Wellcome/CRC Institute, Cambridge CB2 1QR, England; and Department of Biochemistry, Cambridge University, Cambridge CB2 1QW, England
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Abstract |
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Cells can vary their adhesive properties by
modulating the affinity of integrin receptors. The activation and inactivation of integrins by inside-out mechanisms acting on the cytoplasmic domains of the integrin subunits has been demonstrated in platelets, lymphocytes, and keratinocytes. We show that in the
embryo, normal morphogenesis requires the subunit
cytoplasmic domain to control integrin adhesion at the
right times and places. PS2 integrin (
PS2
PS) adhesion
is normally restricted to the muscle termini, where it is
required for attaching the muscles to the ends of other
muscles and to specialized epidermal cells. Replacing
the wild-type
PS2 with mutant forms containing cytoplasmic domain deletions results in the rescue of the
majority of defects associated with the absence of the
PS2 subunit, however, the mutant PS2 integrins are excessively active. Muscles containing these mutant integrins make extra muscle attachments at aberrant positions on the muscle surface, disrupting the muscle
pattern and causing embryonic lethality. A gain-
of-function phenotype is not observed in the visceral
mesoderm, showing that regulation of integrin activity
is tissue-specific. These results suggest that the
PS2 subunit cytoplasmic domain is required for inside-out
regulation of integrin affinity, as has been seen with the
integrin
IIb
3.
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Introduction |
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CHANGES in cell adhesiveness in response to developmental cues are critical in cell migration and differentiation. Cell adhesion is controlled by modulating the binding properties of cell surface receptors and
their ligands. One of the first instances of this regulation
occurring in embryogenesis happens during compaction of
the early mouse embryo to form the blastocyst. The surface expression of the cell adhesion molecule E-cadherin
does not change, but instead is activated at the eight-cell
stage by intracellular events (Fleming and Johnson, 1988).
Another family of cell adhesion receptors that undergoes
modulation of activity is the integrins, which play a major
role in cell-cell contact and in interactions between cells
and the extracellular matrix (Hynes, 1992
). In a number of
cases it has been shown that integrin affinity for extracellular ligands is modulated by an intracellular mechanism
(inside-out signaling), and that this modulation is correlated with changes in cellular behavior. During blood clotting, platelet binding to fibrinogen is achieved by activating the
IIb
3 integrin, which is already present on the cell
surface in an inactive form (Manning and Brass, 1991
;
Shattil and Brugge, 1991
). The
2 integrins expressed on
leukocytes undergo activation in a similar way to that occurring in
IIb
3 integrins (Arnaout, 1990
; Larson and
Springer, 1990
). At sites of inflammation, the
2 integrins,
which are in an inactive state on circulating leukocytes, are
exposed to and activated by inflammatory mediators during rolling, a low-affinity adhesion step mediated by selectins. An analogous situation exists for T lymphocytes,
where cross-linking of CD3 or the costimulating receptor
CD2 leads to activation of
L
2 integrin on the T cells (van
Kooyk et al., 1989
). In both cases, activation of integrins is
mediated by an adhesion and intracellular signaling cascade that involves protein kinase C (Butcher, 1991
; Dustin
and Springer, 1991
). Modulation of
1 integrin activity has
also been found to occur during the final stage of terminal
differentiation in keratinocytes. In this case, keratinocyte
detachment from the basement membrane correlates with
loss in the ability of the
1 integrins to bind ligands, reduced transport of newly synthesized subunits to the cell
surface, and loss of mature integrins from the cell surface
(Adams and Watt, 1990
; Hotchin and Watt, 1992
; Hotchin
et al., 1995
).
It seems vital to ensure that integrins are only activated
at the right times and places. For example, inappropriate
activation of IIb
3 integrin on resting circulatory platelets
may lead to thrombosis (Kieffer and Phillips, 1990
; Kieffer
et al., 1991
), and activation of the
2 integrins at the wrong
places on leukocytes may lead to inflammation (Arnaout,
1990
; Larson and Springer, 1990
). To confirm this hypothesis, it is important to test these models in vivo.
Integrins cytoplasmic domains are likely targets of cytoplasmic signals that alter integrin affinity (reviewed in Sastry and Horwitz, 1993). Requirements for
intracellular
domain function have been demonstrated for cell adhesion (Hayashi et al., 1990
; Hibbs et al., 1991
), cell spreading (Ylanne et al., 1993
), and adhesion-dependent phosphorylation of pp125 focal adhesion kinase (Guan et al.,
1991
; Hanks et al., 1992
). A variety of experiments in cell
culture have shown that the
cytoplasmic domains of integrins also play critical roles in determining adhesive activity. However, the results are somehow contradictory. It
has been shown that deletion of the
2,
4, or
6 tails after
the highly conserved
subunit membrane proximal motif
GFFKR leads to a loss of adhesive activity (Kassner and
Hemler, 1993
; Kawaguchi and Hemler, 1993
; Shaw and
Mercurio, 1993
; Kassner et al., 1994
). Conversely, deletion of the
L,
1, or
5 cytoplasmic domains does not affect the
ability of the mutant
L
2,
1
1, or
5
1 receptors to mediate adhesion to their respective ligands (Hibbs et al., 1991
;
Bauer et al., 1993
; Briesewitz et al., 1993
). In addition, in
the only system where mutations in integrin subunits have
been tested for their effect on inside-out signaling, truncating the
IIb cytoplasmic domain mimics inside-out activation and results in an integrin that constitutively binds its
ligand fibrinogen (O'Toole et al., 1991
). A mutation that
just deletes the
cytoplasmic GFFKR motif also results in
a constitutively high-affinity
IIb
3 integrin (O'Toole et al.,
1994
). Therefore, inside-out signaling appears to occur by
modulating the
cytoplasmic tail, since deleting it mimicks inside-out activation. We were interested in determining whether the cytoplasmic domains of integrin
subunits are required for regulation of integrin activity during
embryogenesis. In Xenopus, changes in cellular adhesion
to fibronectin are correlated with increased migration of
cells during gastrulation, suggesting that inside-out integrin modulation may play an important role in this developmental event (Ramos et al., 1996
). We wished to test
whether the ability of integrins to be modulated by intracellular interactions with the
subunit is not only correlated with morphogenetic events, but is essential for them
to occur.
Identification and characterization of the Drosophila
position-specific (PS)1 integrins provides the opportunity
to examine integrin function in the developing organism.
The two PS integrins, PS1 and PS2, share a common subunit (
PS), but have different
subunits (
PS1 and
PS2).
The Drosophila PS integrins have amino acid sequence homology with vertebrate integrins, and structural and
functional features of the vertebrate integrins are conserved in the fly proteins (Bogaert et al., 1987
; MacKrell et
al., 1988
; Hirano et al., 1991
; Bunch and Brower, 1992
;
Wehrli et al., 1993
). Throughout development, the two PS
integrins are expressed in complementary but closely associated tissues. For example, high levels of PS integrins are
found at sites where the muscles attach to the epidermis, with
PS1
PS (PS1) restricted to the epidermal cells, and
PS2
PS (PS2) to the muscle cells (Bogaert et al., 1987
;
Leptin et al., 1989
; Wehrli et al., 1993
). An analogous situation occurs in the midgut, with PS1 expressed in the gut
endothelium and PS2 in the visceral mesoderm that surrounds the gut (Bogaert et al., 1987
; Leptin et al., 1989
).
Loss of function mutations in inflated (if;
PS2 subunit
gene) have demonstrated that the PS2 integrin is required
in formation and maintenance of somatic muscle attachments to the epidermis, and the attachment of the visceral
muscles to the midgut epithelium (Babrant and Brower,
1993
; Brown, 1994
; Prokop et al., 1998
).
Although the roles of PS integrins during embryonic development have been intensively studied, we do not know
if their function is regulated during development. It has
been shown that the cytoplasmic domain of the PS integrin subunit is essential during development for
PS functions (Grinblat et al., 1994
). Futhermore, we have shown
that this domain is sufficient to localize a chimeric protein to the end of the muscles, demonstrating the existence of
an inside-out mechanism able to localize the PS2 integrin
(Martin-Bermudo and Brown, 1996
). In this work we test
whether this or other inside-out signaling processes are
used to modulate PS2 integrin activity. Since a deletion of
the
cytoplasmic domain has generated a constitutively active
IIb
3 integrin (O'Toole et al., 1994
), we tested
whether a similar mutation in the
subunit of the PS2 integrin would cause developmental defects, indicating that
modulation of integrin activity is essential for embryonic
development. We show that deletion of the
PS2 cytoplasmic domain leads to formation of a PS2 integrin that mediates formation of an abnormal number and size of muscle
attachments consistent with PS2 being constitutively activated. This result demonstrates that modulation of integrin function through the cytoplasmic domain of the
subunit
is essential for embryonic morphogenesis.
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Materials and Methods |
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Preparation of Mutant Integrins
The UAS-PS2 cytoplasmic tail mutants were generated by site-directed
mutagenesis using the technique described by Picard et al. (1994)
. The two
mutations were made by: (a) inserting a stop codon at the end of exon 11 so a truncated form of the protein would be produced (
cyt); and (b) deleting the highly conserved GFFNR motif (
GFFNR). To make the
cyt
mutant, we amplified a portion of the
PS2 gene from a subclone of
PS2
encoding the COOH end (part of exon 11 and 12) using the PCR. For this
amplification we used three primers: two flanking primers (vector sequence) that were upstream (P1) and downstream (P2) of the mutation
site, and a mutant primer that introduced a stop codon at the end of exon
11 (5'GG CTG CTC TAC AAG TAG GAT CCT TAA CCC TTT CTC
TCG G 3'). The mutant primer and P2 were used to produce a mega
primer, which was then used in conjunction with P1 to produce the desired fragment. This PCR fragment was digested with BglII and SalI, gel-purified, subcloned, and checked by sequencing. This fragment was then used
to replace the corresponding fragment in the wild-type UAS-
PS2c/g construct (Martin-Bermudo et al., 1997
). To obtain the
GFFNR construct,
the same procedure was followed as for
cyt, but in this case the mutant
primer was missing the bases encoding the GFFNR motif at the start of
exon 12 (5' CCT TTA ACC CTA CAG TGC AAC CGG CCA ACG
GAT CAC TCG C 3'). To generate germline transformants, both constructs were injected into flies using standard methods.
The construction of the minigene has been described (Bloor and
Brown, 1998). To generate the
GFFNR mutant form of this minigene, we replaced a SacII-RsrII fragment in the wild-type minigene with that
from the UAS-
PS2
GFFNR.
Drosophila Strains
The integrin mutant allele used in this study is the null allele ifB4 (Brown,
1994) marked with y w f. Since if is on the X chromosome, to select the
mutant embryos we have used two derivatives of the FM6 balancer
a y+
derivative and a lacZ marked derivative
and they have been used as described in Martin-Bermudo et al., (1997). To assay rescue of inflated embryonic lethality, a 4-h collection of embryos at 28°C was transferred to
new apple juice plates in groups of 20 aged for 24 h, and the embryos that
failed to hatch were counted. In these experiments, we distinguished the
mutant embryos with the y marker. We have used the following independent inserts for the different constructs: UAS-
PS2c/g: 3.A (Martin-Bermudo et al., 1997
); UAS-
PS2
cyt: 1, 2, 6; UAS-
PS2
GFFNR: 4, 6; minigene-
PS2: 47, 55; and minigene-
PS2
GFFNR: 76, 96.
Histology
Whole mount staining of embryos was performed using standard procedures. The primary antibodies used were the CF6G11 mouse mAb against
PS (1:1,000; Brower et al., 1984
), the 7A10 PS2 hc/2 rat mAb against
PS2
(1:5; Bogaert, et al., 1987), anti-muscle myosin (Kiehart and Feghali,
1986
), a mouse anti-gp150 (Fashena and Zinn, 1997
), and anti-
-galactosidase (Cappel Laboratories, Malvern, PA). We used a biotin-labeled secondary antibody followed by the Vectastain Elite ABC Kit (Vector Labs,
Inc., Burlingame, CA) enhancement to stain the embryos. To visualize the
visceral mesoderm, dissected guts were fixed in 5% formaldehyde in PBT for 20 min and stained with rhodamine-labeled phalloidin as described in
Xue and Cooley (1993)
. Images were obtained by photography on a Zeiss
Axiophot followed by scanning with a Nikon Coolscan (Nikon Inc., Instrument Group, Melville, NY), or directly from the MRC1000 ConfocalTM microscope (Bio-Rad Laboratories, Hercules, CA). Images were assembled in Photoshop 3.0 (Adobe Systems, Inc., Mountain View, CA)
and labeled in Freehand 5.5TM (Macromedia, San Francisco, CA).
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Results |
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Expression of a PS2 Integrin with
Deletions Within the Cytoplasmic Domain in the
Developing Embryo
Two mutant PS2 subunit genes containing deletions within
the cytoplasmic domain were constructed (Fig. 1). In one
construct, we have deleted the entire cytoplasmic domain
(
PS2
cyt), and in the other we removed the
PS2 variant
form of the highly conserved domain GFFKR (
PS2
GFFNR;
see Materials and Methods). We have already successfully
used the GAL4 system (Brand and Perrimon, 1993
) to rescue the embryonic lethality of inflated mutant embryos that lack the
PS2 subunit by expressing a wild-type
PS2
construct in the mesoderm using a combination of two
GAL4 lines: twist-GAL4 and 24B (Martin-Bermudo et al.,
1997
). We have previously shown that in inflated mutant
embryos, the
PS subunit is not found at the end of muscles, but remains within the endoplasmic reticulum (Martin-Bermudo et al., 1997
; and Fig. 2 b). We have also
shown that we can restore wild-type levels of
PS localization to the ends of muscles in mutant embryos by expressing the
PS2 subunit with the GAL4 system. To analyze the
role of the cytoplasmic domain of the
PS2 subunit in regulating PS2 integrin function during embryogenesis, we
have used the same GAL4 lines
twist-GAL4 and 24B
to express the truncated forms of the
PS2 subunit in the mesoderm of inflated mutant embryos. To examine
whether the mutant
PS2 subunits are able to form heterodimers with the
PS that are properly localized, we
stained the different transgenic embryos with an anti-
PS
antibody. When we express either of the two truncated
forms of the
subunit in these mutant embryos, we find that they can also restore
PS localization (Fig. 2 c and data not shown). From these results we conclude that
PS2 subunits containing deletions in the cytoplasmic domain are
able to form heterodimers with the
PS subunit that are
properly localized to the ends of the muscles. We then
tested whether these mutant integrin heterodimers are
able to function in the developing embryo.
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Regulation of PS2 Integrin Activity
Through the Subunit Cytoplasmic Domain
is Essential for Normal Morphogenesis
Experiments with cells in culture have shown that integrin
cytoplasmic domains can regulate the ligand-binding
function of their extracellular domains (reviewed in Ginsberg, et al., 1992; Hynes, 1992
); however, the results obtained vary depending on the integrin examined. As mentioned in the introduction, deletion of
cytoplasmic
domains can have three different effects on the adhesive
activity of the integrin: to alter it, abolish it, or constitutively active it. Therefore, it was not simple to predict the
consequences that mutating the cytoplasmic domain of the
PS2 subunit would have on embryonic development.
Expression of the PS2
GFFNR mutant subunit in embryos
that lack
PS2 rescues the ifB4 embryonic lethality almost as
well as the wild-type construct (Fig. 3; Martin-Bermudo et al.,
1997
). However, if we express the
PS2
cyt mutant form, we
find that it is unable to rescue the lethality caused by the
absence of
PS2 (Fig. 3). This result is not due to lower levels of expression of this mutant integrin, since using two
copies of the UAS-
PS2
cyt transgene does not significantly
improve the ability of this mutant form to rescue ifB4 (Fig.
3). The failure of the
cyt truncated form of the
PS2 subunit to rescue the inflated embryonic lethality could be due
to the fact that deletion of the cytoplasmic domain creates
a nonfunctional
PS2 subunit. Alternatively, this deletion
could create a constitutively active receptor, which causes
the lethal phenotype. To distinguish between these two
possibilities, we examined the muscle attachment phenotype of embryos in which the wild-type
PS2 subunit has
been replaced with the truncated forms of the
PS2 subunit.
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In a wild-type Drosophila embryo, mononucleated myoblasts fuse to form myotubes, and during this process they
elongate and attach to specific sites on the basal surface of
the epidermis (apodemes). Initially, one pole of the muscle is attached to the epidermis at one apodeme, and the
other consists of a growth cone-like structure that migrates towards the other apodeme (Bate, 1990). Additional membrane projections are also observed on the lateral surfaces of the muscles, but these decrease as the
growth cone is established. By the end of stage 15, approximately half way through embryogenesis, most of the muscles have reached their mature size, their surfaces no
longer show any membrane projections, and both ends are
stably associated with their respective apodemes. In embryos mutant for the PS2 integrin, the muscles then begin to detach from the apodemes, showing that the PS2 integrin is essential for formation of strong muscle attachments, but not for their initial formation (Wright, 1960
;
Brown, 1994
).
To look at the ability of the truncated forms of the PS2
subunit to rescue this muscle detachment phenotype, we
have stained if mutant embryos carrying the different
transgenes with an anti-muscle myosin antibody (Fig. 4).
In the absence of any UAS construct, the if mutant embryos show a muscle detachment phenotype, as expected (Fig. 4 b). The expression of a wild-type UAS-
PS2 construct completely rescues the muscle phenotype (Martin-Bermudo et al., 1997
). if mutant embryos carrying the
UAS-
PS2
GFFNR construct are almost completely rescued,
but they have a very mild muscle detachment phenotype,
with just one muscle detached in 15% of the mutant embryos (not shown). In contrast, we could not detect any
muscle detachment phenotype in those if mutant embryos
carrying the UAS-
PS2
cyt construct (Fig. 4 c), despite the
failure of this construct to rescue embryonic lethality.
Closer examination revealed that both mutant forms of
the PS2 subunit cause two new phenotypes. First, the muscles make attachments that are larger than in wild-type
embryos, as can be seen particularly clearly for the transverse muscles, which have broader tips (compare enlargements in Fig. 4, a and c). Second, the muscles of these embryos form abnormal attachments along the lateral
surfaces. For example, the wild-type ventral longitudinal
muscles (VL) normally extend the whole length of each
segment and attach to apodemes at the segment boundary,
while ventral longitudinal muscles containing the mutant
PS2 integrins make new attachments to the ventral acute muscles (VA) in the middle of the segment (arrowheads in
Fig. 4, d and e). Another aspect of this phenotype is the
aberrant processes that are found extending from the lateral surfaces of the VL towards the lateral transverse muscles (LT), as seen with an antibody against the transmembrane glycoprotein gp150 (Fashena and Zinn, 1997
) that
stains the surface of muscles (Fig. 5). The mutant
PS2
cyt
PS integrin is found at the ectopic attachment sites
(Fig. 4 d), and the extracellular matrix protein Tiggrin (Fogerty et al., 1994
), a PS2 ligand, is also recruited (Fig. 4 e).
Although staining of both PS2 and Tiggrin at the ectopic site appears modest, it is the amount expected for the end
of a single muscle, compared with the segment border
where many muscles attach (three layers from external to
internal). These phenotypes are never observed in if mutant embryos carrying the wild-type UAS-
PS2 transgene.
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The fraction of embryos that have broader tips and ectopic attachment sites varies with the different constructs
(Fig. 3). Using one copy of the UAS-PS2
cyt construct, we
find that ~20% of the mutant embryos have both phenotypes (Fig. 3), and two copies increase the fraction to 38%
(Fig. 3). Enlargement of the tips of the transverse muscles
occurs in all of the segments analyzed (T2-A8). Ectopic
attachments or abnormal processes are found in 3.8 segments per embryo on average (from 27 embryos with a
phenotype), usually in segments A2-A5. As mentioned
before, the UAS-
PS2
GFFNR construct is also able to generate these phenotypes, although less frequently, with only
10% of the embryos having these phenotypes when a single copy of the construct is expressed in them, which increases to 18% when using two copies (Fig. 3). Ectopic
attachments or abnormal processes are found in 1.7 segments per embryo (from 31 embryos with a phenotype), while broadening of the tips of the transverse muscles is
found in all segments examined.
In summary, PS2 integrins containing mutant subunit
cytoplasmic domains are able to mediate adhesion of the
embryonic muscles. However, deletion of the cytoplasmic
domain, and to a lesser degree the GFFNR motif, causes
enhanced adhesive activity such that the extent of the
muscle surface that forms an attachment is increased, and
abnormal attachments are formed along the lateral surfaces of the muscles. When the entire cytoplasmic domain
is deleted, excessive adhesion by the muscles is so extensive that it causes embryonic lethality. These results suggest that there is a mechanism that normally limits PS2 integrin adhesion to the discrete regions at the ends of the
muscles, acting on the cytoplasmic domain of the
PS2 subunit. One way this mechanism could be achieved is by inside-out activation of PS2 integrin affinity only at the muscle termini. Excessive adhesion of the muscles is consistent with the possibility that the
PS2 subunit cytoplasmic domain deletions cause constitutive activation of the PS2 integrin, converting the integrin to a high-affinity form, as
has been shown for the integrin
IIb
3 (O'Toole et al.,
1994
).
Neither of the new phenotypes, larger or ectopic muscle
attachments, are observed when we express the mutant
transgenes in a wild-type genetic background, demonstrating that this phenotype is only caused in the absence of the
endogenous PS2 gene. This observation suggests that if the
wild-type
PS2 subunit is available, the
PS subunit will
form heterodimers with it in preference to the
PS2 subunits that have mutant or absent cytoplasmic tails, even
when the mutant forms are overexpressed (see below).
This suggestion may explain why increasing the number of
copies of the UAS-mutant
PS2 construct increases the frequency of the phenotype, even when a single copy appears
to produce excess
PS2 protein. Therefore, heterodimer
formation may be normally initiated by interactions between the cytoplasmic domains, as suggested by previous
work (Briesewitz et al., 1995
).
The other possible explanation of these results is that excessive muscle adhesion only occurs when all the PS2 integrins on the surface are mutants. This explanation suggests a model where wild-type integrin can send signals to downregulate adhesion from the lateral surfaces.
Different Requirements for Inside-out PS2 Signaling in Different Embryonic Tissues
We next tested the requirements for the cytoplasmic domain in another main site of PS2 integrin function: gut development. We have examined the midgut, the associated
gastric caeca, and the proventriculus, which is part of the
foregut. There are several phenotypes in these tissues associated with the loss of the PS2 integrin: (a) morphogenesis of the gastric caeca does not progress normally, with
only two blunt gastric caeca being formed instead of the
four long gastric caeca formed in a wild-type larvae (see Fig. 7 and Martin-Bermudo et al., 1997
); (b) the midgut
does not elongate properly (Figs. 6 and 7); and (c) the continuity of the visceral mesoderm layer surrounding the gut
is disrupted, as seen by phalloidin staining of filamentous
actin in the visceral muscles (Fig. 6). Development of the
proventriculus is normal until late stage 16 in the absence
of the
PS2 integrin subunit (Pankratz and Hoch, 1995
), but
we have found defects later in embryogenesis (Fig. 7): the
keyhole region of the esophagus migrates inwards to form
the inner layer of the esophagus, but becomes pulled out in PS2 mutant guts (Fig. 7, a and b; arrowheads; Pankratz
and Hoch, 1995
). This, like the muscle attachments, is an
adhesion defect, since the structure initially forms normally (Pankratz and Hoch, 1995
). When we express either
of the two truncated forms of the
PS2 subunit, we observe
a complete rescue of the morphogenesis of the gastric
caeca and elongation of the midgut (Fig. 6 and 7), as we
have seen with the wild-type subunit (Martin-Bermudo et al., 1997
). In addition, the visceral mesoderm phenotype
is almost completely rescued. It is only at late stage 17 that
the visceral mesoderm becomes mildly disrupted at only
one position along the gut of embryos carrying the
PS2
subunit mutant forms (Fig. 6). Both mutant PS2 integrins
fail to rescue the proventriculus phenotype (Fig. 7). Since
these phenotypes occur at the very end of embryogenesis,
they could be due to the reduced stability of the truncated
forms of the
PS2 subunit, or to the fact that the GAL4
drivers that we have used do not result in wild-type levels of PS2 integrin expression in these particular tissues, even
though the wild-type UAS-
PS2 is able to rescue these phenotypes completely. This latter explanation is reinforced
by the fact that genetically defined weak (hypomorphic)
mutations in the
PS2 subunit show identical phenotypes
(Bloor and Brown, 1998
, and our unpublished observations). We have not observed any gain of function phenotypes in the developing gut.
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Does the GFFNR Motif of the PS2
Cytoplasmic Tail Play a Role in Synthesis or Stability
of the PS2 Integrin?
In some cases the cytoplasmic tail of the subunit is essential for effective translation or surface expression of the integrin heterodimer (e.g., Bauer et al., 1993
). Our results in
the Drosophila embryo using the GAL4 system show that
deletions within the
PS2 cytoplasmic domain do not prevent surface expression of the PS2 heterodimer. However,
using the GAL4 system leads to much higher levels of expression of the
PS2 subunits compared with the endogenous level of PS2 integrin expression (Fig. 8 b), and the
excess remains in the endoplasmic reticulum. This overexpression could mask any requirement of the cytoplasmic
domain in making a stable PS2 integrin at the end of the
muscles. In fact, despite this overexpression there appears
to be less PS2 heterodimer at the end of the muscles, with
GAL4-expressed mutants compared with the GAL4-
expressed wild-type
PS2 subunit, judging by the staining
with an antibody against the
subunit (Fig. 1 and data not
shown), although this is hard to quantify. Therefore, to
clarify whether deletion of the GFFNR within the cytoplasmic domain affects the ability of the
PS2 subunit to
form a stable heterodimer with the
PS subunit, we constructed a gene that will express
PS2
GFFNR at levels similar to those of the wild-type gene. To do this we used a
shortened version of
PS2 gene, a minigene containing 24 kb
of the
PS2 genomic DNA, that is able to rescue completely
the embryonic lethality of an if null mutation (Bloor and
Brown, 1998
). This construct is expressed in the somatic
muscles at levels very similar to those of the wild-type
PS2
gene (Fig. 8 c). Deletion of the GFFNR motif within the
cytoplasmic domain of this minigene leads to very low levels of PS2 expression in the muscles (compare Fig. 8, d
with c and a), and it does not rescue the embryonic lethality associated with an if null mutation. Independent lines
of each construct yielded similar results, ruling out the
possibility that the difference in the levels of expression
are due to the site of insertion of the transgenes. From
these results we conclude that the highly conserved
GFFNR motif is indeed required for effective synthesis,
assembly, and/or stability of the PS2 integrin at the muscle
attachment sites.
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Discussion |
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In this work we have examined the requirements for the
integrin PS2 subunit cytoplasmic domain in modulating integrin activity in the developing embryo. We have shown
that the cytoplasmic domain is not required for PS2 integrin adhesion, but instead is required to prevent adhesion
at the wrong locations. In the wild-type Drosophila embryo, PS2 integrin adhesion is tightly localized to specific sites at the ends of the somatic muscles. In contrast, when
the wild-type PS2 integrin is replaced with mutant forms
of the PS2 integrin lacking portions of the cytoplasmic domain, then PS2 integrin-mediated adhesion is no longer
tightly localized. The muscles make ectopic attachments
and send out growth cone-like processes, not only from
the ends of the muscles, but also from their lateral surfaces. This indicates that we have created a PS2 integrin
that is functional around the entire surface of the muscles
rather than just at the ends. Therefore, we conclude that the cytoplasmic domain of the
PS2 subunit is required to
keep the PS2 integrin inactive along the lateral surfaces of
the muscles. Normally this inhibition is only released at
the ends of the muscles where strong attachment is
needed.
Control of PS2 integrin adhesion at the ends of the muscles is part of a multistep process that results in a precise
pattern of strong muscle-muscle and muscle-epidermal
cell adhesion. This process starts with an initial recognition/adhesion step, where the muscles make their contact
with and attach to specific epidermal cells. This first step is
independent of integrins and involves formation of short
regions of close membrane contact that are not by themselves strong enough to withstand the force of muscle contraction in the absence of integrin adhesion (Prokop et al., 1998). The muscle-epidermis and muscle-muscle attachments then differentiate by forming extensive hemiadherens junctions, and strong PS integrin-dependent adhesion
develops. The data we have presented here suggests that
part of muscle attachment differentiation is activation of
PS2 integrin adhesion, specifically at the ends of the muscles. This activation could be controlled by an inherent intracellular polarity of the muscles that localizes a protein to the ends of the muscles, which activates the PS2 integrin
through the cytoplasmic domain of the
PS2 subunit. However, it seems more likely that PS2 integrin adhesion
should only occur after successful attachment of the muscle to the correct cell, and therefore be triggered by an extracellular signal. This mechanism would avoid the kind of
ectopic attachments that we have observed when the PS2
integrin cytoplasmic domain mutants are present in the muscles. The extracellular signal could be transmitted by
an unknown transmembrane receptor that sends an intracellular signal, acting on the
PS2 subunit cytoplasmic tail
to initiate adhesion. It is also possible that the PS2 integrin
itself transmits the signal that results in integrin activation.
The low-affinity interaction between PS2 and a localized
extracellular ligand might convert PS2 into a high-affinity
conformation that binds more strongly to a variety of extracellular ligands, including Tiggrin. A similar ligand-dependent activation has been described for the
IIb
3 integrin
(Du et al., 1991
). Interaction of PS2 with ligands could also
stabilize the active state at the ends of the muscles, as has
been proposed for other integrins (Keizer et al., 1988
; van Kooyk et al., 1991
). If this last model is true, then deletion of the cytoplasmic tail of the
PS2 subunit mimics an activation that normally occurs by extracellular interaction of a
ligand with the integrin.
Our experiments have not determined the mechanistic
basis of the activation that occurs when the PS2 cytoplasmic tail is deleted. If the PS2 integrin behaves like the
platelet integrin
IIb
3, then activation occurs by a conformational change in the protein that increases the affinity of
the integrin for its ligands, which may be combined with an
increase in avidity promoted by clustering of the integrin.
As the cytoplasmic domain mutants of the PS2 integrin are
expressed at modestly lower levels than the wild-type PS2
integrin, it seems unlikely that the gain-of-function phenotypes are caused by the mutations, leading to excessive
quantities of the integrin, or one that turns over less rapidly. Indeed, the gain-of-function phenotypes do not occur
when the PS2 heterodimer is overexpressed by GAL4,
driving expression of both UAS-
PS2 and UAS-
PS constructs
(our unpublished observations). In addition, the process of
muscle attachment occurs very rapidly (within 4 h), making changes in the rate of turnover or assembly less likely to contribute to the gain-of-function phenotypes. It is
therefore tempting to speculate that, similar to the integrin
IIb
3, the affinity of PS2 integrin for extracellular ligands
is modulated by the
PS2 cytoplasmic domain.
We have also analyzed the effect that deletions within
the cytoplasmic tail of the PS2 subunit have on the function of the PS2 integrin in other tissues. In general, the
mutant PS2 integrins can replace the wild-type PS2 integrin in the visceral mesoderm and promote normal morphogenesis of the midgut. We have not detected any gain-of-function phenotypes that would indicate that control of
the activation state of the PS2 integrin in the visceral mesoderm is as vital as it is in the somatic muscles. This result
demonstrates that the different embryonic tissues have
different requirements for the
PS2 subunit cytoplasmic
domain, consistent with previous studies showing that different cell types have different ways of regulating integrin
activity. If information processing by the signaling machinery of a cell is dependent on cell type, then one consequence might be for one cell type to possess integrins in a
higher activation state than other types. Therefore, there are two possible ways to explain how the
PS2 cytoplasmic
domain can contribute in different ways to PS2 function:
(a) there could be tissues in which the PS2 integrin is required to be active all the time, as in the visceral mesoderm, while in other tissues like the somatic muscles its activity needs to be regulated. This difference may reflect
the importance for the large multinucleate somatic muscles to have discrete sites of integrin adhesion, while the
visceral muscles may have more uniform adhesion along
the cell surface. (b) the PS2 integrin might perform different functions in the different tissues (e.g. mediating adhesion, migration, or differentiation), and the
PS2 cytoplasmic tail might not be essential for all of these cellular
functions. This latter hypothesis is consistent with data
showing that deletion of the
5 cytoplasmic domain of the
5
1 integrin still permits efficient adhesion and increases
in tyrosine phosphorylation, but causes reduced motility and cell spreading (Bauer et al., 1993
). Identification of
proteins that interact with the
subunit cytoplasmic domains will provide insight into the mechanisms of this regulation.
Finally, our results have shown that deletion of the entire cytoplasmic domain has a stronger effect than deletion
of the highly conserved motif GFFNR, suggesting that
there must be other regions within the PS2 cytoplasmic
domain that contribute to PS2 integrin function regulation. A more detailed analysis of small deletions within the
cytoplasmic domain will help to identify the different regions involved in regulating PS2 integrin function.
In summary, our results show that during embryogenesis
it is essential to have a mechanism to regulate integrin
function at the right places. We have found that being able
to keep integrin adhesion off at the right moment and
place is just as important as having active integrins. We
also show that a mechanism to regulate integrin activity
exists in the embryo, and that activation of the integrin by
this mechanism can be mimicked by deletion of the PS2
subunit cytoplasmic tail, suggesting that the mechanism acts on this domain. A further characterization of this
mechanism will allow us to test whether it is only required
in some tissues during embryogenesis, or whether a similar
mechanism is used in different tissues.
![]() |
Footnotes |
---|
Received for publication 1 December 1997 and in revised form 6 February 1998.
Address all correspondence to Nicholas Brown, Wellcome/CRC Institute, Tennis Court Road, Cambridge CB2 1QR, England. Tel.: 44-1223-334-128; Fax: 44-1223-334-089; E-mail: nb117{at}mole.bio.cam.ac.ukWe would like to thank A. Brand, D. Kiehart, and K. Zinn for providing reagents and fly stocks, and John Overton for technical assistance. We thank A. Gonzalez Reyes, S. Gregory, and Reviewer 1 for helpful comments on the manuscript.
This work was supported by fellowships from the Spanish Ministerio de Educacion y Ciencia and the European Economic Community to M.D. Martin-Bermudo and a Wellcome Trust Senior Fellowship to N.H. Brown.
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