Download Monomeric alpha-catenin links cadherin to the actin cytoskeleton

ARTICLES
Monomeric α-catenin links cadherin to the actin
cytoskeleton
Ridhdhi Desai1,3 , Ritu Sarpal1,3 , Noboru Ishiyama2 , Milena Pellikka1 , Mitsuhiko Ikura2 and Ulrich Tepass1,4
The linkage of adherens junctions to the actin cytoskeleton is essential for cell adhesion. The contribution of the cadherin–catenin
complex to the interaction between actin and the adherens junction remains an intensely investigated subject that centres on the
function of α-catenin, which binds to cadherin through β-catenin and can bind F-actin directly or indirectly. Here, we delineate
regions within Drosophila α-Catenin (α-Cat) that are important for adherens junction performance in static epithelia and dynamic
morphogenetic processes. Moreover, we address whether persistent α-catenin-mediated physical linkage between cadherin and
F-actin is crucial for cell adhesion and characterize the functions of α-catenin monomers and dimers at adherens junctions. Our
data support the view that monomeric α-catenin acts as an essential physical linker between the cadherin–β-catenin complex and
the actin cytoskeleton, whereas α-catenin dimers are cytoplasmic and form an equilibrium with monomeric junctional α-catenin.
Adherens junctions and their core constituents, the classic cadherin
adhesion molecules, contribute significantly to animal development
and tissue homeostasis1–3 . Adherens junction defects can lead to
various human pathologies, including cancer4–6 . Adherens junction
function relies on the association of cadherins with the microtubule
and actin cytoskeleton through their cytoplasmic binding partners,
the catenins1 . Elucidating the function of α-catenin, which operates
at the interface of the cadherin–β-catenin complex and F-actin, is a
major goal in the field7–9 .
Studies on mammalian αE-catenin have given rise to two models for
α-catenin function: the physical linkage and the allosteric regulation
model. αE-catenin can bind both β-catenin and F-actin suggesting
that it can physically link the cadherin–β-catenin complex directly
to F-actin10–12 . This simple model lacks direct experimental support
because a quaternary complex between cadherin, β-catenin, αE-catenin
and F-actin could not be documented13 . Complex formation with
F-actin could be demonstrated in vitro only in the presence of
EPLIN (ref. 14), one of several F-actin-associated proteins that
bind to αE-catenin, such as vinculin, α-actinin, afadin, ZO-1 and
formin9,15 . Thus, a more complex physical linkage model poses that
αE-catenin links the cadherin/β-catenin complex to F-actin indirectly
by interacting with actin-binding proteins. A role for αE-catenin as
a physical linker between cadherin and actin is consistent with the
discovery that αE-catenin acts as a tension sensor that is responsive to
actomyosin contraction at adherens junctions16–18 .
Alternatively, α-catenin was proposed to regulate actin organization
to support adherens junction formation, rather than act as a physical
linker13,19 . αE-catenin binds β-catenin as a monomer but shows high
affinity for F-actin only as a homodimer11,19 . The β-catenin binding site
and homodimerization domain of αE-catenin overlap, suggesting that
it cannot interact with β-catenin and F-actin simultaneously12,13,19,20 .
These findings precipitated the view that αE-catenin may act allosterically by binding β-catenin to increase its own local concentration at
adherens junctions, which is required to promote αE-catenin dimerization after dissociation from β-catenin required for F-actin interaction
and modulation19 . This model does not adequately address how
adherens junctions are physically linked to actin and resist tensile forces.
One question that results from these contradictory models is whether
α-catenin dimerization is critical for adherens junction function.
We performed an in vivo structure–function analysis of Drosophila
α-Catenin (α-Cat) to assess the roles of its domains in several
developmental processes and to distinguish between the physical
linkage and allosteric regulation models for α-catenin function.
RESULTS
Drosophila α-Cat is approximately 60% identical to mouse or human
αE- and αN-catenin. Although the structure of any full-length
α-catenin protein remains unresolved, structures of αE-catenin
fragments, structure predictions and comparison to the related protein
vinculin indicate that α-catenin is a multi-domain protein composed
1
Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario, M5S 3G5, Canada. 2 Ontario Cancer Institute and Department of
Medical Biophysics, University of Toronto, MaRS Toronto Medical Discovery Tower, Room 4-804, 101 College Street, Toronto, Ontario, M5G 1L7, Canada. 3 These
authors contributed equally to this work.
4
Correspondence should be addressed to U.T. (e-mail: [email protected])
Received 12 June 2012; accepted 8 January 2013; published online 17 February 2013; DOI: 10.1038/ncb2685
NATURE CELL BIOLOGY VOLUME 15 | NUMBER 3 | MARCH 2013
© 2013 Macmillan Publishers Limited. All rights reserved.
261
ARTICLES
VH2
Mr (K)
α-Cat
VH3
HA
αCat::HA
95
IP: anti-HA
IB: anti-Arm
αCatΔVH1
95
IP: No Ab
IB: anti-Arm
αCatΔVH3
95
Input (40 μg)
IB: anti-Arm
αCatΔCTD
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Arm α-Spec GFP
Expression of transgenes in α -Cat mutant follicle cells
HA α-Spec GFP
HA
α-Spec
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αC
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at A
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at H3 TD
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Late pupa
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Second instar larva
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Normal head
Weak head
Head defect
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αCatΔVH1
α-Spec
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C
trl
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αCatΔCTD
1. α-Cat
2. αCat::HA
3. αCatΔVH1
4. αCatΔVH3–CTD
5. αCatΔVH3
6. αCatΔCTD
Figure 1 Arm- and F-actin-binding domains are essential for α-Cat function
in Drosophila. (a) Schematic representation of α-Cat constructs (details
are available in Supplementary Table S1). (b) Co-immunoprecipitation
(IP) experiments using anti-HA antibodies indicating that all constructs
listed in a except αCat1VH1 show robust physical interactions with Arm.
IB, immunoblot. (c) Face on (top rows) and side views (bottom rows)
of positively marked α-Cat mutant follicle cell clones express either no
α-Cat transgene (left column) or αCat::HA, αCat1VH1, αCat1VH3 or
αCat1CTD. Follicles are stained for HA to detect the α-Cat constructs, GFP
to positively mark α-Cat mutant cells, and α-Spectrin (α-Spec). Scale bars,
10 µm. (d) Quantification of rescue performance of α-Cat constructs in the
follicular epithelium (FE), border cell (BC) migration and embryos. Follicular
epithelium cells: percentage of follicular epithelium cells that show rescue
of α-Cat mutant (n = 26 clones with 1,228 cells) defects on expression
of α-Cat (n = 24 clones with 1,802 cells), αCat::HA (n = 44 clones with
2,713 cells), αCat1VH1 (n = 24 clones with 743 cells), αCat1VH3–CTD
(n = 20 clones with 688 cells), αCat1VH3 (n = 23 clones with 900 cells)
or αCat1CTD (n = 18 clones with 911 cells). Data are presented as the
mean ± s.e.m. Border cell migration: quantification of border cell migration
rescue on expression of α-Cat (n = 12), αCat::HA (n = 18), αCat1VH1
(n = 23), αCat1VH3–CTD (n = 17), αCat1VH3 (n = 21) or αCat1CTD
(n = 13). Border cell clusters that contain 5 or more α-Cat mutant cells
were examined. The data are presented as the mean ± s.d. Embryo: aligned
dot plot showing the average (thick horizontal line), s.d. (whiskers) and total
range (triangles) of rescue activity of α-Cat (n = 120), αCat::HA (n = 72),
αCat1VH1 (n = 155), αCat1VH3–CTD (n = 263), αCat1VH3 (n = 246) or
αCat1CTD (n = 250) when expressed in α-Cat1 (n = 140) zygotic mutant
embryos. The red triangle indicates rescued animals that hatch as adults.
Data are presented as mean ± s.d. See Methods for scoring criteria. The
red asterisks indicate a significant difference from α-Cat1 mutant follicular
epithelium cells or embryos (P < 0.0001). Uncropped images of blots/gels
are shown in Supplementary Fig. S9.
of a series of α-helices, most of which organize into five helical bundles
connected by linker regions12,21–23 . The first two α-helical bundles
overlap with the vinculin-homology region 1 (VH1), and the last
one with the VH3 region of α-catenin. The linker between bundles
2 and 3 contains two α-helices that bind to vinculin24,25 . The central
VH2 region (or M-fragment21 ) comprises α-helical bundles 3 and 4.
The linker between bundles 3 and 4 is flexible and could facilitate
conformational change in response to actomyosin tension16,21,22 . We
generated α-Cat deletion constructs designed to prevent disruption of
adjacent helical bundles.
Constructs were expressed from the same genomic insertion site,
and tested in an α-Cat null mutant background26 . α-Cat proteins
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ARTICLES
were analysed in the relatively static follicular epithelium, the highly
dynamic morphogenetic movements of border cell migration and
embryonic head morphogenesis, and we determined whether they
supported adult viability. Cells of the follicular epithelium show little
cell rearrangement27 . α-Cat mutant follicle cells lose cell–cell contacts
and adopt a flattened, rounded shape, with adherens junction markers
DE-cadherin (DEcad) and Armadillo (Arm), the Drosophila β-catenin
homologue, lost from the membrane26 (Fig. 1c). α-Spectrin is also
lost from the cortex and forms cytoplasmic aggregates that become
enriched in several basolateral and apical proteins26 . α-Cat constructs
were expressed in α-Cat mutant follicle cell clones, and rescue was
quantified by counting the cells that exhibited any such defects among
GFP-labelled mutant cells in stage 8 to 10 follicles. α-Cat-related defects
are absent from wild-type follicle cells but are present in 95% of α-Cat
mutant cells26 . Expression of full-length α-Cat alone or with an HA
tag (αCat::HA) showed ∼95% rescue (Fig. 1a,c,d and Supplementary
Table S1), with both proteins localizing normally to adherens junctions
(Supplementary Fig. S1; data on all constructs are summarized in
Supplementary Table S1).
The amino- and carboxy-terminal domains are essential for
α-Cat function
The N- and C-terminal regions of α-catenin bind β-catenin/Arm
and F-actin, respectively11,12,20,28–31 . Expression of αCat1VH1, lacking
amino acids 1–233, which contain the Arm-binding site and the
overlapping α-Cat homodimerization domain12 , did not rescue the
mutant phenotype (Fig. 1a,c,d). Rather than localizing to adherens
junctions, αCat1VH1 was cytoplasmic in both wild-type and α-Cat
mutant cells (Fig. 1c and Supplementary Fig. S1). Similarly, constructs
that lacked amino acids 709–917 (αCat1VH3–CTD) or amino
acids 709–868 (αCat1VH3), the sequences mediating direct F-actin
binding, lacked significant rescue activity (Fig. 1a,c,d). In contrast
to αCat1VH1, αCat1VH3 and αCat1VH3–CTD were enriched at
adherens junctions in wild-type follicle cells (Supplementary Fig. S1).
An α-Cat construct lacking the C-terminal domain (CTD; amino acids
865–917; αCat1CTD) localized to adherens junctions in wild-type
cells (Supplementary Fig. S1) and fully rescued α-Cat mutant follicle
cells (Fig. 1a,c,d). DEcad and Arm were normally distributed in α-Cat
mutant follicle cells expressing αCat::HA or αCat1CTD, whereas
adherens junction markers were cytoplasmic in mutant cells expressing
αCat1VH1, αCat1VH3 or αCat1VH3–CTD (Supplementary Fig. S2).
Although the CTD was previously implicated in F-actin binding of
αE-catenin22,31 , its loss did not compromise α-Cat function.
α-Cat constructs were further analysed in border cells. Border
cells undergo collective migration that requires the cadherin–catenin
complex in border cells and the migration substrate, the germline cells32 .
Border cells show a higher turnover of DEcad than follicular epithelium
cells (Supplementary Fig. S3). In the wild type, all border cell clusters
completed migration by stage 10a whereas α-Cat mutant cells failed to
migrate and remained at the anterior tip of the follicle26 . Migration was
scored as complete, incomplete (when border cells were still en route)
or none (when border cells had not detached from anterior follicle
cells) at stage 10b. αCat1VH1, αCat1VH3 and αCat1VH3–CTD did
not support migration, whereas αCat1CTD did in most cases, with
only 3 out of 13 border cell clusters showing incomplete migration
(Fig. 1d and Supplementary Fig. S3 and Table S1).
α-Cat zygotic mutants are embryonic lethal with defects in head
morphogenesis26 . Following a scoring system described previously26
(see Methods), where a score of 8 would indicate that all animals
survived to adulthood, α-Cat mutants attained a score of 0, whereas
α-Cat mutants expressing either α-Cat or αCat::HA attained a score
of 7.9, indicating survival to adults in nearly all animals (Fig. 1d).
αCat1VH1, αCat1VH3 and αCat1VH3–CTD expression in α-Cat
mutants produced scores similar to α-Cat mutants without a transgene
(Fig. 1d). αCat1CTD expression in α-Cat mutants produced a score
of 3.6, with most animals dying during larval development.
αCat::HA, αCat1VH3, αCat1VH3–CTD and αCat1CTD robustly
interacted with Arm in embryonic lysate immunoprecipitates whereas,
as expected, αCat1VH1 did not (Fig. 1b). Together, our findings
suggest that the α-Cat VH1 and VH3 regions are essential for adherens
junction formation. In contrast, the α-Cat CTD is dispensable for
α-Cat function in the follicular epithelium and embryonic head
morphogenesis, but contributed to border cell migration and larval
survival. Deletion of the CTD from αE-catenin strongly reduced F-actin
binding in vitro22,31 and failed to support adherens junctions16,31 ,
suggesting that direct interaction with F-actin may be less important
for Drosophila α-Cat than mouse αE-catenin.
The central region of α-Cat is a complex modulator of
cell adhesion
Expression of αCat1VH2, which lacks VH2 (amino acids 373–627)
in α-Cat mutant follicle cells caused an intermediate level of rescue
(62.1% normal cells), with no cells exhibiting α-Spectrin aggregates
(Fig. 2a,c,d). Despite retaining the Arm-binding domain, αCat1VH2
was poorly recruited to adherens junctions (Fig. 2c), and accumulated
there only when expressed in α-Cat mutant cells (Fig. 2c). Lower
than normal levels of DEcad and Arm were found at adherens
junctions in α-Cat mutant cells expressing αCat1VH2 (Supplementary
Fig. S4). These data suggest that αCat1VH2 is outcompeted by
endogenous α-Cat at adherens junctions and that, in addition to the
VH1-mediated interactions with Arm, VH2 facilitates interactions that
contribute either to recruitment of α-Cat to adherens junctions or to
adherens junction integrity.
We next examined two constructs that removed either the Nterminal (amino acids 398–509; αCat1VH2N) or C-terminal (amino
acids 510–627; αCat1VH2C) half of VH2, deleting α-helical bundles 3
or 4, respectively. αCat1VH2N and αCat1VH2C expression fully
rescued α-Cat mutant follicle cells (Fig. 2a,c,d). Both constructs
localized to adherens junctions in mutant and wild-type cells although
cytoplasmic levels of αCat1VH2N remained higher in wild-type cells
than αCat1VH2C or αCat::HA (Fig. 2c).
αCat1VH2 poorly supported border cell migration and
αCat1VH2N was only slightly better, but migration was almost
normal in border cells expressing αCat1VH2C instead of endogenous
protein (Fig. 2d and Supplementary Fig. S3). In whole-animal rescue
assays, αCat1VH2 supported embryonic head morphogenesis in most
animals, but not larval development (Fig. 2d). Both αCat1VH2N
and αCat1VH2C performed significantly better than αCat1VH2
(Fig. 2d). These findings suggest that α-helical bundles 3 and 4
of α-Cat have overlapping and unique functions. Both contribute
independently to the integrity of adherens junctions, to support
normal epithelial structure or enhance embryonic survival of α-Cat
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263
b
Mr (K)
VH1
VH2
VH3
HA
IP: anti-HA
IB: anti-Arm
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αCat::HA
αCatΔVIN
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IP: No Ab
IB: anti-Arm
95
Input (40 μg)
IB: anti-Arm
αCatΔVH2C
P < 0.05
P < 0.001
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cells
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at
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ARTICLES
1 2 3 4 5
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Expression of transgenes in α -Cat mutant follicle cells
Expression of transgenes in wild-type follicle cells
c
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HA α-Cat
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Weak head
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P < 0.0001
1 2 3 4 5
1. αCat::HA
2. αCatΔVIN
3. αCatΔVH2
4. αCatΔVH2N
5. αCatΔVH2C
Figure 2 The central region of α-Cat acts as a complex positive modulator
of cell adhesion. (a) Schematic representation of α-Cat constructs (details
are available in Supplementary Table S1). (b) Co-immunoprecipitation
(IP) experiments using anti-HA antibodies indicating that αCat1VH2
and αCat1VH2N show a reduced level of physical interactions with
Arm in comparison with αCat::HA or αCat1VH2C. IB, immunoblot.
(c) Face on (top rows) and side views (bottom rows) of wild-type
(left) and α-Cat mutant (right) follicular epithelium cells expressing
αCat1VIN, αCat1VH2, αCat1VH2N or αCat1VH2C. α-Cat constructs
are labelled with HA, cells are counterstained for α-Cat or α-Spectrin
(α-Spec) and α-Cat mutant cells are marked by GFP. Scale bars, 10 µm.
(d) Quantification of rescue performance of α-Cat constructs in the
follicular epithelium (FE), border cell (BC) migration and embryos.
Follicular epithelium cells: percentage of follicular epithelium cells that
show rescue of α-Cat mutant (n = 26 clones with 1,228 cells) defects
on expression of αCat::HA (n = 44 clones with 2,713 cells), αCat1VIN
(n = 33 clones with 1,511 cells), αCat1VH2 (n = 40 clones with 1,642
cells), αCat1VH2N (n = 20 clones with 1,553 cells) or αCat1VH2C
(n = 15 clones with 1,299 cells). Data are presented as the mean±s.e.m.
Border cell migration: quantification of border cell migration rescue on
expression of αCat::HA (n = 18), αCat1VIN (n = 16), αCat1VH2 (n = 21),
αCat1VH2N (n = 22) or αCat1VH2C (n = 15). Border cell clusters that
contain 5 or more α-Cat mutant cells were examined. The data are
presented as the mean ± s.d. Embryo: aligned dot plot showing the
average (thick horizontal line), s.d. (whiskers) and total range (triangles)
of rescue activity of αCat::HA (n = 72), αCat1VIN (n = 96), αCat1VH2
(n = 212), αCat1VH2N (n = 224) or αCat1VH2C (n = 82) when expressed
in α-Cat1 (n = 140) zygotic mutant embryos. The red triangle indicates
rescued animals that hatch as adults. See Methods for scoring criteria.
The red asterisks indicate a significant difference from α-Cat1 mutant
follicular epithelium cells or embryos (P < 0.0001). Uncropped images
of blots/gels are shown in Supplementary Fig. S9.
mutants, but bundle 3 is more important than bundle 4 in stabilizing
α-Cat at adherens junctions and in supporting border cell migration.
The differences in rescue activity of αCat1VH2, αCat1VH2N and
αCat1VH2C correlate with their propensity to form complexes
with Arm (Fig. 2b).
Deletion of the region upstream of VH2 that binds vinculin
in αE-catenin (amino acids 283–373; αCat1VIN; refs 16,24,25,33)
supported complex formation with Arm and adherens junction
localization, although cytoplasm levels were higher than with αCat::HA
(Figs 1c and 2b,c). αCat1VIN supported normal localization of
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ARTICLES
DEcad and Arm (Supplementary Fig. S4) but slightly underperformed
when compared with αCat::HA in follicular epithelium rescue and
animal survival tests (Fig. 2d and Supplementary Table S1), indicating
that the vinculin-binding region makes an essential but minor
contribution to α-Cat activity.
DEcad–α-Cat fusion proteins demonstrate the significance of
the α-Cat central region in adherens junction function
A chimaeric DEcad and α-Cat protein (DEcad::αCat) can rescue the
loss of DEcad, Arm or α-Cat in follicle and border cells26,34 . Fusion
of αCat1VH1 to DEcad (DEcad::αCat1VH1) gave a similar result
(Fig. 3 and Supplementary Fig. S5). To test the function of the α-Cat
central region, we fused VH3–CTD (amino acids 629–917) to DEcad
(DEcad::αCat-VH3–CTD) and observed an intermediate level of rescue
(Fig. 3 and Supplementary Fig. S5). Border cell migration was strongly
inhibited but not abolished (Fig. 3c). Fusion protein assessment
in whole-animal rescue assays was less informative as expression
of chimaeras in wild type caused early larval lethality (Fig. 3c),
possibly due to interference with Arm-dependent Wnt signaling35 .
However, all fusion proteins rescued embryonic head morphogenesis
(Fig. 3c). The rescue activity of DEcad::αCat-VH3–CTD is similar to
αCat1VH2, suggesting that the central region does not assist in the
initial recruitment of α-Cat to adherens junctions, which is probably
facilitated solely by the VH1-mediated interaction with Arm. Thus,
in addition to the VH1-mediated interaction with Arm, the VIN,
VH2N and VH2C regions undergo secondary interactions that stabilize
α-Cat at adherens junctions and consequently adherens junction
integrity and function.
Uncoupling α-Cat recruitment to adherens junctions from
α-Cat function
The high affinity of αE-catenin homodimers, but not monomers,
for F-actin, and the mutual exclusion of dimerization and β-catenin
binding, support an allosteric function of α-catenin, in which its
β-catenin-dependent recruitment to adherens junctions serves to
locally concentrate α-catenin in that region, but its association with
actin occurs only after it dissociates from β-catenin and dimerization19 .
To address this model in Drosophila, we investigated whether an
adherens junction-associated homodimer, rather than the physical
linkage to the DEcad–Arm complex, is crucial for α-Cat function.
We generated constructs that dimerize α-Cat, localize it to adherens
junctions but compromise its interaction with Arm. αCat1VH1 was
fused to the oligomerization domain (OD) of Bazooka/Par3 (Baz;
BazOD::αCat1VH1; Fig. 4a), which facilitates multimerization including dimerization36,37 . Baz is enriched at adherens junctions independently of the cadherin–catenin complex26,38 . BazOD::αCat1VH1 was
recruited to adherens junctions (Fig. 4b,c), and physically interacted
with endogenous Baz, suggesting that BazOD retained its multimerization function (Fig. 4d). Similar to Baz, BazOD::αCat1VH1
accumulated at adherens junctions in α-Cat zygotic mutant embryos
that are strongly depleted of α-Cat protein26 (Fig. 4c).
BazOD::αCat1VH1 did not rescue embryonic head defects,
follicular epithelium integrity or border cell migration (Figs 4e,
5a,c and Supplementary Fig. S6), in contrast to the full rescue
observed with αCat1VH1 recruitment to adherens junctions in the
context of DEcad::αCat1VH1. Only minor interactions with the
DEcad–Arm complex were observed (Fig. 5b), which may result from
BazOD::αCat1VH1 interacting with endogenous Baz (Fig. 4d), a
known binding partner of Arm39 . These findings suggest that the
Arm/α-Cat interaction does not recruit α-Cat to adherens junctions
to enhance its local concentration for homodimerization. Instead,
the poor binding of BazOD::αCat1VH1 and Arm together with
the poor rescue performance of BazOD::αCat1VH1 suggests that
the persistent physical interaction with the DEcad–Arm complex is
crucial for α-Cat function.
We generated three other Baz–α-Cat chimaeras to assess the
consequences of enhancing the interactions with Arm (Fig. 4a).
Full-length Baz, which can bind directly to Arm39 , fused to
α-Cat1VH1 (Baz::αCat1VH1) showed robust localization to adherens junctions, reduced cytoplasmic localization when compared
with BazOD::αCat1VH1 (Fig. 4b) and precipitated more Arm and
DEcad than BazOD::αCat1VH1 but less than αCat::HA (Fig. 5b).
Baz::αCat1VH1 showed a noticeable but minor rescue of α-Cat
mutant follicular epithelium cells (Fig. 5a,c and Supplementary Fig. S6).
We next reinstated the Arm-binding/dimerization domain of
α-Cat to BazOD::αCat1VH1 by inserting BazOD between VH1 and
VH2 of α-Cat (αCatN::BazOD::C; Fig. 4a). This construct showed
improved adherens junction recruitment, complex formation with
Arm and DEcad, and adherens junction integrity when compared
with BazOD::αCat1VH1 (Figs 4b, 5a–c and Supplementary Fig. S6).
Interaction with Arm remained weak despite the presence of the α-Cat
Arm-binding domain, suggesting that BazOD interferes with Arm
binding presumably through forced dimerization/oligomerization of
α-Cat. αCatN::BazOD::C achieved intermediate levels of rescue of
α-Cat mutant follicle cells, but did not support border cell migration
(Fig. 5c). Finally, a full-length Baz and α-Cat fusion protein (Baz::αCat)
was enriched at adherens junctions, interacted with Arm and DEcad,
although less than α-Cat, and exhibited better rescue activity in follicle
cells than αCatN::BazOD::C (Figs 4a,b, 5 and Supplementary Fig. S6).
Baz::αCat was the only Baz chimaera supporting border cell migration,
albeit slow/incomplete (Fig. 5c). The ability of the four Baz-α-Cat
chimaeras to rescue follicular epithelium integrity correlated with the
strength of binding to the DEcad–Arm complex but not with their
localization to adherens junctions, as all constructs were able to localize
there. This argues against the conclusion that the interaction of α-Cat
with Arm acts to enrich α-Cat dimers at adherens junctions, and
supports that persistent physical linkage with the DEcad–Arm complex
is important for adherens junction performance.
Although αCatN::BazOD::C and Baz::αCat substantially rescued
follicular epithelium integrity of α-Cat mutants, these two proteins
and also BazOD::αCat1VH1 and Baz::αCat1VH1 showed little rescue
of the embryonic head, and only Baz::αCat showed limited support of
border cell migration (Figs 4e and 5c). Differences in the turnover rate
of adhesive complexes between the relatively static follicular epithelium
and the head ectoderm and border cells (Supplementary Fig. S3
and ref. 26) are probably responsible for the different rescue levels
observed. However, all four Baz–α-Cat chimaeras are poor substitutes
for endogenous α-Cat. Baz–α-Cat chimaeras may show a different
nanoscale distribution from α-Cat that is not resolvable by confocal
microscopy. Alternatively, the forced dimerization or oligomerization
of Baz–α–Cat chimaeras through BazOD could be interfering with the
normal function of α-Cat.
NATURE CELL BIOLOGY VOLUME 15 | NUMBER 3 | MARCH 2013
© 2013 Macmillan Publishers Limited. All rights reserved.
265
ARTICLES
a
DEcad
DEcad::αCat
VH1
VH2
VH3
DEcad::αCatΔVH1
HA
DEcad::αCat-VH3–CTD
b
α-Cat α-Spec GFP
DEcad
α-Spec
DEcad α-Spec GFP
DEcad::αCatΔVH1
α-Spec
DEcad::αCat-VH3–CTD
α-Cat
DEcad
DEcad::αCat
Expression of transgenes in α-Cat mutant follicle cells
α-Cat
α-Spec
α-Cat α-Spec GFP
DEcad
α-Spec
DEcad α-Spec GFP
c
100
α -Cat mutant FE cells
∗ ∗
∗
α -Cat mutant BCs
Complete
∗
80
60
Slow/
incomplete
40
20
No migration
2
3
4
5
1. αCat::HA
2. DEcad
4. DEcad::αCatΔVH1
1
2
3
4
5
α -Cat mutants
∗
Adult
Late pupa
Early pupa
Third instar larva
Second instar larva
First instar larva
Normal head
Weak head
Head defect
Dorsal hole
∗ ∗ ∗
C
trl
1
C
trl
C
trl
Percentage of rescue
WT
3. DEcad::αCat
1 2 3 4 5
DEcad::αCatΔVH1
5. DEcad::αCat-VH3–CTD
DEcad::αCat
Figure 3 Analysis of DEcad–α-Cat fusion proteins emphasizes the role of
the central region of α-Cat in adherens junction integrity. (a) Schematic
representation of DEcad–α-Cat fusion proteins (details are available in
Supplementary Table S1). (b) Face on (top rows) and side views (bottom
rows) of α-Cat mutant follicle cell clones expressing DEcad::α-Cat, DEcad,
DEcad::α-Cat1VH1 or DEcad::αCatVH3 are labelled with either DEcad
or α-Cat, α-Spectrin (α-Spec), and GFP to mark mutant cells. Scale
bars, 10 µm. (c) Quantification of rescue performance of DEcad–α-Cat
chimaeras in the follicular epithelium (FE), border cell (BC) migration and
embryos. Follicular epithelium cells: percentage of follicular epithelium
cells that show rescue of α-Cat mutant defects on expression of αCat::HA
(n = 44 clones with 2,713 cells), DEcad (n = 12 clones with 394 cells),
DEcad::αCat (n = 22 clones with 1,019 cells), DEcad::αCat1VH1 (n = 16
clones with 600 cells) or DEcad::αCat-VH3–CTD (n = 19 clones with 743
cells). Data are presented as the mean ± s.e.m. Border cell migration:
quantification of border migration rescue on expression of αCat::HA (n = 18),
DEcad (n = 5), DEcad::αCat (n = 10), DEcad::αCat1VH1 (n = 18) or
DEcad::αCat-VH3–CTD (n = 7). Border cell clusters that contain 5 or more
α-Cat mutant cells were examined. The data are presented as the mean ± s.d.
Embryo: aligned dot plot showing the average (thick horizontal line), s.d.
(whiskers) and total range (triangles) of rescue activity of αCat::HA (n = 72),
DEcad (n = 40), DEcad::αCat (n = 170), DEcad::αCat1VH1 (n = 116)
or DEcad::αCat-VH3–CTD (n = 221) when expressed in α-Cat1 (n = 140)
zygotic mutant embryos, and DEcad::αCat (n = 50) and DEcad::αCat1VH1
(n = 71) when expressed in wild-type (WT) embryos. The red triangle
indicates rescued animals that hatch as adults. See Methods for scoring
criteria. The red asterisks indicate a significant difference from α-Cat1
mutant cells or embryos (P < 0.0001).
Recruitment of α-Cat to adherens junctions through Echinoid
does not support adherens junction function
As an alternative means to recruit α-Cat to adherens junctions,
we fused αCat1VH1 to Echinoid (Ed) (Ed::αCat1VH1 and
Fig. 6a), a homophilic adhesion molecule that is highly enriched
at adherens junctions similar to vertebrate nectins39,40 . α-Cat
zygotic mutant embryos retain normal levels of Ed at adherens
junctions despite depletion of the cadherin–catenin complex26 .
Ed::αCat1VH1 concentrated at adherens junctions in wild-type
and α-Cat mutant embryos (Fig. 6b,d), but did not rescue the
α-Cat mutant phenotype (Fig. 6c,d,e), suggesting that recruitment of
αCat1VH1 to adherens junctions in the context of Ed::αCat1VH1,
266
NATURE CELL BIOLOGY VOLUME 15 | NUMBER 3 | MARCH 2013
© 2013 Macmillan Publishers Limited. All rights reserved.
ARTICLES
HA
BazOD::αCatΔVH1
c
α-Cat
Baz::αCatΔVH1
Baz
VH1
VH3
αCatN::BazOD::C
BazOD
Baz::αCat
Baz
d
Arm
Mr (K)
Expression of transgenes in wild-type follicle cells
α-Cat
HA α-Cat CD2
HA
αCatN::BazOD::C
250
IP: anti-HA
IB: anti-GFP
250
IP: No Ab
IB: anti-GFP
250
Input (40 μg)
IB: anti-GFP
135
IP: anti-HA (20%)
IB: anti-HA
Arm
Mr (K)
135
αCatN::BazOD::C
in α -Cat
Baz::αCatΔVH1
BazOD::αCatΔVH1
HA
BazOD::αCatΔVH1 BazOD::αCatΔVH1
in α -Cat
in wild type
b
Ba
zO
Ba D:
zO :αC
+B D
a
az ::α tΔV
::G Ca H
FP tΔV 1
H
1
VH2
Ba
zO
Ba D::
α
z
+B O Ca
az D::α tΔ
::G C VH
FP atΔ 1
VH
1
BazOD
Wild type
a
135
IP: No Ab
IB: anti-HA
135
Input (40 μg)
IB: anti-HA
250
e
Input (40 μg)
IB: anti-GFP
α -Cat mutant embryos
Adult
Late pupa
Early pupa
Third instar larva
Second instar larva
First instar larva
Normal head
Weak head
Head defect
Dorsal hole
2
3
4
αCat::HA
BazOD::αCatΔVH1
Baz::αCatΔVH1
αCatN::BazOD::C
Baz::αCat
5
C
1
1.
2.
3.
4.
5.
*
*
trl
Baz::αCat
IP-anti-GFP
IB: anti-HA
Figure 4 Baz-mediated recruitment of α-Cat to adherens junctions.
(a) Schematic representation of Baz–α-Cat fusion proteins (details are
available in Supplementary Table S1). (b) Face on (top rows) and side views
(bottom rows) of follicle cells that clonally express BazOD::αCat1VH1,
Baz::αCat1VH1, αCatN::BazOD::C or Baz::α-Cat in wild-type follicular
epithelium cells labelled for HA to detect the fusion protein, α-Cat
and CD2, which negatively marks the clone of cells expressing a
fusion protein. Scale bars, 10 µm. (c) Expression of BazOD::αCat1VH1
or αCatN::BazOD::C in the epidermis of early stage 17 wild-type or
α-Cat1 mutant embryos. Scale bars, 10 µm. (d) Co-immunoprecipitation
(IP) experiments showing complex formation of BazOD::αCat1VH1
(tagged with HA) and Baz or Baz::GFP in embryos. IB, immunoblot.
(e) Quantification of rescue performance of Baz–α-Cat chimaeras in
embryos. Aligned dot plot showing the average (thick horizontal line),
s.d. (whiskers) and total range (triangles) of rescue activity of αCat::HA
(n = 72), BazOD::αCat1VH1 (n = 203), Baz::αCat1VH1 (n = 173),
αCatN::BazOD::C (n = 101) or Baz::α-Cat (n = 116) when expressed
in α-Cat1 (n = 140) zygotic mutant embryos. The red triangle indicates
rescued animals that hatch as adults. Data are presented as mean ± s.d.
See Methods for scoring criteria. The red asterisks indicate a significant
difference from α-Cat1 mutant embryos (P < 0.0001). Uncropped images
of blots/gels are shown in Supplementary Fig. S9.
in contrast to DEcad::αCat1VH1, is non-functional. These findings
indicate that physical linkage of α-Cat to cadherin, rather than
an immunoglobulin adhesion molecule at adherens junctions, is
essential for α-Cat function.
consistent with DEcad, Arm and α-Cat forming a 1:1:1 complex at
Drosophila adherens junctions.
To investigate whether α-catenin homodimerization acts positively
or negatively in cell adhesion, we compared different α-catenin
isoforms that form homodimers in vitro with those that do not.
Homodimerization was observed for αE-catenin but not for α-catenin
proteins from Caenorhabditis elegans42 or Dictyostelium discoideum43 .
We confirmed homodimerization of mouse αE-catenin12,19 (Fig. 7a).
Drosophila α-Cat behaved similarly with an even larger dimer
fraction (Fig. 7a). Purified α-Cat monomer and homodimer fractions
(Fig. 7c,d) and αCat-VH3–CTD were able to bind to F-actin in vitro
(Fig. 7e,f). In contrast to αE-catenin and α-Cat, mouse αN-catenin
formed predominantly monomers (Fig. 7a).
The αN-catenin distribution in Drosophila cells was similar to
endogenous α-Cat (Supplementary Fig. S8). αN-catenin fully rescued
αN-catenin can substitute for Drosophila α-Cat
Biochemical evidence suggests that cadherin, β-catenin and α-catenin
occur at adherens junctions as a 1:1:1 complex10 , implying that an
α-catenin homodimer is probably a minor component of adherensjunction-localized α-catenin. This is consistent with the turnover rate
of α-catenin at adherens junctions being similar to that of cadherin
and β-catenin, but much slower than that of actin13 . DEcad and Arm
are present at a 1:1 ratio at adherens junctions in Drosophila embryos41 .
Quantification of fluorescence intensities of αCat::GFP in comparison
to DEcad::GFP also indicate a 1:1 ratio (Supplementary Fig. S7),
NATURE CELL BIOLOGY VOLUME 15 | NUMBER 3 | MARCH 2013
© 2013 Macmillan Publishers Limited. All rights reserved.
267
ARTICLES
α-Spec
HA α-Spec GFP
BazOD::αCatΔVH1
HA
at
::
αC HA
at
ΔV
H
Ba
zO 1
D:
Ba
:α
C
z:
at
:α
Δ
C
αC
at VH
Δ
1
at
V
H
N
1
Ba ::Ba
zO
z:
:α
D:
C
:C
at
b
Expression of transgenes in α -Cat mutant follicle cells
αC
a
Mr (K)
IP: anti-HA
IB: anti-Arm
95
IP: No Ab
IB: anti-Arm
Input (40 μg)
IB: anti-Arm
95
95
DEcad–GFP
Mr (K)
IP: anti-HA
IB: anti-GFP
Baz::αCatΔVH1
95
135
IP: No Ab
IB: anti-GFP
Input (40 μg)
IB: anti-GFP
α -Cat mutant FE cells
∗
100
80
60
40
20
∗
∗
4
5
P < 0.05
1
2
3
C
trl
αCatN::BazOD::C
c
Percentage of rescue
95
α -Cat mutant BCs
Complete
Baz::αCat
Slow/
incomplete
C
trl
No migration
1
1. αCat::HA
2
3
4
5
2. BazOD::αCatΔVH1
3. Baz::αCatΔVH1 4. αCatN::BazOD::C
5. Baz::αCat
Figure 5 α-Cat at adherens junctions not bound to Arm does not support
adherens junction integrity. (a) Face on (top rows) and side views (bottom
rows) of α-Cat mutant follicle cells positively marked by GFP expressing
BazOD::αCat1VH1, Baz::αCat1VH1, αCatN::BazOD::C or Baz::αCat.
Chimaeras are labelled with HA. α-Spectrin (α-Spec) shows cell outlines.
Scale bars, 10 µm. (b) Co-immunoprecipitation (IP) experiments using
anti-HA antibodies showing the amount of complex formation between
Baz–α-Cat fusion proteins and Arm or DEcad compared with αCat::HA
(positive control) and αCat1VH1 (negative control). IB, immunoblot.
(c) Quantification of rescue performance of Baz–α-Cat chimaeras in
the follicular epithelium (FE) and border cell (BC) migration. Follicular
epithelium cells: percentage of follicular epithelium cells that show
rescue of α-Cat mutant defects on expression of αCat::HA (n = 44
clones with 2,713 cells), BazOD::αCat1VH1 (n = 18 clones with 520
cells), Baz::αCat1VH1 (n = 13 clones with 471 cells), αCatN::BazOD::C
(n = 21 clones with 728 cells) or Baz::αCat (n = 16 clones with 818
cells). Data are presented as mean ± s.e.m. The red asterisks indicate a
significant difference from α-Cat1 mutant cells (P < 0.0001). Border cell
migration: quantification of border cell migration rescue on expression of
αCat::HA (n = 18), BazOD::αCat1VH1 (n = 14), Baz::αCat1VH1 (n = 6),
αCatN::BazOD::C (n = 12) or Baz::αCat (n = 19). Border cell clusters
that contain 5 or more α-Cat mutant cells were examined. The data are
presented as the mean ± s.d. Uncropped images of blots/gels are shown
in Supplementary Fig. S9.
α-Cat mutant follicle cells (Fig. 8a,c) and partially rescued border
cell migration (Fig. 8c). Ubiquitous expression of αN-catenin in
α-Cat mutant animals restored embryonic head morphogenesis, and
most animals survived to late larval stages (Fig. 8d). In contrast,
αE-catenin expression only mildly rescued head morphogenesis and
follicle cells, and did not support border cell migration (Fig. 8c,d).
Although expression of the αE-catenin construct was confirmed by
PCR with reverse transcription (RT–PCR), the αE-catenin protein was
undetectable in tissue and by immunoblot analysis, suggesting that
protein levels are very low, precluding a comparison of αN-catenin
and αE-catenin protein performance. Nevertheless, the strong rescue
observed with αN-catenin indicates that α-catenins support adherens
junction function regardless of very different in vitro monomer–dimer
equilibria, as seen with αN-catenin and Drosophila α-Cat.
268
Enhanced dimerization of α-catenin proteins compromises
cell adhesion
We confirmed that homodimerization of αE-catenin could be enhanced
by removing the first 57 amino acids12 (αEcat157; Fig. 7b) and found
that removing the corresponding N terminus from Drosophila α-Cat
NATURE CELL BIOLOGY VOLUME 15 | NUMBER 3 | MARCH 2013
© 2013 Macmillan Publishers Limited. All rights reserved.
ARTICLES
a Ed::αCatΔVH1
Ed
HA
e
20
trl
d
Expression of Ed::αCatΔVH1 in follicle cells
::α
C
C
at
ΔV
H
::α
C
Arm
40
α -Cat mutant BCs
Ed
HA
αC
at
C
trl
::H
A
α -Cat
60
at
::
Arm
80
αC
HA
100
Ed
WT
Adult
Late pupa
Early pupa
Third instar larva
Second instar larva
First instar larva
Normal head
Weak head
Head defect
Dorsal hole
α -Cat mutant FE cells
Percentage of rescue
α -Cat mutant embryos
H
A
at
ΔV
H
1
c
Ed::αCatΔVH1 expression in embryos
1
b
Complete
WT
Incomplete
α-Cat
HA α-Cat CD2
No migration
Ed
αC
α -Cat
C
trl
a
::α t::
C HA
at
ΔV
H
1
HA
HA
α-Spec
HA α-Spec GFP
Figure 6 Recruitment of αCat1VH1 to adherens junctions in
an Ed::αCat1VH1 chimaera does not support adherens junction
integrity. (a) Schematic representation of the Ed::αCat1VH1 construct.
(b) Ed::αCat1VH1 (detected with anti-HA antibodies) co-localizes with
Arm in the epidermis of stage 16 wild-type (WT) and α-Cat1 mutant
embryos. Scale bars, 10 µm. (c) Ed::αCat1VH1 does not rescue α-Cat1
mutant embryos. Quantification of rescue performance of Ed::αCat1VH1.
Aligned dot plot showing the average (thick horizontal line), s.d.
(whiskers) and total range (triangles) of rescue activity of αCat::HA
(n = 72) and Ed::αCat1VH1 (n = 74) when expressed in α-Cat1 (n = 140)
zygotic mutant embryos. The red triangle indicates rescued animals that
hatch as adults. Data are presented as mean ± s.d. See Methods for
scoring criteria. (d) Ed::αCat1VH1 (detected with anti-HA and anti-α-Cat
antibodies) localizes to cell contacts in wild-type follicular epithelium
cells. Ed::αCat1VH1-expressing cells are negatively marked by CD2.
Ed::αCat1VH1 fails to rescue α-Cat mutant follicular epithelium cells.
α-Spec, α-Spectrin. Scale bars, 10 µm. (e) Quantification of rescue
performance of Ed::αCat1VH1 in the follicular epithelium (FE) and
border cell (BC) migration. Follicular epithelium cells: percentage of
follicular epithelium cells that show rescue of α-Cat mutant defects on
expression of αCat::HA (n = 44 clones with 2,713 cells) or Ed::αCat1VH1
(n = 15 clones with 817 cells). Data are presented as mean ± s.e.m.
Border cell migration: quantification of border cell migration rescue on
expression of αCat::HA (n = 18) or Ed::αCat1VH1 (n = 5). Border cell
clusters that contain 5 or more α-Cat mutant cells were examined. The
data are presented as the mean ± s.d.
(αCat164) and αN-catenin (αNcat156) shifted both proteins into
a homodimer configuration (Fig. 7b). The in vivo performance of
αEcat157 was not examined as αE-catenin itself showed little rescue
activity in Drosophila. The performance of αNcat156 and αCat164
was markedly reduced when compared with their corresponding fulllength proteins (Fig. 8a,c,d). αNcat156 protein levels were relatively
low in contrast to αN-catenin and showed an adherens-junctionassociated and enhanced cytoplasmic distribution (Supplementary
Fig. S8). αCat164 was prominently expressed and predominantly
cytoplasmic in both wild-type and α-Cat mutant cells (Fig. 8b).
Complex formation of αCat164 with Arm was robust but reduced
when compared with αCat::HA (Fig. 8e). Thus, when compared
with the respective full-length proteins, αNcat156 and αCat164
underperformed and showed poor localization to adherens junctions,
suggesting that α-catenins with a higher propensity to dimerize are less
active in cell adhesion.
DISCUSSION
The model that α-catenin acts as a physical linker between
cadherin and the actin cytoskeleton seems strongly supported by
the ability of cadherin–α-catenin chimaeras to substitute for the
cadherin–catenin complex, both in tissue culture assays16,44,45 and
during morphogenesis26,34 . The analysis of these fusion proteins
emphasizes that: α-catenin acts in the immediate sub-membranous
space, excluding an essential cytosolic function suggested by other
studies46 in the tissues we have examined (ref. 26 and this work).
Further, a dynamic interaction between α-catenin and the cadherin–βcatenin complex is not required to support normal adherens junctions.
Although recent examples suggest that β-catenin modulation can
contribute to the dynamic regulation of the cadherin–catenin complex
in certain situations47–49 , this is not a basic requirement of adherens
junction assembly and function. That cadherin–α-catenin chimaeras
can replace the endogenous complex suggests instead that much of
NATURE CELL BIOLOGY VOLUME 15 | NUMBER 3 | MARCH 2013
© 2013 Macmillan Publishers Limited. All rights reserved.
269
ARTICLES
a
c
350
Dimer
300
250
Monomer
200
200
α-Cat
150
150
Mr (K)
Mr (K)
250
Mr = 116K ± 2K
100
100
αEcat
50
50
αNcat
0
9
b
10
11
12
Elution volume (ml)
0
13
9
d
250
αNcatΔ56
α-CatΔ64
Mr = 169K ± 3K
150
Mr (K)
Mr (K)
200
100
14
250
200
150
10
11
12
13
Elution volume (ml)
100
αEcatΔ57
50
50
0
0
9
10
11
12
Elution volume (ml)
e
α-Cat (monomer)
α-Cat (dimer)
P
S
P
f
10
11
12
13
Elution volume (ml)
S
P
S
P
14
αCat-VH3–CTD
F-actin
F-actin
S
9
13
F-actin
S
P
S
P
Figure 7 Characterization of purified recombinant α-catenin proteins.
(a) Size-exclusion chromatography (SEC) elution profiles of Drosophila
α-Cat (green), mouse αE-catenin (αEcat, red) and mouse αN-catenin
(αNcat, blue). Lines represent the ultraviolet absorbance (arbitrary unit).
Relative molecular masses of α-catenin proteins in monomeric or multimeric
states are estimated by multi-angle light scattering (shown as dots):
α-Cat forms a monomer (relative molecular mass (Mr ) 133K ± 4K) and
dimer (Mr 239K ± 16K), αE-catenin forms a monomer (Mr 116K ± 9K)
and dimer (Mr 237K ± 10K), and αN-catenin forms a monomer (Mr
98K ± 15K). (b) N-terminal deletion mutants of α-Cat (αCat164),
αE-catenin (αEcat157) and αN-catenin (αNcat156) predominantly exist
as dimers with estimated Mr 165K ± 22K, 151K ± 7K and 181K ± 11K,
respectively. (c,d) Re-purification of previously isolated Drosophila α-Cat
monomer (c) and dimer (d) fractions by SEC. Arrows indicate the emergence
of a dimer population in the monomer fraction (c) and a monomer population
in the dimer fraction (d). (e) Sedimentation of Drosophila α-Cat from the
SEC monomer and dimer fractions alone and in the presence of F-actin
show that both monomer and dimer fractions pellet with F-actin. Note that
SEC analyses of these monomer and dimer fractions (c and d) indicate
the presence of monomer and dimer species in both fractions. The arrow
indicates the position of the actin monomer. (f) Sedimentation of the
αCat-VH3–CTD (amino acids 659–917) alone and in the presence of F-actin
show F-actin binding of αCat-VH3–CTD. The arrow indicates the position of
the actin monomer.
the regulation of cadherin–catenin complex function takes place at the
interface between α-catenin and the actin cytoskeleton. Physical linkage
of α-catenin to cadherin does not interfere with its function. This does
not imply, however, that α-catenin normally needs to be physically
linked to the cadherin–β-catenin complex to function. Indeed, the
estimated dissociation constant (Kd ) of the α-catenin–β-catenin
interaction (∼1 µM) is much weaker than the cadherin–β-catenin
interaction19,50 . The allosteric regulation model for α-catenin suggests
that an α-catenin homodimer modulates actin organization through
interference with the Arp2/3 complex at adherens junctions19,46,51 .
Homodimerization and β-catenin binding require the same binding
interface and are mutually exclusive12 . Cadherins can dimerize or
cluster52 and could therefore promote α-catenin dimerization even
in chimaeric proteins that lack the α-catenin dimerization domain (for
example, DEcad::αCat1VH1). Several membrane-bound regulators
of actin organization exist, including WASp (ref. 53), indicating
that α-catenin could retain its Arp2/3 regulating activity despite
its covalent linkage to cadherin. To gain further evidence into
the molecular mechanism of α-catenin function we investigated
whether α-catenin function at adherens junctions can be decoupled
270
NATURE CELL BIOLOGY VOLUME 15 | NUMBER 3 | MARCH 2013
© 2013 Macmillan Publishers Limited. All rights reserved.
ARTICLES
a
b
DEcad α-Spec GFP
α-Spec
DEcad
WT
αCatΔ64
αN-catenin
α -Cat
α-Cat
HA
HA α-Cat
α -Cat
DEcad α-Spec GFP
e
Lysate
Incomplete
2. αCatΔ64
Mr (K)
3. αE-catenin
135
64
1. αCat::HA
atΔ
Complete
t::H
A
α -Cat mutant BCs
αC
100
80
60
40
20
HA α-Spec GFP
α-Spec
HA
αC
a
α -Cat mutant FE cells
P < 0.05
∗
∗
∗
αCat::HA
αCatΔ64
No
Ab
No
Ab
IP
IP
4. αN-catenin
∗
1
2
3
d
5. αNcatΔ56
No migration
5
4
C
trl
Percentage of rescue
c
αCatΔ64
α-Spec
DEcad
C
trl
αNcatΔ56
α -Cat
1
2
3
4
IP: HA; IB: Arm
f
α -Cat mutant embryos
α-catenin dimer
α-catenin
monomer
∗
Adult
Late pupa
Early pupa
Third instar larva
Second instar larva
First instar larva
Normal head
Weak head
Head defect
Dorsal hole
95
5
∗
∗
1. αCat::HA
?
Cadherin
2. αCatΔ64
∗
∗
F-actin
3. αE-catenin
Several actinbinding proteins
4. αN-catenin
2
3
4
5
p120
β-catenin/Arm
C
trl
5. αNcatΔ56
1
Figure 8 Monomeric α-catenin supports adherens junction integrity. (a) Face
on (top rows) and side views (bottom rows) of α-Cat mutant follicle cells
expressing αN-catenin or αNcat156 are labelled with DEcad, α-Spectrin
(α-Spec) and GFP to mark mutant cells. (b) Face on (top rows) and side views
(bottom rows) of wild-type (WT) or α-Cat mutant follicle cells expressing
αCat164 are labelled for HA and α-Cat or α-Spectrin. Scale bars, 10 µm.
(c) Quantification of rescue performance of αE-catenin, αN-catenin and
αCat164 in the follicular epithelium (FE) and border cell (BC) migration.
Follicular epithelium cells: percentage of follicular epithelium cells that
show rescue of α-Cat mutant defects on expression of αCat::HA (n = 44
clones with 2,713 cells), αCat164 (n = 29 clones with 1,240 cells),
αE-catenin (n = 20 clones with 650 cells), αN-catenin (n = 21 clones
with 1,054 cells) or αNcat156 (n = 27 clones with 1,281 cells). Data are
presented as mean ± s.e.m. Border cell migration: quantification of border
cell migration rescue on expression of αCat::HA (n = 18), αCat164 (n = 21),
αE-catenin (n = 14), αN-catenin (n = 13) or αNcat156 (n = 8). Border cell
clusters that contain 5 or more α-Cat mutant cells were examined. The data
are presented as the mean ± s.d. The red asterisks indicate a significant
difference from α-Cat1 mutant cells (P < 0.0001). (d) Quantification of
rescue performance of αCat::HA, αCat164, αE-catenin, αN-catenin and
αNcat156 in embryos. Aligned dot plot showing the average (thick horizontal
line), s.d. (whiskers) and total range (triangles) of rescue activity of αCat::HA
(n = 72), αCat164 (n = 192), αE-catenin (n = 134), αN-catenin (n = 124)
or αNcat156 (n = 158) when expressed in α-Cat1 (n = 140) zygotic
mutant embryos. The red triangle indicates rescued animals that hatch as
adults. Data are presented as mean ± s.d. See Methods for scoring criteria.
The red asterisks indicate a significant difference from α-Cat1 (n = 140)
mutant cells or embryos (P < 0.0001). (e) Co-immunoprecipitation (IP)
experiments using anti-HA antibodies show a reduced but robust physical
interaction between αCat164 and Arm in comparison with αCat::HA.
(f) Schematic representation illustrating a model for α-catenin dimer and
monomer fractions supported by the data presented in this study. α-catenin
dimers form a cytoplasmic pool. α-catenin is recruited to the adherens
junctions through the interaction with β-catenin/Arm, which stabilizes
α-catenin monomers, and which in turn facilitate interactions with the actin
cytoskeleton. Interactions with F-actin may be direct or mediated through
multiple actin-binding proteins. Uncropped images of blots/gels are shown
in Supplementary Fig. S9.
from β-catenin binding, whether monomeric α-catenin can support
adherens junctions and whether α-catenin dimerization promotes or
inhibits adherens junction function.
To address the first point we fused αCat1VH1 to either BazOD
(BazOD::αCat1VH1) or Baz (Baz::αCat1VH1), which recruited
αCat1VH1 to adherens junctions. αCat1VH1 does not support
adherens junctions alone, but did so when fused to DEcad.
BazOD::αCat1VH1 and Baz::αCat1VH1 showed weak biochemical
interactions with Arm and DEcad and little rescue of adherens junctions.
These findings suggest that the physical link between α-catenin and
β-catenin not only recruits α-catenin to adherens junctions, but
needs to persist for normal adherens junction function. Both the
NATURE CELL BIOLOGY VOLUME 15 | NUMBER 3 | MARCH 2013
© 2013 Macmillan Publishers Limited. All rights reserved.
271
ARTICLES
physical linkage and allosteric regulation models propose that α-catenin
interacts with the actin cytoskeleton at adherens junctions; however,
they differ on whether binding of α-catenin to β-catenin is required to
physically link cadherin and the actin cytoskeleton. Our data argue for
persistent physical linkage as a core requirement for α-catenin function.
We also found that localization of αCat1VH1 to adherens junctions
through fusion to Ed did not support adherens junction integrity, in
contrast to DEcad::αCat1VH1, suggesting that cadherins have distinct
properties that are important for α-catenin function.
Similar to C. elegans42 and D. discoideum43 α-catenin proteins,
αN-catenin was found to be monomeric in solution. αN-catenin can
functionally replace the Drosophila protein, which formed a large dimer
fraction in solution similar to αE-catenin. These results indicate that
monomeric α-catenin can support adherens junction function, and
that the in vitro monomer/dimer ratio may not correlate with the in
vivo function of α-catenins.
To address the relationship between α-catenin dimerization and
adherens junction function we tested the effects of enhanced α-catenin
dimerization. α-Cat or αCat1VH1 fusion to BazOD or Baz probably
causes enhanced multimerization, including dimerization of α-Cat
(refs 36,37). Although these chimaeras localize to adherens junctions
they show reduced interactions with Arm and perform poorly.
Dimerization was also enhanced by removing the N-terminal 56 or
64 amino acids from αN-catenin and α-Cat, respectively. Similar to
αEcat157 (ref. 12), αCat164 interacted with β-catenin/Arm. However,
these constructs performed poorly when compared with their respective
full-length proteins, suggesting that α-catenin dimers are inactive in
adhesion and may represent a cytoplasmic pool that forms a dynamic
equilibrium with α-catenin monomers. Monomers are recruited to
adherens junctions through their interaction with β-catenin/Arm,
which probably stabilizes monomeric α-catenin that links cadherin to
the actin cytoskeleton (Fig. 8f).
αE-catenin operates as a tension sensor and mechanotransducer
at adherens junctions, changing conformation in response to pulling
forces exerted by actomyosin16 . To achieve this, α-catenin needs to be
suspended between two anchor points, which could be cadherin–βcatenin and F-actin, consistent with the physical linkage model.
However, αE-catenin homodimers contain two actin-binding domains
and can bundle actin filaments11 , raising the possibility that actin
filament sliding as a result of myosin activity could apply tension
to α-catenin. As the allosteric regulation model poses that α-catenin
homodimers form preferentially at adherens junctions19,51 , tension
sensing could be restricted to adherens junctions even without
α-catenin linking cadherin to actin. However, actomyosin-generated
tension also applies to E-cadherin and depends on the presence of
αE-catenin54 , suggesting that at least part of the tension exerted
on αE-catenin occurs when it physically links cadherin to the actin
cytoskeleton. These data are complementary to our analysis of
Drosophila α-Cat. Although we and others have not been able to
document a quartenary complex of the cadherin–catenin complex with
F-actin, the data presented here are consistent with α-catenin physically
linking cadherin to the actin cytoskeleton as a core requirement of
α-catenin and cadherin–catenin complex function.
Our results on the function of different regions within Drosophila
α-Cat are in line with data from tissue culture studies on αEcatenin16,33,45,55 . The Arm and actin-binding regions at the N and
272
C termini of α-Cat, respectively, are essential for function. The central
region of α-Cat enhances adherens junction stability but does not
contribute to α-Cat recruitment to adherens junctions, which relies
only on the VH1-dependent binding to Arm. The central region
between the VH1 and VH3 domains includes multiple parts that
make partly independent contributions to α-Cat function, most likely
through interactions with other binding partners. If the central region is
activated by actomyosin pulling forces16,18 , then the tension-mediated
conformational change in α-Cat would be expected to facilitate
multiple interactions.
Although α-catenins can bind directly to F-actin in vitro (refs 11,19,
42,43 and this work), whether this occurs in vivo remains unresolved. It
is possible that interactions with F-actin are indirect and mediated
through F-actin-binding proteins. α-catenin organizes a complex
interface between cadherin and the actin cytoskeleton. Uncovering how
the multiple interactions between α-catenin and actin-binding proteins
such as vinculin, formin, afadin or EPLIN contribute to adherens
junction regulation during morphogenesis remains a major challenge
for future investigation.
METHODS
Methods and any associated references are available in the online
version of the paper.
Note: Supplementary Information is available in the online version of the paper
ACKNOWLEDGEMENTS
We are grateful to M. Takeichi (Riken, Center for Developmental Biology, Japan),
A. Nagafuchi (Nara Medical University, Japan), L. Nilson (McGill University,
Canada), P. Rorth (Institute of Molecular and Cell Biology, Singapore), G. Thomas
(Penn State University, USA), the Vienna Drosophila Research Center, the
Developmental Studies Hybridoma Bank, and the Bloomington Drosophila Stock
Center for supplying reagents. We thank C. Arana for technical assistance and
A. McKinley for help with live-cell imaging. We are grateful to J. Nelson and B.
Weis for valuable discussion, and T. Harris and D. Godt for critical reading of the
manuscript. This work was supported by an Ontario Graduate Scholarship (to R.D.)
and operating grants from the Canadian Cancer Society Research Institute (to M.I.
and U.T.).
AUTHOR CONTRIBUTIONS
R.D. made and analysed the α-Cat structure–function constructs. R.D. and R.S.
made and analysed the Baz–α-Cat chimaeras. R.S. made α-Cat::HA, which was
analysed by R.S. and R.D. R.S. made and analysed the DEcad–α-Cat and Ed–α-Cat
chimaeras. M.I. provided structural biology expertise and N.I. carried out the
biochemical experiments shown in Fig. 7. M.P. generated transgenic lines for
full-length Drosophila and mammalian α-catenin constructs, which were analysed
by R.D. and M.P. All authors contributed to experimental design and data
interpretation. U.T. wrote the paper.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at www.nature.com/doifinder/10.1038/ncb2685
Reprints and permissions information is available online at www.nature.com/reprints
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METHODS
DOI: 10.1038/ncb2685
METHODS
Generation of transgenes. All transgenic constructs are listed in Supplementary
Table S1. Deletions were made in the pBSACAT vector that contained the α-Cat
complementary DNA in which the stop codon was replaced with a NotI site for
subsequent subcloning into the transformation vector. Standard PCR-based cloning
strategies were used to generate individual deletions. Mutated α-Cat cDNAs were
excised from the pBSACAT vector using NotI and subcloned in the pUASP-attB
vector. The pUASP-attB vector also contains a coding sequence for a 2×-HA tag
for C-terminal tagging. Except α-Cat, DEcad, DEcad::αCat and DEcad::aCat1VH1,
all analysed constructs are tagged with 2×-HA. Transgenic animals were produced
by Genetic Services Inc, by using flies carrying an attP2 recombination site on the
third chromosome56 . Immunoblots showing the expression of transgenic proteins
in embryos are shown in Supplementary Fig. S9.
Drosophila genetics. All of the stocks used are in the white background. The
following fly stocks were used to drive expression of transgenes in a wild-type or
α-Cat mutant background: hs–Flp, Act > CD2 > Gal4; ref. 57, da–Gal4, act–Gal4,
hs–Gal4, ubi–α-Cat and α- Cat1 /TM3, Ser twi–Gal4 UAS–GFP (ref. 26).
The following recombinant lines were generated for embryonic rescue experiments: act–Gal4 da–Gal4 α-Cat1 /TM3, Ser twi–Gal4 UAS–GFP; UAS–XXX
α-Cat1 /TM3, Ser twi–Gal4 UAS-GFP—where XXX represents each of the transgenes
listed in Supplementary Table S1.
The following recombinant lines were used for MARCM analysis58 : hs–Flp
FRT40A; da–Gal4 UAS–mCD8::GFP α-Cat1 /TM6B; tub–Gal80 ubi–α-Cat FRT40A;
act–Gal4 UAS–XXX α-Cat1 /TM6b —where XXX represents each of the transgenes
listed in Supplementary Table S1.
The following line was used for expression of BazOD::αCat1VH1 in zygotic
mutants of bazXi106 : BazOD::α-Cat1VH1 da–Gal4.
Live-cell imaging experiments were performed on embryos of the following genotypes: ubi–α-Cat::GFP/ubi–α-Cat::GFP; ubi–DEcad::GFP shgR69 /
ubi–DEcad::GFP shgR69 .
For overexpression analysis in embryos we used da–Gal4, and in the follicular
epithelium we used hs–Flp;Act > CD2 > Gal4. The progeny of this cross were
heat-shocked for 20–30 min each on two consecutive days to induce expression of
FLP. FLP acts to remove CD2 and activate act–Gal4 in a clonal fashion57 .
Immunocytochemistry. For antibody staining embryos were heat-fixed as
previously described59 . Ovaries were fixed in 5% formaldehyde in phosphate
buffer (PB) for 13 min. For staining experiments involving phalloidin, ovaries
were fixed in 4% paraformaldehyde (PFA) in phosphate buffer for 20 min. The
primary antibodies used were: anti-HA (rat monoclonal 3F10, 1:600; Roche),
anti-DEcad (rat monoclonal Dcad2, 1:50; Developmental Studies Hybridoma
Bank (DSHB)), anti-Arm (mouse monoclonal N2 7A1, 1:100; DSHB), anti-αspectrin (mouse monoclonal 3A9, 1:20; DSHB); anti-α-spectrin (rabbit polyclonal,
1:1,000; ref. 60), anti-CD2 (mouse polyclonal, 1:250; Serotec), anti-α-Cat (guinea
pig polyclonal—gp121, 1:1,000; ref. 26), anti-GFP (mouse monoclonal JL-8,
1:400; Clontech), anti-GFP-AlexaFluor488 (mouse polyclonal—1:500; Invitrogen),
anti-αN-catenin (rat monoclonal NCAT2, 1:100; DSHB), and anti-Ed (rabbit
polyclonal, 1:1,000; ref. 61). Secondary antibodies were conjugated either to
Alexa Fluor-488, Alexa Fluor-555, Alexa Fluor-647 or to Cy3 (Invitrogen; Jackson
Immunoresearch Laboratories). F-actin was visualized with rhodamine–phalloidin
(1:40; Invitrogen). Stained embryos and ovaries were mounted in Vectashield
(Vector Labs). Fixed embryos and ovaries were imaged on an LSM510 confocal
microscope (Carl Zeiss) at room temperature using a ×40 objective lens. Captured
images were processed in Adobe Photoshop CS5 and/or Adobe Illustrator
CS5.
Immunoblotting and immunoprecipitation. For immunoblots, dissected
ovaries and dechorionated embryos were homogenized in 2× SDS (made from 4×
SDS—250 mM Tris at pH 6.8, 9.2% SDS, 40% glycerol and 0.02% bromophenol
blue) sample buffer. Protein from ovary lysates (12.5 µg) and protein from
embryonic lysates (40 µg) were loaded into each well and proteins were resolved
by SDS–PAGE.
For immunoprecipitation experiments, transgenic lines were crossed to da–Gal4.
An overnight collection of embryos was dechorionated and subsequently homogenized in chilled lysis buffer (50 mM Tris, at pH 7.6, 5% glycerol, 5 mM
EDTA, 100 mM NaCl, 50 mM NaF, 1% Triton X-100, 40 mM β-glycerophosphate,
and containing protease inhibitors (Roche)). Lysates were cleared of debris by
centrifugation. Anti-HA (3F10) antibody (8 µl) was added to 1 mg of lysate and
incubated for 1 h at 4 ◦ C under agitation. For immunoprecipitation experiments
using anti-Arm, 10 µl of anti-Arm was used to precipitate Arm complexes from
the prepared lysate. Protein G–Sepharose beads (∼30 µg; Sigma) were added and
incubated overnight at 4 ◦ C under agitation. Immunocomplexes were collected
by centrifugation and washed five times with ice-cold lysis buffer. Proteins were
solubilized with 2× SDS and separated by SDS–PAGE. For immunoprecipitation
experiments using anti-Arm, embryonic lysates were prepared as mentioned above.
Anti-Arm (10 µl; N27A1) was used to precipitate Arm from 1 mg of embryonic lysate.
The chemi-illuminescence intensity measurements were made using ImageJ for a
total of two immunoprecipitation experiments.
Proteins resolved by SDS–PAGE were transferred onto nitrocellulose membranes
(Amersham). Membranes were blocked in phosphate-buffered saline (PBS)
containing 5% milk powder and 0.05% Tween-20 for at least 1 h at 25 ◦ C.
Membranes were incubated overnight with primary antibodies at 4 ◦ C and then
with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room
temperature. After extensive washes in PBS containing 0.05% Tween-20, bands were
visualized using the ECL system (Biosciences). The following primary antibodies
were used: anti-HA (rat monoclonal 3F10, 1:1,000; Roche), anti-Arm (mouse
monoclonal N74A1, 1:1,000; DSHB), anti-α-Cat (guineapig polyclonal gp121,
1:2,500; ref. 26), anti-GFP (rabbit-polyclonal ab290, 1:1,000; Abcam), anti-GFP
(mouse-monoclonal JL-8, 1:500; Clontech), anti-β-tubulin (mouse monoclonal E7,
1:1,000; DSHB). HRP-coupled secondary antibodies (Jackson Immunoresearch)
were used at a dilution of 1:1,000.
Whole-animal and ovary rescue experiments. For evaluating the ability of
transgenes to rescue α-Cat1 mutant defects, transgenic insertions in the attP2 target
site were recombined with α-Cat1 and balanced over TM3, Ser twiGal4 UAS–GFP.
These flies were crossed to act–Gal4 da–Gal4 α-Cat1 /TM3, Ser twi–Gal4 UAS–GFP
and eggs were collected on apple juice agar plates at 25 ◦ C. A total of 100–300
fertilized non-GFP embryos were collected and allowed to develop at 25 ◦ C and
monitored daily. Those that did not hatch were collected and mounted in Hoyer’s
medium and lactic acid (1:1 ratio) for examination of the cuticle. Hatched larvae
were subjected to lethality counts at each of the subsequent stages of Drosophila
development.
For quantification of rescue experiments in the embryos and throughout
development, a specific score was allocated for the level of rescue from the embryonic
stage to adulthood for each animal in a collection of n eggs26 . The following scoring
numbers were used for calculating the level of rescue—(0) embryonic lethal with
severe head defects (this phenotype is exhibited by most α-Cat1 zygotic mutants26 );
(1) embryonic lethal with weak head defects; (2) embryonic lethal with normal
head; (3) first instar larvae; (4) second instar larvae; (5) third instar larvae; (6) early
pupa; (7) late pupa; (8) adult. The data were then presented as the mean ± standard
deviation (s.d.).
For quantifying rescue performance in follicular epithelium cells, the percentage
of rescued cells per follicle cell clone was calculated for n number of follicular
epithelium cell clones. The data were then plotted as the mean ± standard error of
the mean (s.e.m.). For quantification of border cell migration, only those border
cell clusters with at least five GFP-positive (that is, α-Cat mutant) cells were
analysed. Stage 10b egg chambers were scored as follows: (0) no migration, (1)
incomplete migration, (2) complete migration. The data were then presented as
mean ± s.d. Statistical significance was assessed using Student’s t -test in Prism
software (GraphPad).
Protein expression and purification. The αE-catenin, αN-catenin and Dαcatenin cDNA were amplified by PCR and subcloned into the pET-28a, pGEX-4T1
and pET-SUMO vectors, respectively. The mouse αE- and αN-catenin cDNA were
gifts from A. Nagafuchi, Kumamoto University, Japan and M. Takeichi, Riken, Japan.
His–αE-catenin, GST–αN-catenin and His–SUMO–Dα-catenin were individually
expressed in a BL21-CodonPlus (Stratagene) strain overnight at 15 ◦ C. After
collection by centrifugation, cells were lysed by sonication on ice and proteins were
isolated either by using the Ni2+ -chelating resin (Qiagen) or glutathione–Sepharose
resin (GE Healthcare). Affinity tags were cleaved by either thrombin or SUMO
protease (Ulp-1) and the cleaved protein was further purified by size-exclusion
chromatography (SEC) using Superdex 200 (GE Healthcare) in the running buffer
(50 mM Tris–HCl, at pH 8.0, 300 mM NaCl, 10 mM β-mercaptoethanol and 1 mM
TCEP-HCl).
Light scattering. Multi-angle light scattering measurements were done in-line
with SEC by using a three-angle (45◦ , 90◦ and 135◦ ) miniDawn light-scattering
instrument and an Optilab rEX differential refractometer (Wyatt Technologies).
Molecular mass was calculated by using the ASTRA software (Wyatt Technologies).
NATURE CELL BIOLOGY
© 2013 Macmillan Publishers Limited. All rights reserved.
METHODS
DOI: 10.1038/ncb2685
Actin-pelleting assays. Monomeric rabbit skeletal muscle actin (Cytoskeleton)
was polymerized in the polymerization buffer (5 mM Tris–HCl, at pH 8.0,
50 mM KCl, 2 mM MgCl2 , 1 mM ATP, 0.2 mM CaCl2 and 0.5 mM dithiothreitol) for 1 h. Equal volumes of F-actin (10 M) and 10 M α-catenin sample were mixed in the binding buffer (20 mM Tris–HCl, at pH 8.0, 50 mM
KCl, 75 mM NaCl, 2 mM MgCl2 , 1 mM ATP, 0.2 mM CaCl2 , 0.5 mM dithiothreitol and 0.5 mM TCEP) for 1 h at 277 K. F-actin with bound protein
samples were co-sedimented by centrifugation at 100,000g for 20 min. Supernatant and pellet fractions were analysed by SDS–PAGE with Coomassie blue
stain.
Live-cell imaging and post acquisition image analysis. Live-cell imaging of
dechorionated embryos undergoing dorsal closure was performed on a spinningdisc confocal system (Quorum Technologies) at room temperature using a ×63 plan
Apochromat NA 1.4 objective (Carl Zeiss). Fluorescence intensity measurements
were made using ImageJ. Epithelial cells that were one cell length away from the
leading edge were used to measure the intensity of αCat::GFP and DEcad::GFP at
the plasma membrane. Approximately seven different cell–cell contacts per embryo
were analysed in five different embryos.
56. Groth, A., Fish, M., Nusse, R. & Calos, M. Construction of transgenic
Drosophila by using the site-specific integrase from phage phiC31. Genetics 166,
1775–1782 (2004).
57. Pignoni, F. & Zipursky, S. L. Induction of Drosophila eye development by
decapentaplegic. Development 124, 271–278 (1997).
58. Lee, T. & Luo, L. Mosaic analysis with a repressible cell marker for studies of gene
function in neuronal morphogenesis. Neuron 22, 451–61 (1999).
59. Tepass, U. Crumbs, a component of the apical membrane, is required for
zonula adherens formation in primary epithelia of Drosophila. Dev. Biol. 177,
217–225 (1996).
60. Byers, T.J., Dubreuil, R., Branton, D., Kiehart, D. P. & Goldstein, L. S. Drosophila
spectrin. II. Conserved features of the α-subunit are revealed by analysis of cDNA
clones and fusion proteins. J. Cell Biol.. 105, 2103–2110 (1987).
61. Laplante, C. & Nilson, L. A. Differential expression of the adhesion molecule
Echinoid drives epithelial morphogenesis in Drosophila. Development 2,
3255–3264 (2006).
NATURE CELL BIOLOGY
© 2013 Macmillan Publishers Limited. All rights reserved.
S U P P L E M E N TA R Y I N F O R M AT I O N
DOI: 10.1038/ncb2685
α-Cat
HA α-Cat
αCat∆CTD
αCat∆VH3
αCat∆VH3-CTD
αCat∆VH1
αCat::HA
HA
Desai, Fig. S1
Figure S1 The VH1 domain is required for recruitment of a-Cat to AJs. Clonal expression of aCat::HA, aCat∆VH1, aCat∆VH3-CTD, aCat∆VH3, or aCat∆CTD
in the FE. Cells were labeled for HA to detect transgenic constructs and α-Cat. Scale bars, 10µm.
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1
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S U P P L E M E N TA R Y I N F O R M AT I O N
Arm
GFP
DEcad Arm GFP
αCat∆CTD
αCat∆VH3
αCat∆VH3-CTD
αCat∆VH1
αCat::HA
DEcad
Desai, Fig. S2
Figure S2 DEcad and Arm localization to AJs is not supported by α-Cat proteins lacking VH1 or VH3. α-Cat mutant FE cell clones (labeled with GFP)
expressing aCat::HA, aCat∆VH1, aCat∆VH3-CTD, aCat∆VH3, or aCat∆CTD are marked for DEcad and Arm. Only aCat::HA and aCat∆CTD support normal
DEcad and Arm localization. Scale bar, 10µm.
2
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S U P P L E M E N TA R Y I N F O R M AT I O N
A
2 hours
4 hours
DEcad::GFP
8 hours
B
10 hours
Complete migration
No migration
αCatN::BazOD::C
αCat::HA
Slbo Arm GFP
HA α-Spec GFP
Slow/Incomplete migration
αΝcat
αCat∆VH2
HA α-Spec GFP
Slbo αN-cat GFP
αCat∆CTD
αCat∆64
HA α-spec GFP
HA α-spec GFP
Figure S3 DEcad turn-over and α-Cat function in BCs. (A) Follicles
of the genotype hs-Gal4, UAS-DEcad::GFP where subjected to a
brief heatshock to express DEcad::GFP. The distribution and levels
of DEcad::GFP was monitored 2, 4, 8 and 10 hours after heat shock.
Note that DEcad::GFP levels steadily decline in the BC cluster while
remaining high in the FE, suggesting a higher turn-over of DEcad in
Desai, Fig. S3
BC during migration compared to the cells of the FE. Scale bar, 10µm.
(B) Example of a-Cat mutant border cell clusters expressing various
constructs as indicated on the individual panels. a-Cat mutant cells are
positively marked with GFP. Other markers are HA to detect the α-Cat
constructs, Slbo to label BCs, α-Spectrin (α-Spec), and αN-catenin
(αNcat). Scale bar, 10µm.
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S U P P L E M E N TA R Y I N F O R M AT I O N
Arm
GFP
DEcad Arm GFP
αCat∆VH2-C
αCat∆VH2-N
αCat∆VH2
αCat∆VIN
DEcad
Desai, Fig. S4
Figure S4 DEcad and Arm proteins are reduced at AJs in α-Cat mutant FE cells expressing αCatΔVH2. a-Cat mutant follicle cell clones expressing aCat∆VIN,
aCat∆VH2, aCat∆VH2N, a-Cat∆VH2C are labeled for DEcad and Arm. Mutant cells are marked by GFP. Scale bar, 10µm.
4
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S U P P L E M E N TA R Y I N F O R M AT I O N
Arm
DEcad Arm GFP
DEcad::αCatVH3-CTD
DEcad::αCat∆VH1
DEcad::αCat
DEcad
DEcad
Desai, Fig. S5
Figure S5 Characterization of DEcad/α-Cat fusion proteins in FE cells. a-Cat mutant follicle cell clones expressing DEcad, DEcad::aCat, DEcad::aCat∆VH1,
or DEcad::aCat-VH3-CTD are labeled with DEcad, Arm and GFP, which marks mutant cells. Scale bar, 10µm.
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5
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S U P P L E M E N TA R Y I N F O R M AT I O N
Arm
GFP
DEcad Arm GFP
Baz::αCat
αCatN::BazOD::C
Baz::αCat∆VH1
BazOD::αCat∆VH1
DEcad
Desai, Fig. S6
Figure S6 Characterization of Baz/α-Cat fusion proteins in FE cells. a-Cat mutant follicle cell clones expressing BazOD::aCat∆VH1, Baz::aCat∆VH1,
aCatN::BazOD::C, or Baz::aCat are labeled with DEcad, Arm and GFP, which marks mutant cells. Scale bar, 10µm.
6
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S U P P L E M E N TA R Y I N F O R M AT I O N
A
ate
ds
s
Lys
ea
l
ad
a
+b
t
b
o
Be
T
A
60000
50000
260 kDa
40000
αCat::GFP
135 kDa
30000
α-Cat
20000
95 kDa
10000
IP: Anti-Arm
72 kDa
0
B
ubi-αCat::GFP
P
αC
ubi-DEcad::GFP, shg/shg
t
Ca
GF
::
at
IB: Anti-α-Cat
α-
1.75
1.50
1.25
1.00
0.75
0.50
/s
FP
,s
C
i-α
ub
i-D
Ec
ad
ub
FP
G
::
at
hg
0.00
::G
DEcad
GFP/R69
DEca
ad GF
P/R6
/R69
R 9
hg
0.25
Ubi-acatGFP
acat
aca
catGFP
Desai, Fig. S7
Figure S7 Drosophila α-Cat shows 1:1:1 ratio with DEcad and Arm at
AJs. Current reagents did not allow us to replace endogenous α-Cat with
αCat::GFP. We therefore expressed αCat::GFP at approximately endogenous
levels in the presence of normal levels of endogenous α-Cat and confirmed
through Co-IP with Arm that approximately half of the α-Cat bound to Arm is
αCat::GFP. We then compared fluorescent intensities of αCat::GFP at AJs in
the epidermis of live embryos with those of DEcad::GFP, where DEcad::GFP
is the only cadherin present. As expected, fluorescent intensities for
αCat::GFP were approximately half of those found for DEcad::GFP. (A)
Embryos expressing αCat::GFP and endogenous α-Cat at approximately
equal levels (genotype: ubi-αCat::GFP/ubi-αCat::GFP). Co-IP using anti-Arm
shows that Arm form a complex with a-Cat and aCat::GFP. Quantification of
band intensity measurements (arbitrary units; n=2) reveals that Arm binds
to a-Cat and aCat::GFP in a ~1:1 ratio. (B) Live imaging of ubi-αCat::GFP/
ubi-αCat::GFP and ubi-DEcad::GFP/ubi-DEcad::GFP; shgR69/shgR69 embryos
at stage 15. Quantification of fluorescence intensities revealed that the total
amount of aCat::GFP (n=34) found at the membrane is approximately half of
the total amount of DEcad::GFP (n=33) as expected for a 1:1 ratio of α-Cat
to DEcad given that half of the α-Cat but all of the DEcad molecules are
labeled with GFP. Arbitrary units; error bars show s.d.
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S U P P L E M E N TA R Y I N F O R M AT I O N
A
αN-catenin
Expression of transgenes in wildtype embryos
Arm
α-Cat
mαNcat Arm α-Cat
mαN-cat
Arm
α-Cat
mαNcat Arm α-Cat
αN-cat∆56
mαN-cat
Desai, Fig. S8
Figure S8 Expression of mouse αN-catenin and αNcatΔ56 in Drosophila epithelia. Stage 14 embryos expressing αN-catenin or αNcatΔ56. Shown is part of
the dorsal epidermis and the amnioserosa, which can be recognized by their large cell profiles. Embryos are stained of mouse αN-catenin, Arm, and α-Cat.
Scale bars, 10μm.
8
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S U P P L E M E N TA R Y I N F O R M AT I O N
DE
ca
d::
αC
DE
at
ca
d::
αC
at∆
VH
1
DE
ca
d::
αC
atV
H3
VH
2
at∆
VH
2-N
αC
at∆
VH
2-C
αC
at∆
VH
3-C
αC
at∆
TD
VH
3
αC
at∆
CT
D
VIN
αC
αC
at∆
VH
at∆
αC
at∆
αC
135kDa
αC
at:
:H
A
1
Fig. S9A: Immunoblots showing transgenic proteins
expressed in embryonic lysates
260kDa
95kDa
135kDa
72kDa
Anti-HA
95kDa
Anti-αCat
52kDa
Anti-HA
52kDa
β-tubulin
β-tubulin
Wild-type embryo lysates (30µg)
C
260kDa
1
VH
Ed
αC
at∆
at∆
αC
z::
Ba
Ba
z::
αC
αC
at∆
at
64
t
VH
1
D::
Ca
Ba
z::α
H1
azO
::B
t∆V
atN
αC
Ba
z::α
Ca
αC
D::
zO
Ba
αC
at:
:HA
at∆
VH
1
Wild-type embryo
lysates (30µg)
260kDa
135kDa
135kDa
95kDa
95kDa
Anti-HA
Anti-HA
52kDa
β-tubulin
52kDa
β-tubulin
Wild-type
ovary lysates (30µg)
Wild-type embryo lysates (30µg)
Figure S9 Immunoblots showing expression of transgenic proteins used in this study and uncropped images of blots shown in Figs 1, 2, 4, 5, and 8.
(A) Immunoblots showing expression of transgenic proteins used in this study. Arrows point to protein bands of predicted size for each construct. (B-E)
Uncropped images of blots shown in Figs 1, 2, 4, 5, and 8.
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S U P P L E M E N TA R Y I N F O R M AT I O N
135 kDa
135 kDa
αC
at
:
αC :HA
at
∆V
H
1
250 kDa
135 kDa
135 kDa
95 kDa
95 kDa
72 kDa
72 kDa
250 kDa
135 kDa
95 kDa
95 kDa
250 kDa
αC
at
::
αC HA
at
∆V
H
αC
1
at
∆V
IN
H
A
+
B
αC ea
at ds
∆V
αC H3
-C
at
TD
∆V
αC H3
at
∆C
TD
250 kDa
250 kDa
αC
at
:
αC :HA
at
∆V
H
1
αC
at
:
αC :HA
at
∆V
H
1
αC
at
:
αC :HA
at
∆V
H
1
Fig. S9B: Uncropped images of immunoblots shown
in Figs 1 and 2
72 kDa
95 kDa
72 kDa
72 kDa
55 kDa
55 kDa
55 kDa
55 kDa
55 kDa
IP: anti-HA (20%)
IB: anti-HA
IP: anti-HA
IB: anti-Arm
250 kDa
135 kDa
95 kDa
250 kDa
TD
at
∆C
3
3-
H
αC
H
∆V
at
∆V
at
αC
αC
at
::
αC HA
at
∆V
H
αC
1
at
∆V
IN
αC
at
::
αC HA
at
∆V
H
αC
1
at
∆V
IN
αC
at
∆V
αC H3
-C
at
TD
∆V
αC H3
at
∆C
TD
250 kDa
αC
αC
at
::
αC HA
at
∆V
H
αC
1
at
∆V
IN
αC
at
∆V
αC H3
-C
at
TD
∆V
αC H3
at
∆C
TD
C
Input (40 µg)
IB: anti-Arm
TD
IP: No Ab
IB: anti-Arm
IP: anti-HA
IB: anti-Arm
135 kDa
135 kDa
95 kDa
95 kDa
72 kDa
72 kDa
72 kDa
55 kDa
55 kDa
55 kDa
C
N
2-
2-
H
H
∆V
at
αC
H
at
∆V
at
αC
αC
∆V
2
C
N
2H
H
at
∆V
2
H
∆V
∆V
at
αC
H
2-
at
αC
∆V
at
αC
2-
C
N
2-
2
H
H
∆V
at
αC
at
C
α
A
H
∆V
ad
Be
2H
+
∆V
at
αC
s
C
N
2H
2
H
∆V
at
αC
∆V
at
αC
IP: anti-HA (20%)
IB: anti-HA
IP: anti-HA (20%)
IB: anti-HA
αC
Input (40 µg)
IB: anti-Arm
IP: No Ab
IB: anti-Arm
250 kDa
250 kDa
250 kDa
135 kDa
135 kDa
135 kDa
95 kDa
72 kDa
95 kDa
95 kDa
72 kDa
72 kDa
55 kDa
55 kDa
55 kDa
35 kDa
35 kDa
35 kDa
IP: anti-HA
IB: anti-Arm
IP: No Ab
IB: anti-Arm
Input (40 µg)
IB: anti-Arm
IP: anti-HA (20%)
IB: anti-HA
Figure S9 continued
10 WWW.NATURE.COM/NATURECELLBIOLOGY
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S U P P L E M E N TA R Y I N F O R M AT I O N
C
at
∆
:: α
:: α
Ba
zO
D
IP-anti-HA
Ba
zO
No Ab
D
C
Input (40µg)
VH
1
C
+B at∆
V
az H
G 1
FP
D
:: α
D
Ba
zO
Ba
zO
:: α
at
∆
VH
1
C
+B at∆
Ba
az VH
zO
G 1
FP
D
:: α
Ba
C
at
zO
∆V
D
H
:: α
1
C
Ba
at
+
zO B ∆V
D az H
:: α G 1
Ba
C FP
at
zO
∆V
D
H
:: α
1
C
H
A+
+B at∆
be
az VH
ad
G 1
s
FP
Fig. S9C: Uncropped images of immunoblots shown in Fig. 4
Input (40µg)
250 kDa
250 kDa
135 kDa
135 kDa
95 kDa
95 kDa
72 kDa
72 kDa
IP-anti-GFP
No Ab
IP-anti-GFP
H
1
C
+B at∆
V
az H
G 1
FP
∆V
at
:: α
D
zO
Ba
Ba
zO
D
:: α
C
:: α
D
zO
Ba
Ba
zO
D
:: α
:: α
D
zO
No Ab
C
at
at
C
:: α
D
zO
Ba
Ba
Input (40µg)
∆V
H
1
C
a
zO +B t∆V
D azG H
1
:: α
Ba
C FP
at
zO
∆V
D
H
:: α
1
C
G
FP
+B at∆
V
+b
az H
ea
G 1
FP
ds
IP-anti-HA (20%)
IB: anti-HA
∆V
H
1
C
+B at∆
az VH
Ba
G 1
FP
zO
D
:
:α
Ba
C
zO
at
∆V
D
:: α
H
1
C
Ba
a
zO +B t∆V
D azG H
1
:: α
Ba
C FP
at
zO
∆V
D
H
:: α
1
C
G
FP
+B at∆
V
+b
az H
ea
G 1
FP
ds
IP-anti-HA
IB: anti-GFP
Ba
IP-anti-HA
IB: anti-GFP
Input (40µg)
250 kDa
250 kDa
250 kDa
135 kDa
135 kDa
135 kDa
95 kDa
95 kDa
95 kDa
72 kDa
72 kDa
72 kDa
IP-anti-GFP
IP-anti-GFP
IB: anti-GFP
IB: anti-HA
IB: anti-HA
Figure S9 continued
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11
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S U P P L E M E N TA R Y I N F O R M AT I O N
Ba
1
zO
D
Ba ::α
C
z:
:α at∆
VH
C
at
αC
∆V 1
at
H
N
1
::B
Ba
az
O
z:
D
:α
::C
C
H
A+ at
be
ad
s
A
VH
H
at
∆
at
::
αC
αC
at
∆
αC
αC
at
::
H
A
VH
Ba
1
zO
D
:
:
αC
αC
at
at
∆V
N
::B
H
Ba
1
az
O
z:
D
:α
::C
Ba Ca
t
z:
:α
C
a
αC
t∆
VH
at
∆6
1
4
H
A+
be
ad
s
Fig. S9D: Uncropped images of immunoblots shown in Fig. 5
250 kDa
250 kDa
135 kDa
135 kDa
Arm
95 kDa
95 kDa
72 kDa
72 kDa
55 kDa
IP-anti-HA
IB: anti-GFP
H
O
D
1
H
N
at
C
Ba
z:
at
:α
::B
az
∆V
at
C
αC
D
Ba
z:
:α
H
∆V
Ba
zO
at
A
αC
::H
at
αC
::α
1
C
at
1
H
∆V
∆6
αC
at
:α
z:
Ba
4
at
at
C
C
:α
z:
Ba
∆V
1
::C
H
D
∆V
O
at
az
C
N
D
at
αC
Ba
::B
::α
1
H
∆V
at
αC
zO
A
::H
at
αC
::C
1
IP: anti-HA
IB: anti-Arm
250 kDa
250 kDa
135 kDa
Arm
95 kDa
135 kDa
72 kDa
95 kDa
55 kDa
72 kDa
Input (40µg)
IB: anti-GFP
1
::C
1
D
O
Ba
z:
:α
C
at
az
::B
N
C
αC
:α
z:
D
Ba
zO
at
C
::α
1
H
∆V
Ba
at
A
αC
::H
at
αC
135 kDa
1
∆V
H
αC
at
VH
C
4
VH
at
∆6
at
at
C
:α
z:
Ba
250 kDa
at
D
Ba
z:
:α
::B
N
αC
at
D
az
C
::α
1
H
Ba
zO
∆V
at
A
αC
::H
at
αC
O
at
∆V
::C
H
1
Input (40 µg)
IB: anti-Arm
250 kDa
95 kDa
72 kDa
135 kDa
95 kDa
55 kDa
72 kDa
IP: anti-HA (No Ab)
IB: anti-Arm
No Ab
IB: anti-GFP
Figure S9 continued
12 WWW.NATURE.COM/NATURECELLBIOLOGY
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S U P P L E M E N TA R Y I N F O R M AT I O N
αC
at
::H
A
αC
at
∆6
4
Fig. S9E: Uncropped image of immunoblot shown in Fig. 8
Input (40µg)
αCat::HA
No
Ab IP: anti-HA
αCat∆64
No
IP:
Ab anti-HA
250 kDa
135 kDa
Arm
95 kDa
72 kDa
IP: anti-HA
IB: anti-Arm
Figure S9 continued
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S U P P L E M E N TA R Y I N F O R M AT I O N
Table S1 Construct data, rescue activity and protein distribution.
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