1 Department of Biophysics and Biochemistry, Graduate School of Science,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
2 UPBSB, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-0033, Japan
* Author for correspondence (e-mail: saigo{at}biochem.s.u-tokyo.ac.jp)
Accepted 30 March 2005
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SUMMARY |
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Key words: Dorsal closure, Adherens junction, Src42A, arm, shotgun, E-cadherin
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Introduction |
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The major autophosphorylation site in focal adhesion kinase (FAK) serves as
a binding site for Src homology 2 (SH2)-containing proteins
(Chen et al., 1996;
Schaller et al., 1994
;
Schlaepfer et al., 1999
). The
FAK-Src complex mediates the phosphorylation of paxillin and
p130-Crk-associated substrate, both of which are major scaffolding proteins
capable of recruiting other molecules for integrin-based cell-substratum
adhesions and which regulate cytoskeleton organization
(Bellis et al., 1995
;
Cary et al., 1998
;
Honda et al., 1998
;
Honda et al., 1999
; Schaller
et al., 1995; Turner, 2000
).
The absence of FAK gave rise to increase in the number and extent of
cell-substratum adhesions (Ilic et al.,
1995
). Recently, quantitative assay of the rate of incorporation
of proteins into cell-substratum adhesion and departure of these proteins from
this adhesion was conducted (Webb et al.,
2004
). Src and FAK were shown to be crucial for adhesion turnover
at the cell front. Thus, the rates of formation, disassembly and/or maturation
of cell-substratum-adhesion appear controlled by FAK-Src activity.
Homophilic cadherin interaction is essential for cell-cell adhesion in
vertebrates (Hinck et al.,
1994). The loss of E-cadherin (E-cad) expression has been shown
related to invasive and metastatic cancers
(Denk et al., 1997
;
Van Aken et al., 2001
).
ß-Catenin binds to
-catenin and the cytoplasmic domain of E-cad
and is essential for linking E-cad to the actin cytoskeleton
(Nagafuchi and Takeichi, 1988
;
Ozawa et al., 1989
).
Tyrosine-phosphorylation of ß-catenin or other
adherens-junction-associated proteins is one means by which cadherin-mediated
cell-cell adhesions may be altered (Lilien
et al., 2002
; Takeda et al.,
1995
). Enhanced tyrosine-phosphorylation of ß-catenin causes
weakening of cadherin-actin interaction with consequent loss of cell
adhesiveness. Src may be one of the tyrosine kinases responsible for this
tyrosine-phosphorylation, because, in cells transformed with Src, loss of
epithelial cell differentiation, gain in invasiveness and cadherin-mediated
adhesion detachment are all correlated with tyrosine-phosphorylation of the
E-cad/ß-catenin complex (Behrens et
al., 1993
; Hamaguchi et al.,
1993
; Lilien et al.,
2002
).
Nonetheless, precise determination of the functional roles of individual
Src family kinases in vertebrates may be attended with considerable difficulty
in that compensatory interactions may occur among nine vertebrate Src kinase
members. By contrast, Drosophila possesses only two Src kinases,
Src64 and Src42A (Simon et al.,
1985; Takahashi et al.,
1996
) and, accordingly, may provide a better and simpler system
for clarifying Src functions in development.
Mutations in Src64 lead to reduction in female fertility, which is
associated with nurse cell fusion and ring canal defects
(Dodson et al., 1998).
Src64-mutant ring canals fail to undergo extensive tyrosine
phosphorylation which normally occurs. Tec29 dominantly enhances the
Src64 ring canal phenotype and loss of Tec29 results in a
phenotype strikingly similar to that noted following loss of Src64
function (Guarnieri et al.,
1998
). Tec29 kinase is localized in the ring canal, and this
subcellular localization requires Src64 function, indicating that
Tec29 is a downstream target of Src64.
Src42A is the closest relative of vertebrate Src in
Drosophila. By localized expression of gain-of-function and
dominant-negative forms of Src42A, it was demonstrated that Src42A
may be involved in the regulation of cytoskeleton organization and cell-cell
contacts in developing ommatidia and that both dominant-negative and
gain-of-function mutations of Src42A cause formation of supernumerary
R7-type neurons, which is suppressible by one-dose reduction of various
components involved in the Ras/MAPK pathway
(Takahashi et al., 1996). Lu
and Li (Lu and Li, 1999
)
isolated a Src42A mutant as an extragenic suppressor of Raf
and with this and other mild Src42A mutants found that Src42A may
serve as negative regulator of receptor tyrosine kinases in a Ras1-independent
manner. Their genetic data for Src functions in ommatidium formation appeared
somewhat at variance with those of Takahashi et al.
(Takahashi et al., 1996
) using
gain-of-function and dominant-negative types of Src42A
transgenes.
As with Src64, Src42A may function in a synergistic manner with
Tec29. A Tec29 mutation was noted to enhance the lethality
of Src42A mutants dominantly
(Tateno et al., 2000).
Although these authors found no dorsal open phenotype in their Src42A
or Tec29 mutants, the double mutant embryos exhibited the dorsal open
phenotype. Src42A has been shown to be functionally redundant to
Src64 at least in the dorsal closure
(Tateno et al., 2000
). Both
dorsal closure of the embryonic epidermis and thorax closure of the pupal
epidermis require the Jun amino-terminal kinase (JNK) homolog Basket (Bsk)
(Zeitlinger and Bohmann, 1999
;
Tateno et al., 2000
). The
severity of the epidermal closure defect in Src42A mutants was found
to depend on the degree of Bsk activity, and this extent to depend on
that of Src42A (Tateno et al.,
2000
), thus indicating that JNK-pathway activation is required
downstream of Src42A.
This paper first presents dynamic changes in cellular and subcellular localization of Src42A and then describes phenotypes of a Src42A protein-null and Scr42A Src64 mutants. Genetic and biochemical analyses indicate that E-cad and Armadillo (Arm) form a complex with Src in the membrane and the resultant putative adherens junction complex is required for proper regulation of F-actin accumulation and actin cytoskeleton dynamics in leading edge cells during dorsal closure.
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Materials and methods |
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Screening of dominant enhancers for sev-Src42A[KR]
Enhancers were searched for using P[sev-Src42A[KR]]
(Takahashi et al., 1996)
inserted into CyO or TM3 balancers. E(7A-1), which is incapable of
complementing shgR64a, was isolated from 20,000
EMS-mutagenized flies.
Antibodies
Histochemical reagents and primary antibodies used were: TUNEL reagents
(Roche Diagnostics), rhodamine-phalloidin, SYTOX (Molecular Probes), mouse
anti-tracheal lumen (2A12) (Manning and
Krasnow, 1993), mouse anti-Fas3
(Patel et al., 1987
), mouse
anti-Fas2 (Hummel et al.,
2000
), mouse anti-Arm
(Riggleman et al., 1990
), rat
anti-E-cad (Oda et al., 1994
),
rat anti-Src64 (Dodson et al.,
1998
), biotinylated mouse anti-phosphotyrosine (pTyr)
(Glenney et al., 1988
), mouse
anti-
-Tubulin (ICN), mouse anti-lacZ (Promega), rabbit
anti-GST (glutathione S-transferase; Sigma), rabbit anti-MBP (maltose binding
protein; New England BioLabs), mouse anti-Elav
(Robinow and White, 1991
),
mouse anti-Engrailed (Patel et al.,
1989
) and rabbit anti-Clawless (Cll)
(Kojima et al., 2005
)
antibodies. Src42A antiserum (rabbit) was raised against GST-Src42A (amino
acids 1-252) fusion protein [see other details in Suzuki and Saigo
(Suzuki and Saigo, 2000
) and
Hayashi et al. (Hayashi et al.,
1998
)].
Cell culture, RNA interference (RNAi), immunoprecipitation and pull-down assay
Transfection and RNAi of Drosophila S2 cells was carried out as
described previously (Ui-Tei et al.,
2000). Membrane and cytosolic fractions for immunoprecipitation
were prepared from stage 13-15 embryos according to Peifer
(Peifer, 1993
). The pellet
(membrane fraction) was resuspended in buffer with the same volume of the
cytosolic fraction. Immunoprecipitates were size-fractionated and
immunoblotted. Pull-down assay was carried out as follows. MBP-tagged
proteins, bacterially expressed, were bound to amylose resin. After PBT
washing, resins were incubated with bacterially expressed GST-tagged proteins.
The MBP/GST fusion protein complex was eluted with maltose (10 mM) and
analyzed with western blotting with anti-GST or anti-MBP antibodies.
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Results |
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At the start of oogenesis, the cystoblast undergoes four rounds of mitotic
divisions with incomplete cytokinesis to generate 16 cystocytes interconnected
via ring canals (Cooley and Robinson,
1996). Follicle cells subsequently separate off individual cysts
to form egg chambers.
Transient but very strong Src42A signals, not associated with strong E-cad
signals, were found in cystocytes in germarium region 2a/b
(Fig. 1A). E-cad signals became
evident at slightly later stages. In stage 1-7 egg chambers, relatively strong
Src42A signals were apparent along the nurse/follicle cell boundary
(Fig. 1A,B) as noted for E-cad
(Niewiadomska et al., 1999);
Src42A signals on the basal follicle-cell surface were very weak. In the
middle of oogenesis, relatively strong Src42A signals were evident in polar
and invading border cells (Fig.
1B,D,E). Middle-stage ring-canals were marked by Src42A enclosed
by weak E-cad (Fig. 1C1,C2). By
stage 7, cytoplasmic Src42A became evident in oocytes. At stage 8, Src42A
unassociated with E-cad started being deposited on the oocyte surface and were
conspicuous by stage 10b, at which time strong Src42A and E-cad signals could
be seen in centripetal cells (Fig.
1F1,F2).
Embryogenesis starts with cleavage (stages 1-4), in which the nucleus
undergoes 13 divisions and the nuclei thus produced become arranged in a
single layer beneath the egg surface
(Hartenstein, 1993).
Membranous Src42A signals were evident
(Fig. 1I). During
cellularization, not only Src42A but also E-cad signals were apparent on the
surface of eggs and the membrane extending inwardly
(Fig. 1G1-G3). The leading
edges of invading membranes are always marked by Src42A but not E-cad. At
stage 6, mesoderm generation started by invagination. In stage 7 dorsal cells
lying anterior to the cephalic furrow, Src42A distribution was virtually the
same as at stage 5 (Fig.
1G3,H1,J), but in more posterior dorsal cells involved in
transient furrow formation or posterior midgut invagination, strong Src42A
expression associated with strong E-cad signals could be confirmed only in the
apical region (Fig. 1H2,J,K).
Src42A unassociated with E-cad expression persisted in invaginated mesodermal
cells and were evident on the ectoderm/mesoderm interface at stage 9
(Fig. 1L). Apical tips of
mesectodermal cells, situated along the ventral midline, showed strong E-cad
and Src42A signals (Fig.
1M1,2).
In late developmental stage embryos, there were strong signals of E-cad and
Src42A in some tubular structures (Fig.
1N-R). In all cases, E-cad was present only in apical regions,
while Src42A varied in location according to tube type
(Fig. 1N,P,R). In hindguts
covered with thin visceral mesodermal cells
(Fig. 1R, inset), Src42A
signals were evident in both apical and basal regions
(Fig. 1R), whereas Malpighian
tubules protruding from hindguts (Fig.
1R) (Skaer, 1993),
salivary glands (Fig. 1P) and
stomodeum opening (Fig. 1N),
none of which having any mesoderm association, all displayed Src42A signals
only in apical regions. Basal strong Src42A signals were eliminated when
hindgut cells acquired Malpighian-tubule fate (see arrows in
Fig. 1R). Similarly, strong
basal Src42A signals, separating the ectoderm from mesoderm at the basal
clypeus cortex, had varnished with stomodeum formation
(Fig. 1N).
During stage 12, tracheal branches develop from invaginated tracheal pits
(Manning and Krasnow, 1993).
At stage 13, the dorsal trunk anterior has fused with the dorsal trunk
posterior of the anterior neighbor to form a long tubular structure. The
arrowheads in Fig. 1S show
Src42A to be co-localized with juxtaposition E-cad signals. Strong Src42A
signals unassociated with E-cad are present in tendon cells to which the
muscle system is attached (see the arrows). Dorsal closure is a major
morphogenic process in which two epithelial sheets converge to enclose the
embryo. At the leading edge, moderate Src42A signals colocalized with strong
E-cad (Fig. 1O).
Strong Src42A signals were evident in CNS
(Fig. 1T). Longitudinal
connectives and commissures stained strongly with anti-Src42A antibody. E-cad
signals could be seen only in midline glial cells, mesectodermal derivatives
(see the arrowheads). In CNS, nervous system-specific N-cadherin appeared
co-expressed with Src42A (Iwai et al.,
1997) (data not shown). Strong Src42A signals were present in the
brain (Fig. 1Q) and the axon
linking the larval eye (Bolwig's organ) to the optic lobe
(Fig. 1U). Strong Src42A
signals, occasionally associated with E-cad signals, were seen in the gonad
(Fig. 1V) (Jenkins et al., 2003
).
Isolation of a protein-null Src42A mutant and functional redundancy of Src42A and Src64
As Src possesses multiple functional domains, the isolation of protein-null
mutants may be required to clarify the roles of Src in development.
Short Src42A deletion mutants were thus generated through imprecise
P-element excision of Src42Ak10108 (enhancer trap line)
and a protein-null lethal mutant, Src42A26-1, was
identified using anti-Src42A antibody (Fig.
2A2,A3). In Src42A26-1, a 1.9 kb region
containing the putative TATA box, RNA start, the first exon of Src42A
and the entire P-lacZ sequence were deleted
(Fig. 2A1).
Src42A26-1 embryos showed mild dorsal closure defects
(Fig. 2B1,B2). Close inspection
of mutant embryos stained for Engrailed revealed occasional segmental
misalignment (Fig. 2B3).
Lethality and morphological defects in Src42A26-1 were
eliminated by introducing the wild-type Src42A transgene driven by
arm-GAL4 (data not shown).
Using Src64P1 and Src42AE1, Src42A
and Src64 have been shown to be functionally redundant to each other
with respect to the dorsal closure (Tateno
et al., 2000). Using a newly isolated protein-null Src42A
mutant, we demonstrate that these two Src genes are functionally redundant not
only in dorsal closure but in many other development contexts as well.
The dorsal open phenotype associated with head involution defects was exhibited by 34% of Src42A26-1;Src64P1/+ embryos (Fig. 2B4). Src42A26-1;Src64P1 embryos showed much severer phenotypes with no apparent germ band retraction (Fig. 2B5). No defects could be found in Src64P1 (data not shown). CNS morphology was extensively affected by the simultaneous elimination of Src42A and Src64 activity. In Src double mutant embryos, longitudinal tracts and commissures were frequently broken without significant loss of Elav-positive neuronal cells (Fig. 2C1-C4). In Src double mutants, optic lobe/Bolwig's organ (Fig. 2E1-E3) and trachea formation (Fig. 2D1-D4) was significantly disrupted, while no apparent defect was detected in Src42A26-1.
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Src may thus be considered to exercise central roles in many normal developmental processes of oogenesis and embryogenesis. Src42A and Src64 contribution to total Src activity would depend on some particular aspect of development.
shotgun and arm as enhancers of Src42A
To identify genes that may interact with Src42A genetically, a
search was made for fly mutants that enhance the eye phenotype induced by
misexpression of the dominant-negative form of Src42A
(Src42A[KR]) (Takahashi et al.,
1996). As previously noted, eyes of flies heterozygous for a
P[Src42A[KR]] insertion were almost entirely normal
(Fig. 3A). Seven putative
enhancer lines were obtained and E(7A-1), a line with the strongest enhancing
activity (Fig. 3B), was
selected for subsequent experiments.
Flies heterozygous for E(7A-1) were viable and not associated with any
apparent morphological eye defects (data not shown), whereas E(7A-1)
homozygotes were embryonic lethal. Complementation tests indicated the E(7A-1)
lethal lesion to be present in 57B5-14 on the second chromosome, which
contains shotgun (shg), a gene encoding E-cad
(Tepass et al., 1996;
Uemura et al., 1996
).
shgR64a (a null allele) failed to complement E(7A-1). As
with E(7A-1), shgR64a enhanced the eye phenotype of flies
heterozygous for P[Src42A[KR]] insertion
(Fig. 3C). Virtually no E-cad
signals could be found in E(7A-1) homozygous stage 13 embryos
(Fig. 3D,E). Thus, we conclude
that E(7A-1) harbors a lethal mutation in shg
(shgE(7A-1)) and that shg activity reduction
enhances Src42A eye phenotypes.
Subsequent experiments indicated shg also capable of enhancing
Src42A mutant phenotypes in various developmental contexts other than
eye morphogenesis. shgR6 and Src42A6-1
are hypomorphic alleles of shg and Src42A, respectively,
(Niewiadomska et al., 1999)
(this work) and dorsal closure of either Src42A6-1 or
shgR6 embryos appeared essentially normal
(Fig. 3F,G). But most
shgR6; Src42A6-1 embryos were
associated with the dorsal open phenotype
(Fig. 3I), indicating that
shg-Src42A interactions are required for normal dorsal
closure.
Src42A-shg interactions may also be involved in normal
thorax closure in pupal stages. Fig.
3K-N shows defects in thorax closure in escapers and pharate
adults of Src42A6-1. Similar defects have been reported
for Src42Ajp45 and classified into three classes
(Tateno et al., 2000).
Src42A6-1 notum phenotypes were found considerably
enhanced in the genetic background of shgg317/+
(Fig. 3O). Two thirds of class
1 were converted to severer classes, while a fraction of class 3 was doubled.
In some double mutant flies, right and left halves of the notum appeared
completely separated from each other (class 4;
Fig. 3N).
E-cad regulates cell-cell adhesion via homophilic association
(Oda et al., 1994). Arm
interacts directly with the cytoplasmic domain of E-cad and
-catenin.
The latter is thought to associate with the actin network
(Oda et al., 1993
). Strong
hypomorphic alleles of arm have defects in the dorsal closure
(Grevengoed et al., 2001
;
McEwen et al., 2000
), so we
sought to determine whether Src42A interacts genetically with
arm in dorsal closure and eye morphogenesis. As with embryos
homozygous for Src42A6-1, virtually all embryos
heterozygous for armYD35 (null allele) and those
homozygous for armH8.6 (hypomorph) were normal in dorsal
closure (Fig. 3H, data not
shown). By contrast, most Src42A26-1 embryos heterozygous
for armYD35 were associated with the dorsal open phenotype
(Fig. 3J).
armH8.6/+ eyes were normal in appearance, but
Src42A[KR]/armH8.6 flies possessed rough eyes, as
also noted for Src42A[KR]/shg (data not shown). It thus
follows that arm-Src interactions are essential for normal dorsal
closure and eye morphogenesis.
Requirements of Src activity for thick F-actin accumulation and adherens-junction maintenance at the leading edge
During early-mid stages of the dorsal closure, dorsal-most epidermal (DME)
cells and epidermal cells located more ventrally elongate along the
dorsoventral axis (Fig. 4A) and
F-actin thickly accumulates at the leading edge
(Fig. 5A). In the zippering
stage, actin-based processes are essential for zippering epithelial sheets
together (Jacinto et al.,
2000). DME-cell elongation is associated with the redistribution
of many proteins such as those involved in planar polarity and cytoskeleton
(Kaltschmidt et al., 2002
).
Genetic experiments showed interactions between Src, shg and
arm to be involved in the dorsal closure and thus examination was
made of temporal change in the locations of E-cad, Arm, Fas3 and F-actin
during dorsal closure in Src and shg mutants as well as wild
type.
In wild type, not only F-actin but also E-cad and Arm signals increased at
the leading edge from 9 hours after egg laying (AEL;
Fig. 4B and
Fig. 5A) and polarized Fas3
expression and tubulin bundling occurred with dorsoventral elongation of
dorsal epidermal cells (Fig.
4A) (Kaltschmidt et al.,
2002). As stated above, no germ band retraction occurs in
Src42A26-1;Src64P1 embryos
(Fig. 2B5) and so examination
was made of the effects of reduction in Src-activity on protein distribution
at the leading edge of
Src42A26-1;Src64P1/+ and
Src42A26-1 embryos. In
Src42A26-1;Src64P1/+ embryos, DME-cell
elongation and polarized deposition of Fas3 and tubulin bundling appeared to
proceed normally (Fig. 4A).
But, unlike wild-type embryos,
Src42A26-1;Src64P1/+ embryos exhibited
significant reduction in E-cad and F-actin deposition at the leading edge at
11-12 hours AEL (Fig. 4B6,B8
and Fig. 5A4,A6). Arm signals
appeared reduced throughout the entire membrane region, including the leading
edge and accumulated in the cytoplasm. The leading edge of
Src42A26-1;Src64P1/+ embryos,
initially smooth in appearance (Fig.
4B2), frequently kinked with partial DME-cell deformation from 10
hours AEL onwards (Fig.
4B4,B6,B8). Kinking of the actin cable at the zipper front is
thought most likely due to lamellae traction
(Jacinto et al., 2000
), and,
accordingly, Src activity reduction in
Src42A26-1;Src64P1/+ embryos may
possibly give rise to defects in the cytoskeletal machinery that are essential
for driving the dorsal closure. In Src42A26-1 single
mutant embryos, DME-cell elongation and the leading-edge structure appeared
virtually normal (Fig. 4A9,B9
and Fig. 5B4) but morphological
defects could sometimes be seen along the zippered midline as mentioned above
(see Fig. 2B3).
Fig. 5B1-2 also shows that
F-actin signals are also significantly reduced in shgR64a
mutant embryos.
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Taken together, our results indicate that the leading-edge adherens junction containing E-cad, Arm and actin may serve as a cytoskeletal and/or regulatory machinery for properly driving the dorsal closure, and that interactions between Src42A, shg and arm would be essential for membrane localization of their own protein products.
Src-activity-dependent induction of cell migration and cell-shape change
To further clarify Src function, activated (Src42A[YF]),
dominant-negative (Src42A[KR]) and wild-type (Src42A[WT])
forms of Src42A were driven by pnr-GAL4 to determine any
change in Arm, E-cad or Src42A signals
(Fig. 6A1-D3). Immunostaining
of embryos collected at 10-14 hours AEL showed that, as with wild type
(control; Fig. 6A1-A2), nearly
all Arm and E-cad signals localize in the plasma membrane when kinase-inactive
Src42A[KR] is driven (Fig.
6B1-B3), while considerable cytoplasmic E-cad and Arm signals are
evident in cells overexpressing Src42A[WT] or [YF]
(Fig. 6C1-3,D1-3). It may thus
follow that activated Src stimulates cytosolic Arm stabilization and/or
arm expression. Alternatively, Src42A may be involved in regulating
possible cadherin endocytosis.
Overexpression of activated Src42A may also cause change in cell
morphology. As shown in Fig.
6A1-D1, forced expression of UAS-Src42A[WT] and
[YF] prevented dorsal epithelial cells from elongating normally.
Occasionally, cells that strongly expressed Src42A and were separated from the
amnioserosa or dorsal epidermis plane could be found in embryos transformed
with Src42A[YF] (Fig.
6E-G). These cells were frequently associated with the expression
of Cll (Kojima et al., 2005)
or Fas3, which are maker proteins for amnioserosa and epidermis, respectively
(Fig. 6H-K) (Jagla et al., 2001
;
Kaltschmidt et al., 2002
;
Kojima et al., 2005
), but not
with TUNEL signals (Booth et al.,
2000
) at least up to the end of stage 13
(Fig. 6L,M,O,P), suggesting
that they are live cells dissociated from amnioserosa or dorsal epidermis
because of elevated Src activity. Most released cells degenerated at stage 16
via apoptosis (Fig. 6N,Q). We
conclude that Src42A is essential for proper cell migration and
cell-shape regulation.
Physical binding of Src42A to Arm through kinase-domain/Arm-repeat interactions
To determine the molecular basis for genetic interactions between
Src42A, shg and arm, study was made as to whether or not
protein products of these genes come together to form complexes within cells
was examined using fractionated embryonic extracts. Membrane and cytosolic
fractions were prepared from wild-type embryos and embryos with
pnr-GAL4-dependent forced expression of either UAS-Src42A[WT],
[KR] or [YF]. Here, we describe only endogenous interactions in
wild-type embryos, whereas physical interactions in embryos with forced
Src expression are described in the next section.
Membrane and cytosolic fractions of wild-type embryos were treated with anti-Arm or anti-E-cad antibodies and the resultant immunoprecipitates were analyzed by SDS-PAGE and subsequent western blotting (Fig. 7B-C). Any appreciable Src42A/Arm signals were detected in the E-cad immunoprecipitates of untransfected S2 cells, which expresses only a low level of E-cad (Fig. 7A, lane 1). Fig. 7B also shows that unrelated anti-Fas3 antibody gave no Src42A/Arm signals.
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Arm protein possesses 13 repeats referred to as Arm repeats, which provide
binding sites for many Arm/ß-catenin-binding proteins
(Provost and Rimm, 1999). To
determine whether Src42A binds to Arm directly and if so which part of Src42A
is responsible for the Src-Arm interaction, pull-down assay was carried out
(Fig. 7D). Strong
GST-Arm-repeat (GST-ArmR) signals were recognized in the lanes for
MBP-Src42A[Kinase] and the 14 amino acid autophosphorylation site-containing
peptide (Fig. 7D, lanes 6,8).
But no signals could be found in lanes for MBP-Src42A[SH3SH2],
MBP-Src42A[(Kinase)-(391/404)] or MBP (Fig.
7D). Src42A is thus shown to bind to Arm through interaction of
the 14 amino acid kinase domain peptide with Arm repeats.
Requirements of Src for Arm phosphorylation
In vertebrates, tyrosine phosphorylation has been shown to cause the
binding of ß-catenin to E-cad to diminish significantly. Interactions
between ß- and -catenin may also be negatively regulated by
tyrosine phosphorylation (reviewed by
Lilien et al., 2002
). We
therefore studies whether Arm phosphorylation requires Src activity. Arm was
overexpressed in Drosophila S2 cells and RNAi was carried out to
clarify any Src42A and/or Src64 involvement in Arm
tyrosine-phosphorylation (Fig.
7E). dsRNAs used specifically abolished protein expression of the
corresponding target genes. Change in the degree of Arm tyrosine residue
phosphorylation was monitored with Arm western blotting of anti-pTyr antibody
immunoprecipitates of whole or E-cad-free cell extracts. The levels of
Tyr-phosphorylated Arm were reduced by transfection with Src64 and/or
Src42A dsRNAs (Fig.
7E, parts d,e, lanes 2,5-7), indicating that redundant Src
function is required for Arm phosphorylation.
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Discussion |
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In ectodermal cells, strong Src42A signals in apical or apicolateral
regions were always associated with strong E-cad signals
(Fig. 1J). E-cad is a core
component of the adherens junction that is responsible for cell-cell adhesion
(reviewed by Takeichi, 1990)
and, hence, most, if not all, E-cad-associated membranous Src42A are probably
related to adherens junction-dependent cell-cell adhesion.
A considerable fraction of ectodermal cells were also found associated with
the second type of basal Src42A free of E-cad
(Fig. 1L,N,R). E-cad-free
Src42A was localized on the ectoderm/mesoderm interface and eliminated from
ectodermal cells, which had evaginated or invaginated without mesoderm
association (Fig. 1N,P,R). The
extracellular matrix (ECM) comprises several groups of secreted proteins such
as integrin ligands. During embryogenesis, different cell layers become
properly connected, most probably via cell adhesion to ECM
(Yurchenco, 1994). E-cad-free
Src42A may thus be related to integrin-mediated cell-matrix adhesion. Cell-ECM
adhesion may not be restricted to the interface between ectodermal and
mesodermal cell layers. Strong Src42A signals have actually been found present
on the interface between mesodermal and endodermal cell layers.
Requirements of Src incorporated into putative adherens junction for Drosophila development
JNK signaling, which includes hemipterous (hep) and
bsk, is essential for dorsal closure of the embryonic epidermis in
Drosophila (reviewed by Goberdhan
and Wilson, 1998). Based on examination of Tec29 Src42A
mutant phenotypes, it was considered that Src42A may act upstream of
bsk (Tateno et al.,
2000
). Consistently, our study showed that, as with JNK signaling
genes (Kaltschmidt et al.,
2002
), Src is required not only for thick F-actin
accumulation at the leading edge (Fig.
5A) but proper cell-cell matching along the midline seam as well
(Fig. 2B3).
In vertebrates, JNK is considered to be situated downstream of Src in
integrin signaling (Oktay et al.,
1999; Schlaepfer et al.,
1999
). Our genetic experiments
(Fig. 3) would indicate that
interactions between Src and arm/shg, genes
encoding the core components of the adherens junction are essential for JNK
signaling regulation required for dorsal closure. A pull-down assay
(Fig. 7D) also showed that Src
protein is capable of directly binding to Arm. Both putative adherens-junction
Src and integrin-associated Src thus would appear involved in the regulation
of JNK signaling.
The adherens junction is necessary for cell-cell adhesion (reviewed by
Takeichi, 1990) and thick
F-actin accumulation occurs at the level of the adherens junction at the
leading edge (Kaltschmidt et al.,
2002
). As E-cad and Arm signals along with actin signals were
reduced significantly at the leading edge in
Src42A26-1;Src64P1/+ embryos
(Fig. 4B8) and the leading edge
of the mutants was significantly kinked
(Fig. 4B4,B6,B8), the absence
of Src protein from the adherens junction may possibly result in destruction
of structural integrity, implying that adherens junction is also involved in
dorsal closure regulation in a structural way.
Dorsal closure and CNS defects similar to those in Src mutants
were previously observed in abl mutants
(Grevengoed et al., 2001). In
vertebrates, Abl is tyrosine-phosphorylated with Src
(Plattner et al., 1999
) and is
capable of interacting with
-catenin, an E-cad-binding protein
(Lu et al., 2002
). Abl may
thus function as well downstream of Src signaling in Drosophila.
Germ-band retraction and possibly too, head involution, both of which require
Src activity (this work), may be regulated by the two above distinct Src
functions.
1,2-laminin and
PS3ßPS integrin have clearly
shown to be essential for spreading a small group of amnioserosa epithelium
cells over the tail end of the germ band during germ-band retraction
(Schoeck and Perrimon, 2003
).
Our unpublished data indicate that shg activity is essential for
normal germ-band retraction and head involution.
Src-dependent dynamical regulation of E-cad-dependent cell-cell adhesion
may also necessary for visual system formation. E-cad overexpression or
elimination of EGFR activity have been shown to render optic placode cells
incapable of invaginating and prevent the separation of Bolwig's organ
precursors from the optic lobe (Dumstrei
et al., 2002). Virtually identical phenotypes were induced by loss
of Src activity (this work), suggesting involvement of at least the
adherens junction Src in larval visual system formation and that Src should
function either upstream or downstream of EGFR signaling.
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ACKNOWLEDGMENTS |
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