1 Department of Biology, University of North Carolina at Chapel Hill, Chapel
Hill, NC 27599 USA
2 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel
Hill, Chapel Hill, NC 27599, USA
* Author for correspondence (e-mail: peifer{at}unc.edu)
Accepted 24 August 2005
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SUMMARY |
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Key words: Cadherin, Morphogenesis, RhoGEF2, Shotgun
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Introduction |
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In addition to these core AJ components, regulatory proteins modulate both
AJ stability and connections to the cytoskeleton (reviewed by
Gumbiner, 2000). Identifying
how these regulators modify AJs during development is crucial to understanding
morphogenesis. Studies in cultured mammalian cells and other systems
identified many candidate AJ regulators, including the catenin p120 and the
small GTPase Rho (mammalian RhoA or Drosophila Rho1).
p120 binds the juxtamembrane region of cadherins (reviewed by
Anastasiadis and Reynolds,
2000). Initially, the regulatory relationship between p120 and AJs
was unclear. Overexpression of mutant E-cadherins lacking the juxtamembrane
domain in different mammalian cell types gave opposing results suggesting that
p120 either promotes (Yap et al.,
1998
) or downregulates adhesion
(Ozawa and Kemler, 1998
).
siRNA knockdown of p120 in mammalian cells clarified this, showing that p120
promotes AJ stability by blocking E-cadherin endocytosis
(Davis et al., 2003
;
Xiao et al., 2003
).
In invertebrates, p120 also promotes adhesion but may be dispensable for
viability. In Caenorhabditis elegans, p120/jac-1 RNAi enhances the
hmp-1/-catenin phenotype, but jac-1 RNAi alone does
not disrupt morphogenesis (Pettitt et al.,
2003
). Similarly, loss of p120 enhances the phenotype of
Drosophila E-Cadherin (DE-Cad; shotgun FlyBase)
mutants but loss of p120 alone (Myster et
al., 2003
) or expression of p120 RNAi transgenes
(Pacquelet et al., 2003
) do
not affect viability or cell adhesion. However, injection of p120
double-stranded RNA (dsRNA) in embryos was reported to disrupt morphogenesis
(Magie et al., 2002
). This
suggested that rapid depletion of p120 might have more severe consequences
than chronic depletion.
In mammalian cells p120 also may function outside of AJs
(Anastasiadis and Reynolds,
2000). In the cytoplasm, p120 can negatively regulate RhoA. Rho
regulates many cellular processes, including actin organization, cell
migration and cell polarity (reviewed by
Etienne-Manneville and Hall,
2002
). siRNA knockdown of mammalian p120 increases RhoA activity
and promotes stress fiber formation
(Shibata et al., 2004
).
Conversely, p120 overexpression causes fibroblasts to lose stress fibers
(Anastasiadis et al., 2000
;
Noren et al., 2000
) and
contractility (Grosheva et al.,
2001
), both RhoA-dependent processes
(Ridley and Hall, 1992
;
Chrzanowska-Wodnicka and Burridge,
1996
).
Recently, the relationship between p120 and Rho has begun to be tested in
vivo, but the results do not yield a consistent mechanistic picture. Embryonic
defects caused by knockdown of p120 family members in Xenopus can be
partially rescued by both dominant-negative
(Fang et al., 2004) and
wild-type RhoA (Ciesiolka et al.,
2004
). These contrasting results are consistent with p120
activating or repressing RhoA, respectively. In Drosophila, p120
preferentially binds Rho1-GDP and regulates Rho1 localization. Overexpression
of p120 enhances the Rho1 phenotype
(Magie et al., 2002
),
suggesting that fly p120 negatively regulates Rho1. Thus p120 may regulate
morphogenesis by regulating AJs and/or Rho.
Additionally, Rho itself regulates AJ stability. Blocking RhoA function in
keratinocytes prevents the formation of stable AJs
(Braga et al., 1997). In
Drosophila, both Rho1 loss of function and expression of
dominant-negative Rho1 during embryogenesis alter DE-Cad localization
(Magie et al., 2002
;
Bloor and Kiehart, 2002
).
However, the regulation of AJs by Rho is probably complex, as manipulation of
different RhoA effectors can promote or decrease AJ stability in mammalian
cells (Sahai and Marshall,
2002
; Vaezi et al.,
2002
). Additionally, AJs may regulate Rho activity, as
cadherin-cadherin engagement can either activate
(Charrasse et al., 2002
;
Nelson et al., 2004
) or
inhibit (Noren et al., 2001
;
Noren et al., 2003
) RhoA
activity in vitro.
Work in cultured cells supports roles for both p120 and Rho as AJ regulators, but the interactions among p120, Rho and AJs are complex. Ultimately, we want to understand how Rho and p120 regulate AJs during the intricate events of embryonic morphogenesis. One key question is whether Rho and p120 act together in this process. Here we use Drosophila to investigate this.
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Materials and methods |
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Fly stocks
Mutations are described at
http://flybase.bio.indiana.edu/.
Rho1rev220 and Rho11B
(Magie et al., 2002; Magie et
al., 2005) were from S. Parkhurst (Fred Hutchison). Homozygous Rho1
mutants were identified using a Kr::gfp balancer
(Casso et al., 1999
); controls
were Kr::gfp positive siblings. For Rho1 localization in p120,
histone::gfp was the control. Otherwise it was yellow white.
Recombinant genotypes were confirmed by failure to complement an independently
derived allele; p120308 was confirmed by PCR. Cuticle
preparations were as in Wieschaus and Nüsslein-Volhard
(Wieschaus and Nüsslein-Volhard,
1986
). Unless noted, experiments were done at 25°C. Live
imaging utilized wild-type or p120308 mutants expressing
moesin::GFP. Follicle cell clones: heat-shock-FLP/+;
FRT42DshgR69/FRT42D gfp females were heat-shocked for 1
hour at 37°C for 2 consecutive days before dissection.
Immunofluorescence
Ovaries were treated as in Magie et al.
(Magie et al., 2002). Embryos
were fixed in 1:1 PBS+3.7% formaldehyde:heptane for 20 minutes, except for
Rho1 staining, which was as in Padash-Barmchi et al.
(Padash-Barmchi et al., 2005
).
Embryos were methanol-devitellinized (or hand-devitellinized for phalloidin),
blocked and stained in PBS/1% goat serum/0.1%Triton-X-100. Antibodies:
anti-Rho1p1D9 (1:50), anti-ßPS1 integrin (1:3), anti DE-Cad2 (1:200),
anti-ArmN2 (1:200; all Developmental Studies Hybridoma Bank), anti-DRhoGEF2
(1:500) (Rogers et al., 2004
),
anti-phosphotyrosine (1:1000, Upstate Biotechnology). Alexa-phalloidin was
used at 1:1000; secondary antibodies were Alexas 488, 568 and 647 (Molecular
Probes). Embryos were mounted in Aqua-Polymount (Polysciences). Fixed samples
were imaged using a Zeiss LSM510 confocal microscope and LSM software. Live
imaging used a Perkin-Elmer Ultraview spinning-disc confocal, an ORCA-ER
digital camera (Hamamatsu), and Metamorph software. To analyze dorsal closure
timing, we began analysis of all movies when the maximum separation between
the leading edges was 52 µm (as measured in Metamorph), and ended analysis
when the leading edges met along their entire length. All images were acquired
at 40x. Adobe Photoshop 7.0 was used to adjust brightness and contrast.
When comparing wild-type and mutants, images were adjusted identically.
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Results |
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We next examined whether more subtle changes in cell behavior were revealed
by imaging dorsal closure in living embryos expressing a GFP-tagged
F-actin-binding fragment of Moesin that highlights the cytoskeleton
(Moesin::GFP) (Edwards et al.,
1997). Once again, we saw no gross defects in cell shape in
p120 mutants. However, loss of p120 slowed the rate of dorsal
closure. On average, wild-type embryos completed dorsal closure within 75
minutes (Fig. 1O).
p120 mutants were significantly slower, taking 112 minutes
(Fig. 1O). Despite this,
p120 mutants completed dorsal closure without apparent defects. Thus,
loss of p120, while not lethal, alters the efficiency of morphogenesis.
Rho1 exhibits dynamic localization
In mammalian cells, p120 regulates Rho activity. One mechanism to regulate
Rho is by controlling its subcellular localization. Previous workers examined
Rho1 localization in both ovaries and embryos
(Magie et al., 2002;
Padash-Barmchi et al., 2005
).
Drosophila Rho1 was reported to localize to AJs
(Magie et al., 2002
),
suggesting that this might be a mechanism by which it both regulates and is
regulated by AJs. We reexamined Rho1 localization compared to that of AJs.
This revealed new information about Rho1 dynamic localization, sometimes
confirming previous work and in other cases contradicting it.
We began with oogenesis (utilizing the same protocol and anti-Rho1
monoclonal antibody used in Magie et al.,
2002). Ovarian follicle cells form an epithelium with its apical
surface inward, providing an excellent place to examine AJs. After egg
chambers formed, Rho1 localized to follicle cell apical and lateral membranes
(Fig. 2A, white arrowhead), and
along lateral membranes of stalk cells
(Fig. 2A, red arrowhead). Rho1
remained enriched at follicle cell lateral membranes
(Fig. 2F,H), but apical
enrichment decreased at later stages (Fig.
2H). DE-Cad was strongly enriched in apical AJs
(Fig. 2C,F,H blue arrowheads).
Rho1 localization sometimes overlapped DE-Cad at AJs, but it was not enriched
there (Fig. 2C,F,H). In grazing
sections at stage 10, Rho1 accumulated in puncta, where three follicle cells
meet (Fig. 2E, arrows;
Fig. 2J); these puncta were
basal to the strongest DE-Cad staining (data not shown) and did not
co-localize with DE-Cad (Fig.
2E). Thus Rho1 was not enriched in AJs of most follicle cells.
However, like AJ proteins (Peifer et al.,
1993
; Oda et al.,
1997
), Rho1 did accumulate at germ cell boundaries
(Magie et al., 2002
)
(Fig. 2B, arrowhead).
|
We also compared Rho1 and DE-Cad localization in embryos, extending earlier
work [(Magie et al., 2002;
Padash-Barmchi et al., 2005
);
the pictures presented use the protocol of Padash-Barmchi et al.
(Padash-Barmchi et al., 2005
),
but similar results were also seen with the protocol of Magie et al.
(Magie et al., 2002
)]. Rho1
localization was very dynamic. During cellularization, Rho1 was enriched at
invaginating furrow canals (Fig.
3A, blue arrowhead), as previously observed
(Padash-Barmchi et al., 2005
),
while DE-Cad localized both to basal (Fig.
3A, yellow arrowhead) and developing apical AJs
(Fig. 3A, white arrowhead). At
gastrulation, DE-Cad was enriched in AJs of posterior midgut cells
(Fig. 3E, blue arrowhead),
while Rho1 accumulated in basal puncta
(Fig. 3D,E white arrowheads)
that may be furrow canal remnants. DE-Cad was also enriched in apical AJs of
invaginating cells in the ventral furrow, while Rho1 was only weakly enriched
in the ventral furrow (Fig. 3F,
arrowheads). After mesodermal cells invaginated, they accumulated cortical
Rho1 (Fig. 3H, yellow
arrowheads). In epithelial cells, we observed two general features of Rho1
localization. In several cell types, Rho1 localized basally. After germband
extension, Rho1 accumulated basally where ectoderm meets mesoderm
(Fig. 3G,H arrowheads).
ßPS1-integrin also localized there
(Fig. 3I, arrowheads). Later,
Rho1 localized basally in the hindgut epithelium
(Fig. 3M, blue arrowheads;
DE-Cad accumulated apically). Second, comparison of blastoderm, extended
germband, and dorsal closure-stage embryos showed that cortical enrichment of
Rho1 decreases over time (Fig.
3J-L). Thus Rho1 localization varies in different cell types, but
it is not significantly enriched in AJs of most cells.
Rho1 zygotic mutants have defects during dorsal closure
(Magie et al., 1999). We thus
closely examined Rho1 localization at that stage, collecting sections in the
z-axis through the lateral epithelia. Apically, Rho1 did not localize
to AJs but accumulated at low levels in the cytoplasm
(Fig. 4A,A'). Basal to
AJs (where DE-Cad was seen at AJs of invaginating segmental groove cells;
Fig. 4B,B'), Rho1 levels
increased and were more cortical. Thus in these cells Rho1 was enriched basal
to AJs. Another important input in Rho1 regulation is localized activation by
RhoGEFs. While a comprehensive study is beyond the scope of our work, we
examined RhoGEF2 during dorsal closure, which co-localizes with Rho1 during
cellularization (Padash Barmchi et al.,
2005
). During dorsal closure, RhoGEF2 accumulated basal to AJs
(Fig. 4H,H'), like Rho1.
However, RhoGEF2 was more cortical, poising it to activate a cortical pool of
Rho1. Thus, during oogenesis and embryogenesis Rho1 localization varies among
different cell types, with basal or basolateral accumulation in many
epithelia. Importantly, we found no evidence for preferential Rho1
accumulation at AJs, although the localizations do sometimes overlap.
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|
Drosophila p120 is not a key regulator of Rho1 function
Although p120 is not essential for Rho1 localization, it might regulate
Rho1 function by other mechanisms. Often regulators, even those that are
partially redundant, can be identified by looking for phenotypic effects in a
sensitized genetic background. For example, the supporting role of p120 in AJs
was revealed by the fact that loss of p120 strongly enhances DE-Cad mutants
(Myster et al., 2003). In
zygotic Rho1 mutants, maternal Rho1 is gradually depleted, and thus
we reasoned that altering a Rho1 regulator might modify the consequences of
reduced levels of Rho1.
|
|
As a second test for genetic interactions, we examined whether p120
overexpression modifies the Rho1 phenotype, ubiquitously expressing a
p120 transgene under the control of the GAL4-UAS system
(Myster et al., 2003) (using
actin-GAL4). Magie et al. (Magie et al.,
2002
) previously reported that p120 overexpression using
actin-GAL4 enhanced the Rho1 phenotype. However, using an
independently derived UAS-p120 transgene, we did not observe phenotypic
enhancement. Instead, our results suggested a slight suppression of the
Rho1 phenotype. The overall range of phenotypes was similar, with a
small shift toward the less severe categories
(Table 3; data not shown).
However, this effect was fairly small, and may reflect differences in genetic
background.
We also compared the effect of loss of Rho1 with the loss of both Rho1 and
p120 on F-actin during dorsal closure. As previously observed by Magie et al.
(Magie et al., 1999),
Rho1 mutants were nearly normal during early dorsal closure
(Fig. 5J versus
Fig. 5K), with defects becoming
apparent during late dorsal closure. The Rho1 phenotype was variable
in more severely affected Rho1 mutants, both the leading-edge
actin cable and cell shape changes were less uniform
(Fig. 5M versus
Fig. 5N). In less severely
affected mutants, when leading edges met at the dorsal midline the two
epithelial sheets did not line up or intercalate normally
(Magie et al., 1999
)
(Fig. 5P versus
Fig. 5Q). p120 Rho1
double mutants exhibited the same range of phenotypes as Rho1 single
mutants during early (Fig. 5K
versus Fig. 5L) and later
stages of dorsal closure (Fig.
5N versus Fig. 5O;
Fig. 5Q versus
Fig. 5R). Thus, p120
does not behave genetically as a key regulator of Rho1 function, contrasting
with its strong genetic interactions with DE-Cad
(Myster et al., 2003
).
|
|
|
We also sequenced the two non-null shg alleles.
shgG119 has an in-frame deletion of four conserved amino
acids in the membrane-proximal lamininG domain in the extracellular domain
(Fig. 6D).
shg2 has two mutations: mis-sense changes in a conserved
amino acid in the lamininG domain and in a conserved residue in the
cytoplasmic tail (Fig. 6D;
Fig. 7G) at the C-terminal end
of the Arm-binding site (Pai et al.,
1996; Huber and Weis,
2001
; Pokutta and Weis,
2000
).
Rho1 regulates DE-Cad but not Arm localization
Rho1 was reported to be required for correct localization of AJ proteins
(Magie et al., 2002;
Bloor and Kiehart, 2002
). We
examined embryos lacking zygotic Rho1. During dorsal closure, DE-Cad
accumulated ectopically, as previously reported
(Magie et al., 2002
). Ectopic
DE-Cad accumulated in the cytoplasm of epithelial or amnioserosal cells near
the leading edge (Fig. 8A
versus Fig. 8B). Ectopic DE-Cad
also accumulated in Rho1 embryos prior to
(Fig. 8C versus
Fig. 8D) and following
(Fig. 8E versus
Fig. 8F) dorsal closure.
Importantly, however, ectopic DE-Cad did not co-localize with its binding
partner Arm (Fig. 8B). Finally,
we used the ectopic DE-Cad phenotype of Rho1 mutants to further test
whether p120 regulates Rho1. p120 Rho1 double mutants accumulated
ectopic DE-Cad during dorsal closure in a fashion identical to Rho1
single mutants (Fig. 8G versus
Fig. 8B). Thus, Rho1 regulates
DE-Cad but not Arm localization and the effect of loss of Rho1 is not enhanced
or suppressed by removing p120.
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Discussion |
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|
As Drosophila has a single p120 family member, simple redundancy
does not explain the difference between vertebrates and invertebrates. We
imagine two possible explanations. First, p120 proteins may play fundamentally
different roles in the two groups of animals. Alternatively, the role of p120
in both may be similar, but the relative importance of p120 and unrelated,
partially redundant regulators of cadherin and/or Rho may differ. We favor the
latter possibility, because p120 binds to and promotes the function of AJs in
vertebrates and invertebrates (Anastasiadis
and Reynolds, 2000; Myster et
al., 2003
; Pettitt et al.,
2003
), and p120 has a conserved role in regulating morphogenesis,
contributing to dorsal closure efficiency (these data) and regulating dendrite
morphology (Li et al., 2005
)
in Drosophila and regulating gastrulation and craniofacial
morphogenesis in Xenopus (Fang et
al., 2004
; Ciesiolka et al.,
2004
). One role of p120 is to inhibit cadherin endocytosis.
Perhaps in invertebrates other regulators of cadherin trafficking compensate
for its absence.
|
We also tested the hypothesis that p120 regulates Rho1 localization,
examining several places in which Rho1 exhibits striking subcellular
localization, and examining the place where Rho1 exhibits its zygotic
phenotype: the dorsal closure front. We saw no change in Rho localization in
p120 mutants. Therefore, if p120 regulates Rho1 localization, it must
do so redundantly with other putative Rho1 regulators, such as -catenin
(Magie et al., 2002
). These
data do not rule out the possibility that p120 recruits a pool of active Rho1,
which may be only a small fraction of total cellular Rho1.
p120 appears to regulate RhoA during Xenopus development
(Fang et al., 2004;
Ciesiolka et al., 2004
).
Perhaps redundant Rho regulators act in parallel to p120 in flies.
Alternatively, the role of p120 as a Rho regulator may not be conserved: the
N-terminal domain of p120, which is implicated in regulating transitions
between its adhesive and cytoplasmic roles, is not well conserved between
mammalian and fly p120. As p120
(Myster et al., 2003
) and
Rho1 (Fig. 5)
mutations modify shg mutant phenotypes differently, p120 and Rho1 may
act in separate pathways to regulate AJs in Drosophila.
Rho1 localization and its regulation
We extended previous analyses of Rho1 localization
(Magie et al., 2002;
Padash-Barmchi et al., 2005
).
It is dynamic, with Rho1 accumulating at different subcellular sites in
distinct cell types, some consistent with proposed Rho functions. For example,
mammalian RhoA regulates integrin-based cell-matrix junctions (reviewed by
Burridge and Wennerberg, 2004
).
The basal localization of Rho1 raises the possibility that it may regulate
integrins in Drosophila. Rho1 accumulation in mesodermal cells is
consistent with its role in regulating cell shape during mesoderm spreading
(Wilson et al., 2005
).
Relative levels of cortical Rho1 decrease through development. Perhaps at
later stages Rho1 is activated by localized RhoGEFs. Consistent with this,
RhoGEF2 is more cortically enriched during dorsal closure than Rho1. Thus,
future studies will need to examine the localization of Rho1 regulators and
effectors. Recent advances also allow visualization of active Rho GTPases
(e.g. Benink and Bement, 2005
).
As much of the Rho1 pool may be inactive, application of this approach to
flies will advance our understanding of Rho1 function.
It was previously proposed that Rho1 is enriched at Drosophila AJs
and that this is regulated by core AJ proteins
(Magie et al., 2002). We
examined this in follicle cells and embryos. In follicle cells, Rho1 localized
to lateral and apical membranes in early egg chambers, and to lateral
membranes later. In neither case did we observe enrichment in AJs, although
Rho1 was not excluded from them. In embryonic epithelia, Rho1 sometimes
localized uniformly to the basolateral membrane, while in other places it was
enriched basally. During dorsal closure, when Rho1 exhibits its
zygotic phenotype, Rho1 accumulated basal to AJs. The lack of preferential
Rho1 localization at AJs does not rule out accumulation of a pool of active
Rho1 at AJs: this will require reagents to measure Rho1 activation in vivo. We
also tested the hypothesis that AJs regulate Rho1 localization. In follicle
cells mutant for DE-Cad and embryos mutant for arm or DE-Cad during
dorsal closure, Rho1 localization was not obviously disturbed.
Rho is an important regulator of AJs during embryonic morphogenesis
In cultured cells, Rho and AJs have a complex relationship. Rho regulates
AJ stability, and conversely AJs regulate Rho activity (reviewed by
Yap and Kovacs, 2003).
Further, different Rho effectors can promote or decrease AJ stability in
cultured mammalian cells (Sahai and
Marshall, 2002
; Vaezi et al.,
2002
). We examined this complex relationship during morphogenesis,
using genetic and cell biological assays. Our data support the hypothesis that
Rho1 is an important regulator of cadherin-based adhesion during embryonic
development.
Loss of Rho1 leads to DE-Cad mislocalization
(Magie et al., 2002), while
dominant-negative Rho1 reduces DE-Cad in AJs
(Bloor and Kiehart, 2002
),
implicating Rho1 in regulating DE-Cad localization. Our results support this
hypothesis. Cytoplasmic DE-Cad accumulation is consistent with a role for Rho1
in regulating either DE-Cad transport to or recycling from AJs. We observed
that the ectopic DE-Cad in Rho1 mutants accumulates independently of
its binding partner Arm. In mammalian cells, newly-synthesized E-cadherin must
bind ß-catenin before it can be transported to AJs
(Chen et al., 1999
), while
endocytosed E-cadherin accumulates with either no
(Xiao et al., 2003
) or reduced
(Le et al., 1999
) amounts of
ß-catenin. Thus our data are more consistent with ectopic DE-Cad
accumulating after endocytosis. Consistent with this, mammalian RhoA regulates
clathrin-mediated endocytosis (Lamaze et
al., 1996
), and Drosophila Rho1 regulates endocytosis of
the ligand Wingless (Magie et al., 2005). Further, constitutively active Rac1,
which can inhibit RhoA (Sander et al.,
1999
), triggers E-cadherin recruitment to intracellular vesicles
in keratinocytes (Akhtar and Hotchin,
2001
). As high levels of Rho1 do not accumulate at AJs, either a
small pool of active Rho1 at AJs is sufficient to inhibit cadherin endocytosis
or the effect is more indirect, with Rho1 acting on the actin cytoskeleton or
regulators of endocytic trafficking. The mechanism by which Rho1 regulates
DE-Cad trafficking is an interesting question for future studies.
Mammalian p120 also regulates cadherin endocytosis
(Davis et al., 2003;
Xiao et al., 2003
). The
viability of fly p120 mutants suggests that in flies this role is not
rate limiting, although the enhancement of mutants with reduced DE-Cad by
p120 is consistent with p120 playing a similar role
(Myster et al., 2003
). p120
and Rho could regulate DE-cad trafficking via the same or distinct pathways.
The effect on DE-Cad trafficking in zygotic Rho1 mutants, which
should have limiting levels of maternal Rho1, is not enhanced by removing
p120. This is more consistent with a model in which the two proteins
work in different pathways, and in which p120 acts partially redundantly with
another unknown regulator.
Our analysis of Rho1 and Rho1 shg mutants is consistent
with the hypothesis that Rho1 regulates AJs, but suggests that their
interactions are complex. A weak shg allele was enhanced, but
stronger alleles were suppressed. There are several possible explanations for
these contrasting results. Weak alleles (e.g. shgG119)
make protein with reduced but residual function. If Rho1 negatively regulates
cadherin endocytosis, more mutant DE-Cad protein might be endocytosed in
Rho1's absence, further reducing functional DE-Cad and enhancing the
phenotype. However, null or very strong shg alleles accumulate no
functional DE-Cad at AJs (for shg2 see
Uemura et al., 1996),
rendering regulation of cadherin endocytosis a moot point. The slight
suppression by Rho1 of strong shg alleles may result from a
reduction of morphogenetic movements, reducing cuticle disruption (as in
Tepass et al., 1996
).
Alternatively, some mutant DE-Cad proteins may be capable of coupling to Rho1
while others are not. Rho1 can bind
-catenin
(Magie et al., 2002
), and
active Rho1 may be recruited to AJs by that interaction.
shgG119 has a wild-type cytoplasmic domain and could
presumably couple to Rho1; reducing Rho1 might further impair its function. By
contrast, the shg2 mutation may impair Arm and/or
-catenin binding and thus Rho1 recruitment; if so this mutant protein
would not be further impaired by Rho1 removal. Finally, the complex genetic
interactions might reflect different requirements for Rho1 during neuroblast
delamination and head involution, which are affected by strong or weak
reduction in DE-Cad function, respectively
(Tepass et al., 1996
). Future
studies of Rho regulation of and by AJs will help distinguish between these
possibilities.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/21/4819/DC1
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