapical vertices
in twist and snail mutants, respectively (Fig. 7c,d). In twist and snail mutants, Myo-II,
and presumably Rok, colocalized with apical and subapical AJs, respectively (Fig. 7e).
Thus, Twist and Snail are required to polarize Rok to the medioapical domain and away
from AJs. twist mutants continued to exhibit transient medioapical Myo-II pulses
between Myo-II foci at vertices, but medioapical Myo-II was not stabilized (Fig. 7c).
Thus, medioapical Rok foci appear to stabilize Myo-II in the medioapical cortex of VF
cells. We propose that the function of Twist is not simply to activate apical Rho1 and
Rok, but that Twist polarizes Rho1 signaling in the plane of the apical surface, leading to
the polarization of Rok and Myo-II to the medioapical domain.
Discussion
We have demonstrated a spatial organization to Twist pathway components,
which polarizes Rok/Myo-II and E-Cadherin/AJs to medioapical and junctional domains,
respectively. The enrichment of Rok/Myo-II and E-Cadherin/AJs to separate domains is
similar to the process of cell-cell intercalation during Drosophila germband extension36-40.
However, Rok/Myo-II and AJ components exhibit planar cell polarity (PCP) in germband
cells, whereas they exhibit “radial” cell polarity (RCP) in VF cells (Fig. 8a). Dia-mediated
F-actin polymerization appears to maintain E-Cadherin polarity and link the medioapical
and junctional domains in VF cells, allowing contractile forces to be stably transmitted
across AJs. Thus, Myo-II stabilization, AJ organization, and F-actin assembly are
temporally and spatially coordinated to drive ratchet-like apical constriction (Fig. 8b).
During VF cell apical constriction, Rok becomes concentrated as medioapical
foci and is absent or present at low levels at AJs. The RCP of Rok appears to target
Myo-II accumulation to the medioapical domain. The importance of Rok localization is
strongly supported by the fact that the RCP of both Rok and Myo-II are inverted in twist
and snail mutants, with Rok and Myo-II accumulating inappropriately at AJs. Although
Rok is improperly localized in both mutants, twist mutants still exhibit contractile pulses,
suggesting that the apical Rok at AJs or low levels of Rok in the medioapical domain are
sufficient to promote Myo-II pulses. We propose that Rok’s localization increases Myo-II
phosphorylation when Myo-II reaches the medioapical domain either by diffusion or
contractile flow, leading to increased minifilament assembly and stabilization of Myo-II
structures (Fig. 8b)59,60. The inappropriate localization of Rok in twist mutants suggests
that Myo-II assembly in the medioapical domain, rather than junctions, is critical to
stabilize cell shape between pulses for cells to constrict like a ratchet.
Medioapical contractility and epithelial tension requires that the contractile cortex
be coupled to the junctional domain30,33. Rho1 and Dia localize to both the medioapical
and junctional domains, and we observe F-actin cables originating from both locations in
the absence of Rok and Myo-II activity. Reducing F-actin assembly using CytoD or diaM
mutants destabilizes the connection between the medioapical contractile network and
the junctional domain. Interestingly, CytoD and diaM mutants cause E-Cadherin to
accumulate across the entire apical surface rather than being restricted to the junctional
domain. Dia-mediated F-actin assembly could polarize E-Cadherin through clathrinmediated endocytosis, as was described in germband cells39, thus removing medioapical
E-Cadherin. F-actin-dependent regulation of endocytosis has also been proposed to
establish cortical domains during Drosophila cellularization53,57,58. Thus, Dia could
function to separate E-Cadherin from peak Rok activity, creating discrete anchor points
at cell-cell interfaces that can be pulled inwards by Myo-II. In addition, F-actin assembly
is required to form a continuous meshwork of F-actin cables that connects the
medioapical and junctional domains. twist mutants maintain E-Cadherin RCP but lack a
continuous meshwork of medioapical F-actin cables, suggesting that F-actin assembly
contributes to the actin-myosin ratchet independently from regulating AJ polarity.
Our data suggests that Myo-II localization and F-actin assembly are coordinated
to prevent cell relaxation. First, disruption of F-actin turnover and meshwork formation
using F-actin inhibitors, such as CytoD, does not disrupt apical Myo-II recruitment20.
Second, F-actin cable assembly occurs continuously in Rok inhibitor injected embryos
that lack apical Myo-II. Therefore, we propose that the combination of localizing Myo-II
stabilization to the medioapical cortex while more broadly activating medioapical and
junctional F-actin assembly results in the formation of contractile cables that span the
apical domain of VF cells and become anchored at discrete AJ sites (Fig. 8b). These
actin-myosin cables could function like stress-fibers to prevent cell relaxation after
contractile pulses and form a supracellular meshwork30.
Planar polarized actomyosin and AJ localization results in cell-cell intercalation
whereas radially polarized actomyosin and AJ localization causes apical constriction
(Fig. 8a)36,37,40,48. This is illustrated by the fact that Rok and Dia localize medioapically in
VF cells to drive apical constriction, whereas Dia activation in the germband induces
Myo-II accumulation at vertical junctions that contain Rok48. Therefore, developmental
cues, such as Twist and Snail, must properly orient Myo-II, F-actin assembly, and AJ
organization to elicit the appropriate cell shape change48. Importantly, we show that the
function of Twist is not simply to activate, but to polarize apical signals like Rok to
spatially regulate Myo-II stabilization and F-actin assembly. Determining the mechanism
that establishes RCP will be critical to determine how the common set of signaling and
cytoskeletal proteins are regulated to generate different cell shape changes during
morphogenesis61,62.
Acknowledgements
We thank Jennifer Zallen, Sérgio Simões, Rodrigo Fernandez-Gonzalez, Thomas Lecuit,
Mark Peifer, Steven Wasserman, and the Bloomington Stock Center for providing fly
stocks or antibodies used in this study. In addition, we thank members of the Martin lab
and A. Sokac, V. Hatini, T. Orr-Weaver for their comments on versions of this
manuscript. This work was supported by grant R00GM089826 to A.C.M. from the
National Institute of General Medical Sciences. Adam C. Martin is a Thomas D. and
Virginia W. Cabot Career Development Assistant Professor of Biology.
Author Contributions
F.M.M. designed and performed experiments, analysed data and wrote the manuscript.
M.T. designed and performed experiments. A.C.M. designed and performed
experiments, analysed data, supervised the project and wrote the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Figure Legends
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: 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: 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 Factin 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 Y27632 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: 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: 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 ECadherin 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: 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 Factin 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, Factin 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, twotailed, 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 Factin 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, twotailed, Mann-Whitney test) but not in twist mutants (n.s.).
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 threeway 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 ECadherin. Scale bars are 5µm.
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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Methods
Fly stocks and genetics.
The following fluorescent-fusion protein stocks were used: Utr::GFP, Myo-II::mCherry
(sqhAX3;sqhp-sqh::mCherry(ChFP),sqhp-Utr::GFP/CyO)22, Myo-II::mCherry (sqhpsqh::mCherryA11)20, sqhp-Utr::GFP/CyO22, rho1p-GFP::Rho163, ubi-ECad2::GFP64, UASpVenus::Rok(K116A)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, where sqhp is the sqh promoter and rho1p is the Rho1
promoter. The halo mutant was used to identify homozygous twist or snail mutants
during cellularization65. To visualize membranes and Myo-II, Gap43::mCherry/CyO;
sqhp-sqh::GFP30 was crossed to Myo::GFP (sqhAX3;sqhp-sqh::GFP(II)) to remove
potential phenotypes resulting from the CyO balancer47. UASp-Venus::Rok(K116A)40
females were crossed to males containing the maternal GAL4 driver mat15:GAL4.
Resultant females (UASp-Venus::Rok(K116A)/+;mat15/+) were mated to OregonR
males and their embryos were imaged. Germline clones for dia were generated by heatshocking hsFLP;dia5 FRT40A/ovoD FRT40A;sqhp-sqh::GFP/+ larvae for two hours at
37°C for three to four days. Controls for the germline clones were
hsFLP;dia5,FRT40A/Sco;sqhp-sqh::GFP embryos. To generate halo,twistey53/Utr::GFP
embryos, halo,twistey53/CyO,sqh::GFP were crossed to Utr::GFP/CyO. halo
twistey53/Utr::GFP females were then mated to halo twistey53/CyO,sqh::GFP males and
their embryos were imaged. The same strategy was applied to halo snailIIG05/
CyO,sqh::GFP mutants. To generate twistey53/Gap43::ChFP; sqhp-Venus::Rok(K116A),
halo twistey53/CyO,sqh::GFP was crossed to Gap43::ChFP/CyO; sqhp-GFP::Rok(K116A)
flies. The same strategy applied for snailIIG05/Gap43::ChFP; sqhp-GFP::Rok(K116A).
Controls for twist or snail experiments were halo,twistey53/halo,CyO or
halo,snailIIG05/halo,CyO expressing the appropriate transgene. For Rok germline clones
and rescue, rok2 FRT19A/ovoD FRT19A; hsFLP UAS-Venus::Rok(WT)/+; mat15/+ larvae
were heat shocked at 37°C for two 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 coated with embryo glue (double-sided tape
soaked in heptane). Spacer coverslips (No. 1.5) were attached to glue and a coverslip
was attached to create a chamber. Halocarbon 27 oil was added to chamber. Embryos
were not compressed. All imaging occurred at room temperature (~23°C).
For fixed imaging, embryos were dechorionated with bleach then fixed in 4%
paraformaldehyde in 0.1M phosphate buffer pH 7.4 with 50% heptane for 20-30 minutes
and manually devitellinized. To visualize F-actin, embryos were then incubated with
AlexaFluor568 Phalloidin (Invitrogen) diluted in PBS+0.1% TritonX-100 (PBST)
overnight. Embryos were then blocked with 10% BSA in PBST. Primary and secondary
antibodies were diluted in 5% BSA in PBST. Embryos were then mounted using
AquaPolymount (Polysciences, Inc.). Diaphanous was recognized using antibody (a gift
from S. Wasserman) diluted at 1:5000, E-Cadherin (DCad-2, from Developmental
Studies Hybridoma Bank) at 1:50, and Rho1 (p1D9, from Developmental Studies
Hybridoma Bank) at 1:50. Secondary antibodies used were AlexaFluor488, 568, or 647
(Invitrogen), diluted at 1:500.
All images were acquired on a Zeiss LSM 710 confocal microscope, with a 40x/1.2
Apochromat water objective (Zeiss), an argon ion, 561nm diode, 594nm HeNe, and
633nm HeNe lasers. Pinhole settings ranged from 1-2 airy units. For two-color, live
imaging, simultaneous excitation was used with bandpass filters set at ~499-561nm for
GFP and ~599-696nm for ChFP.
All images shown are representative of at least three embryos, unless otherwise
indicated here or below: 6 rok2 mutant;Venus::Rok(WT) embryos. 10
GFP::Rok(K116A);GAP43::ChFP embryos. 4 GFP::Rok(K116A) embryos stained for ECad. 19 GFP::Rok(K116A) embryos stained for Rho1. 13 embryos stained for Rho1 and
Dia. 6 GFP::Rok(K116A) embryos stained for Dia. 12 Sqh::GFP embryos stained for Dia.
5 Utr::GFP/GAP43::ChFP embryos. 16 mutant (diaM) and 13 control (dia/sco) embryos
expressing Sqh::GFP and stained for E-Cad. 11 mutant (diaM) and 4 control (dia/sco)
embryos expressing Sqh::GFP for live imaging. 5 control (twist/CyO), 7 twist, and 8 snail
embryos expressing GFP::Rok(K116A) and GAP43::ChFP. 2 control (twist/CyO), 6 twist,
and 4 snail embryos expressing Sqh::GFP for live imaging. 10 control (snail/CyO), 26
twist and 28 snail embryos expressing Sqh::GFP and stained for E-Cad. More than 15
embryos expressing Utr::GFP and Sqh::ChFP.
Drug Injections.
Dechorionated embryos were mounted ventral side up for furrow injection or lateral side
up for germband injections then desiccated for 5-10 minutes using Drierite (Drierite
Company). A 3:1 mixture of halocarbon 700:halocarbon 27 oils was added over the
embryo for injection. Embryos were injected laterally during mid-to-late cellularization
(furrow canals at base of nuclei) for ventral furrow cells or injected dorsally after initiation
of pole cell movement for germband cells. Embryos were injected with a drop size
around 40µm, estimating a 1:200-300 dilution of the drug. Y-27632 (Enzo Life Sciences)
was resuspended and injected at 50mM in water. 16 Y-27632 injected embryos were
imaged and quantifications in Fig. 3g are from a representative embryo. CytoD (Enzo
Life Sciences) was resuspended at 5mg/mL in DMSO, diluted in DMSO and injected at
0.125mg/mL (low), 0.25mg/mL (intermediate) and 5mg/mL (high). For CytoD
experiments, 6 control (DMSO solvent), 7 low dose CytoD, and 5 high dose CytoD
GAP43::ChFP, Sqh::GFP embryos were imaged and quantifications in Fig. 4 are from
representative embryos from each condition. Images from E-Cad::GFP injected with
intermediate CytoD are representative from three individual embryos. Embryos were
imaged 3-5 minutes after injection.
Image processing and analysis.
Images were processed using MATLAB (MathWorks), Fiji (http://fiji.sc/wiki/index.php/Fiji)
and Photoshop CS5.1 (Adobe Systems, Inc.). A Gaussian filter (kernel=1 pixel) was
applied to images. Apical images are maximum intensity projections of multiple zsections (~2-3µm). Subapical images are one z slice, ~1-2µm below the apical sections.
Image segmentation for quantification of area and F-actin intensities was performed
using custom MATLAB software titled EDGE (Embryo Development Geometry
Explorer)45. Raw images were processed via background subtraction to remove
cytoplasmic Utr::GFP and/or Myo::ChFP by clipping cytoplasmic intensity values two
standard deviations from the mean. Additionally a rotationally symmetric Gaussian
smoothing filter (kernel=3 pixels, σ=0.5) was applied. We made maximum intensity
projections, summing the top two highest-intensity values. Images were imported into
EDGE, cell membranes were automatically detected and manually corrected. Cell area
and integrated fluorescence intensity of Myo-II and Utr were quantified (Figs. 3, 4, and 6,
and Supplemental Fig. 2). For pulse intensities (Fig. 3d-f), maximum intensity projections
of apical 2-3µm were generated in FIJI. We generated kymographs of pulses and
intensity values were obtained by drawing lines through pulse in kymograph. Pulse data
is from one representative embryo of four individual embryos where pulses were
quantified. Temporal cross-correlation was determined using “crosscoef” function in
MATLAB. To calculate cross-correlation between constriction rate and myosin rate in
CytoD and control injected embryos (Fig. 4c,d), we first smoothed apical area and
myosin intensity curves by averaging values at a given time point with immediately
neighboring time points (3 time points total). We then calculated a rate for each time
point by subtracting the value following a time point by the preceding time point and
dividing by the time interval. Pearson Correlations were calculated using 50 time points
for each cell. Quantifications of F-actin (Utr::GFP) for Figure 4 are from representative
embryo of 9 twist, 10 snail or 3 control (twist/+) embryos imaged. To determine average
pulsing behavior in control (twist/+) and twist mutants (Fig. 6e), individual constriction
pulses (n=51 for control, n=45 for twist) were identified by thresholding area constriction
rate and were then aligned at their peak constriction rate (time zero). Values for each
pulse were normalized to initial and final areas and F-actin levels then averaged.
Linescans of Utr::GFP in cells were generated using FIJI and a line width of 25 (Fig. 6b).
Over 30 cells were analyzed for each genotype, and six representative cells of similar
size were averaged. Ratios of medioapical to junctional F-actin were obtained using FIJI,
by manually drawing a line (width=4) around cell junctions to determine average
intensity (Fig. 6c). Circles or ellipses were drawn over medioapical region to obtain
average intensity. For each cell, junctional intensities were divided by medioapical
intensity and then averaged for each genotype.
Statistical analyses were performed in Prism (Graphpad). Data sets were subjected to
Komolgorov-Smirnov tests to determine distributions. Non-normal distributions resulted
in nonparametric analyses using two-tailed, Mann-Whitney test for average pulse
behavior (Fig. 6e) and medial-to-junctional F-actin ratio (Fig. 6c). Unpaired, two-tailed
test was used to determine significance of mean cross-correlation for CytoD pulsing data
(Fig. 4d). All error bars are standard deviation and results of tests are represented as pvalues.
References
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Rosales-Nieves, A. E. et al. Coordination of microtubule and microfilament
dynamics by Drosophila Rho1, Spire and Cappuccino. Nature cell biology 8, 367376, doi:10.1038/ncb1385 (2006).
Oda, H. & Tsukita, S. Real-time imaging of cell-cell adherens junctions reveals
that Drosophila mesoderm invagination begins with two phases of apical
constriction of cells. J Cell Sci 114, 493-501 (2001).
65
Gross, S. P., Guo, Y., Martinez, J. E. & Welte, M. A. A determinant for
directionality of organelle transport in Drosophila embryos. Current biology : CB
13, 1660-1668 (2003).
Supplemental Figure 1: Venus::Rok(K116A) localizes to medioapical foci during
constriction.
During apical constriction, Venus::Rok(K116A) accumulates in medioapical foci in
ventral furrow cells, similar to Venus::Rok(WT). Images are from a live embryo
expressing Gap43::ChFP (subapical slice) and Venus::Rok(K116A) (apical projection).
Apical constriction appears to be unaffected by expression of Rok(K116A). Scale bar is
5µm.
Supplemental Figure 2: Apical F-actin is dynamic in ventral furrow cells.
(a) Utrophin::GFP (Utr::GFP) labels F-actin throughout the cell, including medioapical,
junctional, and subapical structures. Images are from fixed Utr::GFP embryo and
stained with phalloidin to label F-actin. Scale bar is 5µm. (b) Orthogonal view/crosssection of same embryo from a. Scale bar is 10µm. (c) Total F-actin levels decrease
throughout apical constriction. Quantifications are averages from embryos (n=4
embryos, at least 35 cells per embryo) expressing Utr::GFP and Myo::ChFP. Area was
quantified from sub-apical F-actin images. Total apical F-actin and Myo-II levels were
quantified from apical z-projections. Values for individual cells were averaged within an
embryo and then averaged across multiple embryos. (d) Quantification of total F-actin
levels and area in individual cells from a live embryo expressing Utr::GFP and
Gap43::ChFP. F-actin levels during contractile pulses (highlights) are heterogeneous,
and can increase (yellow), stabilize (blue), or decrease (magenta). F-actin levels most
often increase or remain stable during a contractile pulse. (e) Graph of average
behavior of area and F-actin during contractile pulses (n=44 pulses) from embryo in d.
Values are normalized to highest and lowest values during individual pulses. Error bars
are standard deviation. On average, F-actin levels are briefly stabilized during contractile
pulses. (f) Schematic illustrating the calculation of the time resolved cross-correlation
function. Cross-correlation is calculated between constriction rate and myosin rate for
various temporal shifts (ΔT). A peak cross-correlation at ΔT~0 indicates a significant
correlation with the value of ΔT indicating the time lag between the two signals.
Supplemental Figure 3: E-Cadherin localizes across the medioapical surface in diaM
mutants.
a,b) Serial z-sections of confocal images from two different fixed diaM embryos
expressing Myo::GFP and stained for E-Cadherin. Medioapical E-Cadherin appears to
be associated with the cortex and is localized in the same plane as Myo-II (arrows).
Medioapical E-Cadherin in diaM mutants can be distinguished from subapical E-Cadherin
puncta that are likely to represent endocytic vesicles. Distances indicated are positions
relative to the apical z-slice (0µm). Scale bars are 5µm.
Supplemental Figure 4: Snail is required for dorsal-ventral polarity of apical Rok
localization.
Control (twist/+) and twist mutants have apical GFP::Rok(K116A) (green arrowhead)
enrichment in the ventral furrow (green bracket) prior to accumulation in germband cells
(green arrowheads). White arrowhead marks the vitelline membrane. snail mutants
accumulate GFP::Rok(K116A) in the ventral furrow and in germband cells at the same
time. Images are projections of cross-sections from live control, twist, or snail embryos
expressing GFP::Rok(K116A). Scale bars are 20µm.
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
5µm, 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 zslice and were acquired at 0.78s intervals. The video is displayed at 30 fps. Scale bar is
5µm.
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 5µm.
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 20µm.
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 20µm.
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 5µm.
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 5µm.
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
20µm.
Supplemental Video 9: Snail and Twist are required for a medioapical meshwork of Factin 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 Factin 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 5µm.
Figure 1
a
Junctional
Medioapical
Contraction
Stabilization
b
Twist
Twist
Snail
Fog
Apical
Subapical
Cta
Basal
Myosin
c
T48
Subapical junctions
F-actin
Adherens junctions
Apical junctions
Rho1
Apical constriction
Rok
DRhoGEF2
Dia
(F-actin
assembly)
Myo-II activation
d
f
Rok
Membranes
Merge
g
Rok
E-Cad
Merge
Rok
Myosin
Merge
6.7m
11m
18.1m
surface
rok2 mutant;
Venus::Rok WT
cross-section
2.3min
crosssection
rok2 mutant;
Venus::Rok WT
surface view
e
0min
2.3m
4.3m
6.3m
8.4m
10.6m
h
Figure 2
c
Merge
d
Apical
projection
Dia
Rho1
Rho1
Dia
Merge
Rok
Dia
Merge
Myosin
b
Rok
F-actin
a
Subapical
F-actin
Merge
Figure 3
a
5 min
b
10 min
Apical area (μm2)
(apical)
1500
40
1000
20
500
0
0
1
2
3
F-actin
18s
36s
54s
72s
Membranes
Merge
F-actin
Membranes
0.6
200
0.4
Quantification of
total intensity for
individual pulse
150
0.2
100
0
50
−0.2
0
0
100
−53 −26.5 0 26.5 53
200
Time offset (sec)
200
10
0
0
5
Time (min)
40
600
400
20
200
10
0
5
Time (min)
0
10
Germband
30
0
800
Norm. Myo (a.u./μm2)
+Y-27632
50
0
10
i
Apical F-actin + Y-27632
Merge
Junctional polymerization
400
20
Subapical
F-actin
Ventral Furrow
30
Control
600
+Y-27632
40
800
Apical
F-actin
+Y-27632
50
Norm. Myo (a.u./μm2)
Control
Merge
f
250
Medioapical
h
Apical area (μm )
0
7
Time (sec)
g
Apical area (μm2)
6
5.7 min
e
90s
Merge F-actin Myo-II
0 sec
2
5
r
Merge
5.2 min
Membranes
4.6 min
F-actin
d
4
Quantification of
total apical
intensity
for individual cell
Time (min)
F-actin and Myo (a.u.)
F-actin
F-actin
(subapical)
c
60
F-actin (a.u.)
Myosin
0 min
0s
0sec
20s
40s
60s
80s
100s
4s
8s
Membranes
Merge
Apical Area (μm2)
Myosin Intensity
Apical Area (μm2)
Myosin Intensity
High CytoD Low CytoD
c
e
DMSO
r
1
0
Control
20
25
0
40
50
−2 −1 0 1 2
Time offset (min)
0
High CytoD
−1
60
−1 0 1
Time offset (min)
d
0.4
DMSO
0.2 Low
20
****
r
High
40
0
60
−1 0 1
Time offset (min)
Apical
F-actin
Low CytoD
-0.2
−2 −1 0 1 2
Time offset (min)
Int. CytoD Low CytoD
0
Cell index
b
Apical Area (μm2)
Myosin Intensity(a.u.)
Myosin
Control
Figure 4
a
50
40
30
20
10
Control
0
0 1 2 3 4 5 6 7 8
Time (min)
50
40
30
20
10
Low CytoD
0
0 1 2 3 4 5 6 7 8
Time (min)
50
40
30
20
10
High CytoD
0
0 1 2 3 4 5 6
Time (min)
Subapical
F-actin
Merge
Figure 5
b
dia/+
Myosin
0.6 min
1.3 min
1.8 min
0 min
0.2 min
1.1 min
1.7 min
0 min
0.7 min
2.4 min
3.6 min
Subapical
E-Cad
Merge
Apical E-Cad
Subapical
E-Cad
Merge
c
d
Subapical
E-Cad
Merge
diaM
CytoD
dia/+
Control
Apical E-Cad
diaM
Low CytoD
diaM
0 min
Myosin
dia/+
a
e
Loss of F-actin
assembly
Myosin
Adherens junctions
Diffuse Adherens junctions
F-actin
Adherens junction
depolarization
Network
dissolution
Figure 6
a
Subapical
F-actin
Subapical
F-actin
Apical F-actin
Merge
Merge
Subapical
F-actin
Merge
twist
Apical F-actin
snail
Control
Apical F-actin
0sec
17s
25s
58s
c
0
0.0
0
2.5
5.0
Distance (μm)
7.5
Control
50
0
750
0 1
2 3 4 5
Time (min)
3
6
9
Distance (μm)
6
500
12
0
0
3
6
9
Distance (μm)
twist
60
50
40
700
0
1
2 3 4
Time (min)
5
500
12
1.5
n.s.
0.5
0.0
Control pulse
1.0
****
snail
1.0
twist
twist pulse
n.s.
0.5
0.5
0.0
****
1.0
Control
e
900
F-actin (a.u.)
0
60
1000
25
twist
120
Medial/Junctional
apical F-actin ratio
60
60
snail
Normalized area
and F-actin (a.u.)
120
F-actin (a.u.)
Apical area (μm2)
d
Control
120
Apical area (μm2)
Subapical and apical
F-actin intensity (a.u.)
b
66s
0.0
-30
0
30
Time (sec)
-30
0
30
Time (sec)
Figure 7
Membranes
Control
Rok
Merge
b
c
Myosin
Control
cross-section
Control
a
VM
Rok
d
Control
cross-section
Apical
Myosin
snail
snail
snail
snail
e
Control cross-section
Myosin
E-Cad
Merge
twist
twist
twist
twist
snail cross-section
twist cross-section
Figure 8
a
b
Germband / Cell Intercalation
PCP
Fog/T48
E-Cadherin &
Par3/Baz
Rok & Myosin
Ventral Furrow / Apical Constriction
RCP
Twist
Rho1 localization
Dia localization
Rok localization
F-actin assembly
Myo-II stabilization
E-Cadherin
Rok
twist
RCP inversion
E-Cadherin
Rok
diaM
RCP loss
E-Cadherin
Contraction
F-actin
Stabilization
Myo-II
Stabilized Myo-II
Adherens junctions
Supplemental Figure 1
Rok
Membranes
0 min
1
2
3
4
8.3 min
6.8 min
5.1 min
3.5 min
1.8 min
5
1
2
3
4
5
1
2
3
5
1
3
5
4
2
4
Merge
Supplemental Figure 2
a
Phalloidin
b
Merge
Medioapical
d
600
30
400
20
200
10
0
5
Time (min)
10
1600
1400
40
1200
1000
20
800
0
600
0
1
2
3
4
5
6
7
1800
60
1600
1400
50
1200
40
1000
30
800
0
1
Apical area (μm2)
0.4
0.2
0.0
0
30
1500
40
1000
20
500
0
0
0
Time (sec)
1
2
3
4
5
Time (min)
3
4
5
6
6
7
Cell 6
80
2200
2000
60
1800
40
1600
1400
20
0
1200
0
1
2
3
4
5
6
7
1000
F-actin intensity (a.u.)
0.6
60
F-actin intensity (a.u.)
0.8
2
Time (min)
Cell 13
1.0
Normalized area
and F-actin (a.u.)
70
Time (min)
e
-30
Cell 4
Apical area (μm2)
0
Apical area (μm2)
40
Cell 2
60
F-actin intensity (a.u.)
800
Basal
F-actin intensity (a.u.)
50
F-actin and Myo
intensity (a.u.)
Apical area (μm2)
Apical
Apical area (μm2)
Apical F-actin
z-projection
Subapical F-actin
c
0
Junctional
Orthogonal View Orthogonal View
Utr::GFP
Phalloidin
Utr::GFP
Time (min)
Examples where F-actin levels during pulses are increasing,
increasing stabilizing or decreasing
f
ΔT
ΔT
Cross-Correlation
Constriction Rate
Myosin Rate
40
20
0
−20
−40
0
1
2
3
Time (min)
4
0
Time offset
Supplemental Figure 3
a
b
Myosin
E-Cadherin
Merge
Apical surface
Optical section
-0.54μm
-0.46μm
0μm
-0.08μm
-0.15μm
-0.23μm
-0.31μm
-0.39μm
-0.31μm
-0.23μm
-0.15μm
-0.08μm
0μm
Myosin
E-Cadherin
Merge
Apical surface
Optical section
Supplemental Figure 4
5 min
0 min
Control; GFP::Rok(K116A)
10 min
5 min
0 min
twist; GFP::Rok(K116A)
10 min
5 min
0 min
snail; GFP::Rok(K116A)