Huntsman Cancer Institute and Department of Biology, University of Utah, Salt Lake City, UT 84112, USA
* Author for correspondence (e-mail: mary.beckerle{at}hci.utah.edu)
Accepted 17 March 2003
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SUMMARY |
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Key words: LIM domains, Cell adhesion, Cytoskeleton, Integrins, Drosophila
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INTRODUCTION |
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One cytoplasmic protein that has been postulated to play a role in integrin
function is PINCH, a protein comprising five tandemly arrayed LIM domains
(Rearden, 1994). LIM domains
are double zinc-finger structures that serve as protein-binding interfaces
(Michelsen et al., 1993
;
Schmeichel and Beckerle,
1994
); therefore, PINCH probably functions as a molecular scaffold
that supports the assembly of a multi-protein complex at sites of integrin
enrichment. In agreement with this notion, biochemical studies of human PINCH
have identified Integrin-Linked Kinase (ILK) as a binding partner for the
first LIM domain of PINCH (Tu et al.,
1999
), and the SH2-SH3 adaptor protein NCK2 as a partner for the
fourth LIM domain (Tu et al.,
1998
). Although the complete binding partner repertoire of PINCH
remains to be elucidated, the colocalization of PINCH with integrins and its
capacity to bind ILK and NCK2 provided the first hints that PINCH might play a
role in recruitment of regulatory factors to integrin-rich sites and may thus
contribute to integrin function (Wu,
1999
; Wu and Dedhar,
2001
).
Further support for the view that PINCH is essential for integrin function
came from studies in which PINCH expression in C. elegans was
compromised by RNA interference. Developing embryos that are deficient in
PINCH display a paralyzed-at-twofold (PAT) phenotype, similar to that observed
in integrin mutants (Hobert et al.,
1999). In spite of the comparable developmental arrest when either
integrin or PINCH function is compromised in the worm, this phenotypic
description did not provide mechanistic insight into the relationship between
PINCH and integrins. Recently, however, it was demonstrated that expression of
a dominant-negative form of PINCH in tissue culture cells results in
compromised cell adhesion (Zhang et al.,
2002c
). These findings are consistent with the view that PINCH is
required for integrin-dependent cell adhesion. However, because the LIM domain
is a conserved structural feature found in many modular proteins
(Schmeichel and Beckerle,
1994
; Dawid et al.,
1998
; Bach, 2000
),
it is essential that conclusions from studies using dominant-negative tools be
confirmed using a loss-of-function strategy where specificity is insured.
We have taken a genetic approach in Drosophila to define the
physiological contributions of PINCH to integrin-mediated cellular events in
vivo. Drosophila provides an excellent model system with which to
study integrin function as integrin-dependent cell adhesion is required for
proper organization of multiple embryonic and adult tissues
(MacKrell et al., 1988;
Brower and Jaffe, 1989
;
Leptin et al., 1989
;
Brabant and Brower, 1993
;
Brown, 1994
;
Brower et al., 1995
). Moreover,
Drosophila is a particularly valuable system for assessing PINCH
function because, in contrast with C. elegans and mouse, where
multiple PINCH family members are present
(Hobert et al., 1999
;
Zhang et al., 2002a
), only a
single pinch gene exists in the fly. Our analysis of the cellular and
developmental consequences of mutations in Drosophila pinch
illustrates that PINCH is essential for integrin-dependent cell adhesion
events in embryos and adults and reveals that PINCH is required to stabilize
membrane-cytoskeletal linkages at sites of cell-substratum anchorage.
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MATERIALS AND METHODS |
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Molecular biology
All cloning and other DNA manipulation was carried out essentially as
described (Sambrook, 1989), with exceptions noted below. Genomic DNA from
stck heterozygous flies was subjected to a standard PCR reaction
using primers that would amplify the coding region for pinch. The PCR
reaction, representing a mixed population of amplification products from both
the stck chromosome and the balancer chromosome, was sequenced
directly with pinch primers. The amplification was carried out twice,
and each time the PCR reaction product was sequenced on both strands to ensure
that the lesions detected were not due to a polymerase error propagated during
the PCR amplification. A lesion in pinch was identified when the
trace of the sequencing reaction went out of phase, indicating a point where
the two PCR products differed in their sequence. This phase-shift was seen in
sequencing reactions on both strands. As a control, genomic DNA from
82w+ flies was subjected to the same treatment in
parallel and sequenced.
To generate the pinch genomic rescue construct, genomic DNA encompassing pinch was amplified by PCR, cloned into the TA vector (Invitrogen) and excised with SpeI and NotI. This fragment, which contains the pinch transcription unit plus 2882 bp of DNA 5' to the transcription unit and 436 bp 3', was cloned into the pCaSpER4 vector (kindly provided by Carl Thummel) to generate the P[w+ pCas-pingen] transformation construct.
Northern blot and RT-PCR
Total RNA was isolated from Drosophila at various stages of
development, using Trizol (Gibco BRL), following the manufacturer's
recommendations. Approximately 15 µg RNA from each sample was loaded and
run on a denaturing formaldehyde gel and transferred to Gene Screen nylon
membrane (PerkinElmer Life Sciences). The resulting blot was hybridized in
Ultrahyb (Ambion) and processed according to the manufacturer's instructions.
Band intensities were assessed by scanning the autorads and quantifying pixel
values on a Kodak 440 image station (Kodak); pinch values were
normalized relative to the rp49 signals to control for any unequal
loading.
RT-PCR analysis of the pinch transcription unit was conducted on mRNA samples from 16- to 24-hour-old embryos, third instar larvae and adult females, using primers that would amplify the entire PINCH transcript. In each case, 50 ng of mRNA was used in a reaction according to the manufacturer's instructions (Access RT-PCR introductory kit, Promega). Resulting cDNAs were sequenced with pinch-specific primers.
Antibody production, affinity purification, immunoprecipitation and
immunodetection of proteins
Rabbit polyclonal antisera were generated (Capralogics, Hardwick, MA)
against an 18 amino acid peptide (ELRRRLRTAHEMTMKKNT) corresponding to
residues 318-335 of the predicted PINCH protein. Anti-PINCH antibodies were
affinity-purified prior to use.
PINCH complexes were immunoprecipitated with the affinity-purified
anti-PINCH antibody from Drosophila 0- to 18-hour-old embryo
extracts, prepared from a transgenic line carrying an ILK::GFP genomic
construct (Zervas et al.,
2001). Approximately 1 µg of affinity-purified anti-PINCH
antibody, pre-immune serum or anti-MLP60A antisera
(Stronach et al., 1996
) was
used for each immunoprecipitation. Recovered proteins were resolved by
SDS-PAGE and analyzed by western immunoblots probed with affinity-purified
PINCH polyclonal antibody at a dilution of 1:10,000, anti-GFP mAb (Clontech,
Palo Alto, CA) at a dilution of 1:500, or anti-MLP60A antisera
(Stronach et al., 1996
) at a
dilution of 1:600.
Embryos were collected and prepared for immunofluorescence analysis
essentially as described (Patel,
1993). In some cases (e.g. Fig.
3), embryos were prepared for immunofluorescence by heat fixation
(Miller et al., 1989
), as
opposed to the normal formaldehyde fixation. Developing wings were dissected
from staged pupae (
45 hours after puparium formation), and prepared for
immunofluorescence using published procedures
(Wolff, 2000
). Antibodies to
proteins visualized in this study were used at the following concentrations:
rabbit anti-PINCH (this study) 1:500; mouse anti-ß-galactosidase
(Promega) 1:2000; rabbit anti-ß-galactosidase (Cappel) 1:5000; rabbit
anti-Mlp84B (B50) (Stronach et al.,
1996
) 1:500; mouse anti-ßPS integrin (CF6G11 ascities)
(Brower et al., 1984
) 1:1000;
and rabbit anti-dPak (Harden et al.,
1996
) 1:500. Secondary antibodies were preabsorbed against
w1118 embryos before use. For phalloidin
staining, the fixation procedure was changed such that embryos were
devitellinized in 80% ethanol instead of methanol. Images were obtained from a
LSM-510 confocal microscope (Zeiss).
|
![]() |
RESULTS |
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The lethality associated with homozygous stck mutations can be rescued by introduction of a transgene that encodes wild-type PINCH (data not shown). Further confirmation that PINCH is encoded by the stck locus comes from western immunoblot analysis of PINCH protein levels in stck mutants (Fig. 1D). Affinity-purified antiserum directed against a C-terminal PINCH epitope recognizes a single polypeptide with an apparent molecular mass of 31 kDa in wild-type embryos (Fig. 1D, lane 1). Wild-type PINCH protein levels are significantly reduced in stck zygotic mutants (Fig. 1D, lanes 2 and 3) and the protein is undetectable when maternal PINCH is also eliminated (Fig. 1D, lane 4). Collectively, these data provide compelling evidence that Drosophila pinch is encoded by the stck locus.
Mutations in PINCH destabilize membrane cytoskeletal linkages in
embryonic muscle and compromise cell anchorage
We have characterized the phenotypes associated with the two stck
alleles described above. When examined as hemizygous mutations, greater than
85% of the stck mutant embryos die, indicating a strong requirement
for PINCH during embryonic development. Comparison of wild-type larvae and the
few stck mutant larvae that survive to hatch revealed dramatic
morphological differences. The stck mutant larvae are significantly
shorter than wild-type larvae (0.50±0.05 mm versus 0.72±0.03
mm). Additionally, stck mutant larvae are nearly immobile, a
phenotype that suggests impaired muscle function, and die within 24 hours of
hatching.
pinch transcript is expressed prominently in the developing
somatic muscles of Drosophila embryos
(Hobert et al., 1999),
therefore we examined the mutant embryos more closely for any perturbations in
somatic muscle patterning and development. Initial muscle patterning is not
affected in stck mutants (data not shown), indicating that PINCH is
not required for muscle cell differentiation, fusion or migration. Defects in
muscle morphology are first detected in stck mutants at embryonic
stage 16. By comparing wild-type and mutant embryos that are stained with
antibody directed against Mlp84B, a muscle-specific protein that is associated
with the contractile apparatus and enriched at muscle-attachment sites
(Stronach et al., 1996
), it is
evident that the mutant muscles exhibit a distorted morphology (Fig.
2A,B).
The embryonic musculature is less organized in stck mutants compared
with their wild-type counterparts, and gaps are evident occasionally between
adjacent muscle cells, indicating a failure of some muscle-attachment sites
(Fig. 2B, arrowheads).
|
Both stck17 and stck18 alleles retain some PINCH-coding sequence. In particular, these mutant alleles could theoretically support the production of C-terminally truncated PINCH products that might retain partial function or have dominant-negative activity. In order to assess whether stck17 and stck18 behave as simple loss-of-function alleles, we compared the cellular phenotypes of stck17 and stck18 hemizygotes with embryos that carry a homozygous deletion of the stck locus (l(3)097) and observed a comparable terminal phenotype (Fig. 2F,G). These findings illustrate that the stck17 and stck18 alleles disrupt PINCH function to a similar extent as occurs when PINCH function is completely eliminated by a gene deletion. Thus, stck17 and stck18 do not display any residual PINCH activity that ameliorates the mutant phenotype relative to what is observed in a molecular null. Moreover, neither stck17 nor stck18 heterozygotes display any cellular defects or loss of viability (data not shown) that might be anticipated if the stck17 and stck18 alleles produced a dominant-negative product.
Because pinch transcripts are maternally inherited, we evaluated the phenotype of animals in which both zygotically and maternally derived PINCH were eliminated by construction of germline clones. Analysis of maternal/zygotic stck mutants did not reveal additional phenotypes that were not evident in zygotic stck mutants; however, the disturbance in muscle morphology was evident at an earlier stage than for the zygotic mutants, with actin clumping apparent in some muscle cells by the end of stage 16 (data not shown), consistent with the time of onset of muscle contraction.
PINCH protein is prominently expressed in embryonic muscle where it
localizes at muscle-attachment sites
Since the stck mutants exhibited defects in the anchorage of actin
filaments at the myotendinous junction, we postulated that PINCH might be a
constituent of these cell-substratum attachment sites. Indeed, by
immunocytochemical analysis, we detect PINCH protein in the developing somatic
muscles, with prominent enrichment at the muscle-attachment sites
(Fig. 3A-C). PINCH is also
detected in other musculatures including the dorsal vessel (the heart
equivalent in Drosophila; Fig.
3A,C),
the visceral musculature surrounding the gut (Fig.
3B,D)
and in the pharyngeal muscles (Fig.
3B,C).
There is also prominent staining in the midgut epithelium
(Fig. 3D). The
affinity-purified serum also labels the chordotonal organs, but this appears
to be due to crossreaction with another protein because this staining remains
in stck17 maternal/zygotic mutants, whereas all muscle
attachment site staining is absent (data not shown).
Integrins are necessary for the proper localization of PINCH to the
muscle-attachment sites
The Drosophila integrin subunits PS2 and ßPS are also
enriched at muscle-attachment sites, where they participate in the adhesion of
the muscle termini to a specialized ECM, the tendon cell matrix
(Leptin et al., 1989
;
Brown, 1994
). Using confocal
microscopy, we found that PINCH is precisely colocalized with ßPS
integrin at muscle-attachment sites in the somatic muscle termini (Fig.
4A,B).
PINCH and ßPS integrin proteins also display overlapping patterns of
concentration in other tissues such as the visceral musculature, pharyngeal
muscles and epithelial tissues (data not shown).
|
In complementary experiments, we examined ßPS integrin distribution in
wild-type and stck mutant embryos. Wild-type embryos show a striking
accumulation of ßPS integrin at muscle-attachment sites
(Fig. 4G)
(Leptin et al., 1989).
Although muscle morphology is perturbed in stck mutants, ßPS
retains the capacity to localize at muscle-attachment sites when PINCH
function is compromised by mutation (Fig.
4H). Thus, the appropriate targeting of ßPS integrin to the
cell surface and their concentration at adhesive junctions can occur in the
absence of PINCH.
The lethal phenotype associated with stck mutations does not arise
due to a failure of ILK to localize properly
Based on biochemical studies in vertebrate systems, it has been suggested
that an integrin-ILK-PINCH complex might be necessary for integrin-dependent
cell adhesion (Li et al.,
1999; Tu et al.,
1999
; Wu, 1999
).
Consistent with this view, a recent characterization of Drosophila
ILK revealed that ILK colocalizes with ßPS integrin at muscle-attachment
sites and is required for integrin function
(Zervas et al., 2001
). As can
be seen in Fig. 5A-C, PINCH and
ILK display completely overlapping patterns of localization in
Drosophila muscle, with both proteins prominently enriched at the
muscle-attachment sites. PINCH and ILK are also co-expressed in the visceral
mesoderm and pharyngeal muscles. Thus, PINCH, ILK and ßPS integrin are
co-residents of the same cellular compartments in vivo.
|
The actin phenotypes we describe for stck mutants are similar to
those recently reported for Drosophila ILK mutants, in that the actin
filament linkage appears to be unstable and actin filaments detach from the
muscle membrane (Zervas et al.,
2001). As ILK and PINCH associate in vivo, the stck
mutant phenotype may arise as a result of ILK mislocalization. We explored
this possibility by examining the localization of an ILK::GFP fusion protein
in stck mutant embryos derived from stck17
germline clones (i.e. embryos that lack functional maternally-derived and
zygotic PINCH protein). In stck mutant embryos, ILK::GFP retains the
capacity to localize at muscle-attachment sites
(Fig. 5F). Thus, the phenotypes
seen in a stck mutant cannot be attributed to mislocalization of
ILK.
PINCH function is required for the stable adhesion between epithelial
layers in the wing
pinch transcription is upregulated in pupae and adults
(Fig. 1B), suggesting that
PINCH may have functions during these later developmental stages as well.
Moreover, the two stck alleles that encode PINCH were originally
identified in a genetic screen for potential integrin effectors that relied on
wing blister formation (Prout et al.,
1997). Using mitotic recombination, we confirmed that homozygous
stck mutations cause wing blistering
(Fig. 6A). This observation
suggests that PINCH is expressed in the wing epithelium, and is required for
integrin-dependent adhesion in this tissue, but neither the expression nor the
subcellular localization of PINCH in wing epithelium had been described. To
examine directly the expression and subcellular distribution of PINCH in the
developing Drosophila wing, we performed immunocytochemical analysis
with anti-PINCH and anti-ßPS integrin antibodies. We first examined PINCH
expression in wing discs dissected from wandering third instar larvae and
found that PINCH was associated with wing cell membranes; ßPS integrin
displays a similar pattern at this stage of development (data not shown).
Later in development, when the wing epithelia have become apposed, ßPS
integrin becomes enriched at basal junctions that form between the two layers
(Fristrom et al., 1993
)
(Fig. 6B). We find that PINCH
is enriched at this junction and is also associated with the cell cortex
coincident with sites of integrin accumulation (Fig.
6C,D).
Collectively, our findings support the view that PINCH is required for
integrin function in both embryos and adults.
|
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DISCUSSION |
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A link between PINCH and integrin function
Genetic analyses of the roles of integrins in Drosophila have
clearly highlighted the importance of integrins for adhesion and signaling in
vivo (Martin-Bermudo and Brown,
1999; Bokel and Brown,
2002
). We report that Drosophila PINCH is colocalized
with integrins in both muscle and epithelial cells. Integrins retain the
capacity to accumulate at muscle-attachment sites in stck mutants,
illustrating that PINCH does not have an obligatory role in the proper
processing and membrane targeting of integrins in vivo. The integrin staining
in stck mutants does lack the high degree of order and lateral
registration observed in wild-type embryos. In the Drosophila system,
it is difficult to distinguish whether this modest disorganization simply
reflects the underlying disturbance of the musculature or if it is revealing
some contribution of PINCH to maintenance of spatially restricted integrin
localization. In C. elegans embryos in which PINCH function is
compromised by unc-97 mutation, both integrin and vinculin spread
laterally beyond their normal zones of accumulation in dense plaques,
suggesting a role for PINCH in clustering of adhesive junction components in
this system (Hobert et al.,
1999
).
Interestingly, PINCH depends on the presence of integrins for its stable
accumulation at muscle-attachment sites. The physiological roles of several
other proteins, including Talin, ILK, Myosin II and Short Stop, that
colocalize with ßPS integrin at Drosophila muscle-attachment
sites have recently been characterized
(Gregory and Brown, 1998;
Bloor and Kiehart, 2001
;
Zervas et al., 2001
;
Brown et al., 2002
). These
proteins display variable levels of dependence on integrins for their
localization. Like Talin, a well-established integrin effector
(Horwitz et al., 1986
;
Brown et al., 2002
;
Calderwood et al., 2002
),
PINCH depends on the presence of integrins for its concentration at
muscle-attachment sites. The reliance of PINCH and Talin on integrins for
their spatially restricted accumulation in muscle emphasizes their connection
to the integrin receptors.
Integrins must establish links to both extracellular determinants and to
intracellular cytoskeletal elements in order to support strong adhesion
(Critchley et al., 1999;
Brown et al., 2000
).
Examination of the cellular defects in stck mutant muscle suggests
that PINCH contributes to the stabilization of actin-membrane linkages at
integrin-rich adhesion sites. In a stck mutant muscle cell, the actin
filaments lose their linear organization and eventually accumulate in clumps
at one end of the cell. We interpret these defects to mean that a primary
consequence of disturbed PINCH function is a destabilization of the linkage
between the actin cytoskeleton and the muscle membrane; it appears that the
actin-membrane attachments in stck mutants lack the mechanical
strength to remain intact during cyclic muscle contraction. Because integrin
functionality relies on the ability of the receptors to establish a
transmembrane link between the cytoskeletal elements and the extracellular
matrix, reduced substratum attachment strength and/or stability might also be
expected to occur if membrane cytoskeletal linkages were compromised.
Consistent with this prediction, loss of adhesion is evident in the
stck17-/- wing cell clones and, to some extent,
in muscles of stck mutant embryos.
The relationship between PINCH and integrin-linked kinase
The molecular architecture of PINCH suggests that it may function as a
platform for the docking and/or productive juxtaposition of protein partners.
ILK, a binding partner of PINCH, is thus a candidate to collaborate with PINCH
in the stabilization of integrin-cytoskeletal linkages. Consistent with the
view that PINCH and ILK could cooperate to promote stable actin anchorage at
sites of integrin-mediated adhesion, the phenotypes that result from
compromised function of either protein in Drosophila are very similar
(this report) (Zervas et al.,
2001). Moreover, we show that PINCH and ILK are colocalized in
Drosophila embryos and are recovered in a protein complex isolated
from embryos by immunoprecipitation. Drosophila PINCH also interacts
directly with ILK using two-hybrid methods (J. L. Kadrmas, S. M. Pronovost and
M.C.B., unpublished). These latter results are consistent with findings
reported previously for vertebrate PINCH and ILK
(Li et al., 1999
;
Tu et al., 1999
). Confirmation
that PINCH and ILK interact in Drosophila was important as
biochemical findings in vertebrate systems are not always recapitulated in the
fly (Zervas et al., 2001
).
PINCH and ILK also colocalize at actin-membrane anchorage sites in C.
elegans muscle, and elimination of either gene product was shown to
produce a paralyzed at twofold stage (PAT) phenotype similar to that seen for
b-integrin mutants (Hobert et al.,
1999
; Mackinnon et al.,
2002
). Collectively, results in both invertebrate and vertebrate
systems illustrate that the capacity to form a PINCH/ILK complex has been
conserved through evolution.
Given the fact that ILK and PINCH colocalize, co-precipitate and have similar loss of function phenotypes, it was possible that disturbed PINCH function could adversely affect ILK localization and that such mislocalization might account for the stck mutant phenotype. To explore this possibility we examined the localization of ILK in stck mutant embryos and found that ILK is unperturbed in its ability to accumulate at muscle-attachment sites, even when a dramatic lethal phenotype is evident in stck mutant embryos. As noted above, ßPS integrin also accumulates at muscle-attachment sites in stck mutant embryos. These findings illustrate that the proper localization of integrin and ILK is not sufficient to stabilize actin membrane linkages at sites of integrin-dependent adhesion, and define PINCH as a critical component of the molecular machinery necessary for the tethering of actin to the integrin-rich membranes.
The demonstration that single ilk and stck mutants both
display deficiencies in integrin-dependent processes illustrates that neither
PINCH nor ILK is sufficient on its own to support full integrin function. It
is possible that PINCH acts as a positive regulator of ILK function, either by
modulating ILK function by direct binding or by recruitment of an
ILK-modifying factor. Alternatively, ILK may activate some PINCH function that
is crucial for stabilization of actin-membrane linkages. Finally, a PINCH-ILK
protein complex may be a key component of the platform necessary for the
recruitment of other proteins required to achieve stable actin-membrane
associations. In this regard, it is of interest that PINCH and ILK can be
recovered in a complex with the ILK-binding partner, CH-ILKBP, a calponin
domain-containing protein related to Affixin and Actopaxin that could provide
the link to actin filaments (Tu et al.,
2001; Yamaji et al.,
2001
; Nikolopoulos and Turner,
2002
). Because the localization of Drosophila PINCH is
dependent on integrins, the establishment of PINCH-ILK complexes at
muscle-attachment sites would not be supported in the absence of integrin
function. This dependence of PINCH localization on integrins could provide a
means to couple integrin adhesive function to its role in cytoskeletal
anchorage.
In vertebrate cells, PINCH and ILK appear to be mutually dependent on each
other for their localization to integrin-rich focal adhesions
(Zhang et al., 2002b).
However, as noted above, despite their ability to interact with each other,
PINCH and ILK show distinct requirements for their recruitment to specific
subcellular domains in Drosophila. In particular, we show that PINCH
requires functional integrins for its localization to muscle-attachment sites,
whereas it has previously been demonstrated that Drosophila ILK fails
to bind integrins directly and localizes normally in an integrin mutant
(Zervas et al., 2001
). Rather
than employing an association with integrins, ILK may rely on a protein such
as Paxillin for its targeting to integrin-rich sites
(Nikolopoulos and Turner,
2001
). Although Drosophila PINCH requires integrins for
its stable accumulation at muscle-attachment sites, there is no evidence that
PINCH can associate directly with integrin cytoplasmic domains, therefore
additional proteins probably act as a bridge.
Drosophila as a model system for the study of integrin function
Recently, two laboratories have independently conducted a clever genetic
screen for potential integrin effectors in Drosophila
(Prout et al., 1997;
Walsh and Brown, 1998
). These
screens relied on the fact that loss of integrin function results in a readily
scorable blistering phenotype because of compromised epithelial cell adhesion
in the wing. The screening strategy employed mitotic recombination to allow
examination of homozygous mutant cell clones in an otherwise heterozygous
background. This approach permitted a large number of independent mutations to
be examined for effects on integrin-dependent adhesion. Over 25 loci were
identified that could produce wing blisters when mutated. However, to date,
the molecular lesions associated with these wing blister mutations have only
been identified for a few loci (Prout et
al., 1997
; Gregory and Brown,
1998
; Walsh and Brown,
1998
; Brown et al.,
2002
). The identification of stck as the PINCH gene in
Drosophila gives additional confidence that the genetic screens are
identifying molecules important for integrin function. Moreover, the unique,
but related, phenotypes that result when genes encoding different components
of integrin adhesive membranes are mutated have provided significant new
insight into how various accessory proteins cooperate with integrin. For
example, Short Stop appears to contribute to integrin-dependent cell adhesion
by coupling the microtubule cytoskeleton to the adhesive membrane
(Gregory and Brown, 1998
). Our
characterization of PINCH loss of function phenotypes suggests that PINCH
plays a key role in stabilizing the link between cytoplasmic actin filaments
and the integrin-rich adhesive membrane. Collectively, these analyses allow a
precise molecular dissection of integrin function in Drosophila.
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ACKNOWLEDGMENTS |
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