1 Umeå Center for Molecular Pathogenesis, Building 6L, Umeå
University, Umeå, 901 87, Sweden
2 The Wellcome Trust/Cancer Research UK, Gurdon Institute and Department of
Anatomy, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR,
UK
3 The Salk Institute, Molecular and Cell Biology Laboratory, 10010 North Torrey
Pines Road, La Jolla, CA 92037-1099, USA
Author for correspondence (e-mail:
ruth.palmer{at}ucmp.umu.se)
Accepted 14 September 2004
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SUMMARY |
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Key words: Drosophila, FAK, Integrins, Signal transduction
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Introduction |
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FAK localization to focal adhesions is mediated by the C-terminally located
focal adhesion targeting (FAT) domain
(Hildebrand et al., 1993;
Hildebrand et al., 1995
).
Additionally, the N-terminal region of FAK has been proposed to bind directly
to the cytoplasmic tail of ß-integrins, which are thought to be the major
regulators of FAK activity (Schaller et
al., 1995
), and it has recently been reported that the cytoplasmic
tail of ß1-integrin stimulates FAK activity in vitro
(Cooper et al., 2003
).
Integrins are the major family of cell surface receptors that link the
extracellular matrix (ECM) to the actin cytoskeleton
(Watt, 2002). They are
heterodimeric glycoproteins, which function as receptors for a variety of ECM
proteins such as fibronectin, collagen and laminin. In addition to providing
this structural link of cell adhesion, integrins also activate many
intracellular signaling pathways and influence many intracellular events that
play key roles during, for example, development and immune responses
(Hynes, 2002
). The mechanism
that links integrin clustering to FAK activation and the role of FAK in
integrin signaling pathways has been intensively studied in mammalian cells in
culture, leading to the identification of a number of diverse pathways
downstream of FAK, but the significance of these in the intact animal is as
yet unclear.
An essential role for FAK in mammals has been demonstrated by genetic
studies in mice. The mouse FAK knockout
(Ptk2/ Mouse Genome Informatics)
dies early in embryogenesis (Ilic et al.,
1995), with defects in mesoderm development that are similar to
those caused by the knockout of fibronectin
(George et al., 1993
). Studies
on FAK-null cells derived from these mice have indicated that rather than
being involved in the assembly of integrin adhesive junctions, FAK may be
involved in their remodeling (Webb et al.,
2004
), a process critical for cells to migrate. Thus,
Ptk2/ fibroblasts exhibit a rounded
morphology, with an increased number of focal contact sites and decreased
rates of cell migration (Ilic et al.,
1995
). Interestingly, inhibition of Rho signaling can partially
reverse these morphological and motility defects
(Chen et al., 2002
), while
v-Src transformation of Ptk2/ fibroblasts
rescues the integrin-stimulated motility defects as well as re-introduction of
FAK itself (Hsia et al.,
2003
). Furthermore, studies in
Ptk2/ fibroblasts have demonstrated a role
for FAK in the organization of the fibronectin matrix
(Ilic et al., 2004
). This has
recently been elegantly shown in vivo, where conditional loss of FAK in the
developing dorsal forebrain of mice results in altered basement membrane
organization (Beggs et al.,
2003
).
In contrast to the dramatic phenotypes observed in Ptk2 mutant
mice, Pyk2 mutant animals are viable with no gross defects in
adhesion, instead displaying immune system defects
(Guinamard et al., 2000).
However, the lack of strong phenotypes in Pyk2 mutant mice may be
misleading, since it is unclear whether the presence of the wild-type
FAK locus is able to compensate for the lack of Pyk2 in this case.
This is an important consideration since it has been shown that the targeting
of Pyk2 to ß1-integrin-containing focal contacts can rescue the
fibronectin-stimulated signaling and motility effects observed in FAK-null
cells (Klingbeil et al.,
2001
).
In order to identify functions of FAK that are conserved in metazoan
evolution, we have investigated the role of FAK in Drosophila. A
single protein with the domain structure characteristics of FAK and Pyk2 is
encoded by the Drosophila genome
(Adams et al., 2000;
Fox et al., 1999
;
Fujimoto et al., 1999
;
Palmer et al., 1999
). This
protein, Fak56 (Fak56D FlyBase), exhibits a high overall amino acid
similarity with human FAK. In the fly, Fak56 is ubiquitously expressed with
particularly high levels in the developing CNS and muscle
(Fox et al., 1999
;
Fujimoto et al., 1999
;
Palmer et al., 1999
). It is
phosphorylated on tyrosine in vivo and this phosphorylation is increased upon
plating Drosophila cells onto ECM proteins. Overexpression of Fak56
results in lethality when ubiquitously expressed, and when expressed more
selectively gives phenotypes such as wing blistering, which are characteristic
of loss of integrin function (Palmer et
al., 1999
). Integrins have been found to be essential for diverse
developmental functions in the fly, including mediating strong adhesion
between two layers of cells, particularly at muscle attachment sites and
between the two surfaces of the developing wing, mediating migration, and
regulation of gene expression during differentiation
(Bokel and Brown, 2002
;
Brower, 2003
).
In order to use Drosophila to learn more about FAK function in vivo, we generated mutants in the gene encoding Fak56 and analyzed their phenotype. Given that mutations in Ptk2 are lethal in the mouse, we were surprised to find that flies completely lacking Fak56 were viable and fertile. We examined integrin-dependent processes and the development of cells known to have particularly high levels of FAK expression, but did not detect any defects. This indicates that Fak56, contrary to earlier assumptions, is not critically required for fly development or physiology.
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Materials and methods |
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Generation of Fak56 mutants
The P{SUPor-P} line [KG00304] with a P-element insertion 5' to the
Fak56 gene was mobilized using P[ry+t7.2
2.3](99B) as a transposase source. A total of 750
independent excision lines (both viable and non-viable) were established, and
subjected to Southern blot analysis using two independent Fak56
genomic regions as probes. FAK I probe corresponds to a PCR fragment
encompassing bp 14-743 in the genomic Fak56 sequence (with 1 denoting
the start of transcription) and the FAK II probe, bp 2300-3008
(Fig. 1B). Primers used were:
for FAK I, 5'-GAGCCACCAGTCAAC-3' and
5'-GCTTTGATGTGGCTATCA-3'; for FAK II,
5'-GGATTATCACGTTGGGTT-3' and
5'-AATAATATAGGTCTGAGG-3'.
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Drosophila DNA isolation and Southern hybridization
Genomic DNA was prepared using standard techniques. Genomic DNA, digested
with EcoRI (New England Biolabs), was electrophoresed on 1.0% agarose
gel and blotted onto Hybond N+ filter (Amersham) using standard techniques.
DNA was crosslinked to the filter by UV-exposure. Filters were prehybridized
in prehybridization buffer (0.7% SDS, 50% formamide, 5 x SSC, 50 mM
Na-phosphate buffer pH 7.0, 1% Blocking reagent) for 1-4 h at 42°C prior
to incubation with DIG-labeled Fak56 or Spt5 probes at 42°C overnight.
After washing, the filters were analyzed by the DIG detection chemiluminescent
assay according to manufacturer's recommendations (Roche).
PCR and sequencing
Standard molecular biology protocols were used. PCR over the excision
breakpoint was performed on genomic DNA prepared from
Fak56CG1 flies using the primers
5'-CTTTCCACGCCAGTGGTGG-3' and
5'-GCATAATCATCGGTCAGCATCGG-3'. Similarly, PCR over the deletion
breakpoints of Df(2R)ED3716CG2 was performed as follows:
(1) on the 3' side using the primers
5'-GCAGGTGCTCTTGCGGAC-3' and
5'-TTATGAGTTAATTCAAACCCCAC-3' and (2) on the 5' side using
the primers 5'-CGTACTTTGGAGTACGAAATGC-3' and
5'-CACACATACGCCACAGAGGGAG-3'. Sequencing data were collected using
the BigDyeTM Terminator Sequencing kit v2.0 (Applied Biosystems).
Sequence was determined on both strands.
Drosophila protein isolation and western blotting
Drosophila embryos were dechorionated in 50% sodium hypochlorite
and washed extensively in PBS prior to lysis in lysis buffer (4% SDS, 20%
glycerol, 100 mM Tris/HCl pH 6.8, 40% ß-mercaptoethanol). Lysates were
cleared by centrifugation and protein concentrations determined using the
Bio-Rad protein assay. Protein samples were separated on SDS-PAGE and
transferred to a polyvinylidene difluoride membrane (Millipore). Membranes
were blocked in 5% milk (in 1 x PBS, 0.1% Tween-20) for 1 h, prior to
incubation with primary antibodies overnight, and ECL detection (Amersham
Pharmacia Biotech).
Immunostaining and antibodies
Embryos were fixed and immunostained as described previously
(Patel, 1994). Rabbit
anti-Fak56 was used at 1:1500 (Palmer et
al., 1999
). Phospho-FAKY397 was used at 1:1000
(Biosource). Mouse monoclonal anti-ßPS integrin (CF.6G11) was used at
1:10 (Developmental Hybridoma Bank). Mouse polyclonal anti-Tiggrin
(Fogerty et al., 1994
) was
used at 1:1000 (kind gift from Dr L. Fessler). Mouse monoclonal anti-Talin
(Brown et al., 2002
) was used
at 1:5. Mouse monoclonal anti-
-Tubulin was used at 1:5000 (Sigma).
Mouse monoclonal anti-MHC (muscle myosin heavy chain)
(Bloor and Kiehart, 2001
;
Kiehart et al., 1990
) was used
at 1:5 (kind gift from Dr D. Kiehart). Actin was visualized with
Rhodamine-Phalloidin (Sigma). Staining with Rhodamine-Phalloidin required
embryo devitelinization in 90% ethanol. Immunolocalization was visualized with
fluorescent secondary antibodies (Southern Biotechnology Associates). Imaginal
discs were fixed in 4% PFA in PBS for 1 hour on ice, and stained as for the
embryos.
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Results |
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To confirm that we had generated a null allele, expression of the Fak56
protein in Fak56CG1 mutants was examined using an
antiserum directed against the C-terminal part of Fak56
(Palmer et al., 1999). In
wild-type embryos, this antiserum recognized the endogenous Fak56 protein as a
band of 140 kDa, which was completely absent from Fak56CG1
embryo extracts (Fig. 2A).
Whole-mount staining of embryos with anti-Fak56 antibodies showed that the
ubiquitous Fak56 stain observed in wild-type embryos was absent in the
Fak56CG1 mutant embryos
[Fig. 2B compare (i) and (ii),
with (iv) and (v)]. Furthermore, using a phosphospecific
anti-phospho-FAKY397 antibody developed against mammalian FAK,
which cross-reacts with Fak56 phosphorylated at the equivalent tyrosine 430
(see Figs S1, S2 in the supplementary material), we observed a strong
localization of phosphorylated Fak56 at wild-type embryonic muscle attachment
sites [Fig. 2B(iii)]. This was
strongly reduced in Fak56CG1 mutant animals
[Fig. 2B(vi)], consistent with
these antibodies primarily recognizing phosphorylated Fak56. In summary, by
DNA sequencing, Southern blotting, immunoblotting and immunostainings we
conclude that Fak56CG1 is a null mutant allele of
Fak56.
|
To make absolutely sure that we had completely inactivated Fak56 function
we generated another deletion mutant that completely removes the
Fak56 gene as well as the adjacent Calpain A gene
(Fig. 3A). This was achieved
using the DrosDel isogenic deletion kit approach
(Golic and Golic, 1996;
Ryder et al., 2004
). This
deletion, named Df(2R)ED3716CG2, was confirmed to be 12.8
kb in size and to have deleted Fak56 by Southern, PCR and sequence
analysis of the breakpoints (Fig.
3B,C). This deletion was found to be homozygous lethal, probably
due to the loss of another gene, because: (1) the transheterozygotes
Fak56CG1/Df(2R)ED3716CG2 were null for Fak56
protein (see Fig. S3 in the supplementary material), and viable with no
obvious phenotypes; and (2) the lethality of the larger deficiency was not
rescued by a genomic Fak56 transgene, even though it restores normal
levels of Fak56 protein (see Fig. S3 in the supplementary material).
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Despite the fact that removing Fak56 does not have severe consequences,
overexpressing this protein clearly does. We have seen from earlier
experiments that overexpression of Fak56 results in multiple phenotypes. This
together with the intriguing localization of phosphorylated Fak56 protein at
muscle attachment sites (Fig.
2) led us to investigate further the effect of Fak56 function
using the UAS-GAL4 system (Brand and
Perrimon, 1993) to drive expression in a muscle-specific fashion.
In order to do this we employed Mef2-Gal4, which results in expression
specifically in the muscles. Overexpression of Fak56 resulted in a potent
muscle detachment phenotype (Fig.
6D-F). This phenotype was indistinguishable in severity to that
caused by the absence of integrins in the muscles, due to the lack of
PS2 (Brown, 1994
), and
in both cases development appeared to occur normally prior to detachment.
Moreover, we could observe that in spite of the obvious rounding up of somatic
muscles, integrins, as monitored by anti-ß-integrin antibodies
(Fig. 6A,D), could still be
found at the ends of the muscles that remain attached.
|
|
As Fak56 overexpression results in a loss of integrin phenotype, we wished
to confirm in another way that this was directly inhibiting integrin function.
If this were the case, then reducing the amount of integrins should enhance
the phenotype caused by Fak56 overexpression. Using the Eng-Gal4 driver, which
drives expression in the posterior portion of the wing, we have previously
shown that overexpression of Fak56 induces wing blistering
(Palmer et al., 1999). Null
mutants of the gene encoding the ßPS integrin subunit,
myospheroid (mys), are lethal; however, hypomorphic alleles
exist that are homozygous viable. In order to ask experimentally whether the
wing blistering we observe on overexpression of Fak56 is sensitive to
mutations in the ß-integrin subunit, we employed two different alleles,
mysnj42 (Wilcox et
al., 1989
) and mysb43
(Jannuzi et al., 2004
). In
control flies, at 25°C, overexpression of Fak56 in the wing resulted in
wing blisters in 28% of adult females (Fig.
8B). Under these conditions, 0% of male flies displayed wing
blisters, although minor wing defects could be observed
[Fig. 8A(ii),B]. In a
background of either mysnj42 or mysb43
an enhancement of wing blistering was observed in adult females to 44%
and 40% respectively (in this case females are heterozygous for the respective
mys allele) (Fig. 8B).
However, when we observed adult males, which were hemizygous for the
mys alleles, we observed a dramatic increase in wing blistering
from 0% to 31% (for mysnj42) and 21% (for
mysb43) [Fig.
8A(iii) and B]. Thus, this confirms that overexpressed Fak56 acts
negatively on integrin adhesion in Drosophila.
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Discussion |
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In Drosophila, mutants for many of the proteins known to localize
at the muscle attachment site cause lethality, in many cases due to muscle
detachment, i.e. the integrins themselves (reviewed by
Bokel and Brown, 2002;
Brower, 2003
), ILK
(Zervas et al., 2001
), Talin
(Brown et al., 2002
), Tiggrin
(although 1% of flies eclose) (Bunch et
al., 1998
), Laminin
(Henchcliffe et al., 1993
),
-actinin (Fyrberg et al.,
1990
), and PINCH (Clark et
al., 2003
). Other molecules, such as the Drosophila
Tensin homologue (also known as Blistery), are not lethal, but do display
phenotypes indicative of failure of adhesion, such as wing blistering
(Lee et al., 2003
;
Torgler et al., 2004
).
However, it is interesting to note that another cytoskeletal protein
Vinculin has also been shown to be non-essential in
Drosophila (Alatortsev et al.,
1997
), in contrast with previous results in the mouse, where
animals mutant for the vinculin gene display a lethal phenotype due
to heart and brain defects during embryogenesis
(Xu et al., 1998
). The
non-essential nature of Fak56 in Drosophila perhaps explains why
extensive attempts to target Fak56 through various methods have thus
far been unsuccessful, since the general assumption has been that such mutants
would be lethal. Moreover, this may also be the reason why, despite extensive
genetic screening by several groups with the purpose of identifying molecules
involved in integrin-mediated signaling, no mutations have ever been
identified in Fak56 (C.G. and R.H.P., unpublished), although the
effectiveness of these screens has been demonstrated by the fact that they
have independently identified several common loci in addition to existing PS
integrin genes (Prout et al.,
1997
; Walsh and Brown,
1998
).
While Fak56 protein appears to be ubiquitously expressed
(Fox et al., 1999;
Fujimoto et al., 1999
;
Palmer et al., 1999
),
phospho-Fak56 is strongly localized at muscle attachment sites. This implies
that Fak56 is not only localized, but also activated at these locations, since
phosphorylation of the FAKY397 site (which is conserved in Fak56),
is considered to reflect FAK activation in vivo
(Calalb et al., 1995
). The
anti-phospho-FAKY397 antibodies seem to be specific for
phosphorylated Drosophila Fak56, based on two criteria: (1) loss of
immunoreactivity in the Fak56 mutants, and (2) overexpressed
wild-type Fak56 is recognized by the antiphospho-FAKY397
antibodies, whereas overexpressed Fak56Y430F mutant protein is not
(data not shown). A role for Fak56, albeit an accessory one, at muscle
attachment sites, is endorsed by our finding that phosphorylated Fak56 is
absent from muscle attachment sites in integrin mutants. Thus, while not
required for integrin function, Fak56 appears to be involved through an as yet
undefined mechanism in integrin-mediated events in Drosophila
embryogenesis. In spite of the absence of the Fak56 PTK at muscle attachment
sites in Fak56 mutant embryos, the levels of phosphotyrosine observed
are indistinguishable from wild-type (Table S1 in the supplementary material),
suggesting that another PTK(s) is activated at these sites. This is
interesting in light of a recent report that Src kinases can be activated by
direct interaction with integrin ß-subunit tails
(Arias-Salgado et al., 2003
).
Such a model implies that Src family kinases can be activated by integrins
independently of FAK and could explain the unexpected lack of phenotypes in
the Fak56 mutant. Interestingly, it has recently been shown that
v-Src transformation of Ptk2/ fibroblasts
rescues the motility defects observed in
Ptk2/ fibroblasts, which is in keeping with
these results (Hsia et al.,
2003
; Moissoglu and Gelman,
2003
).
Consistent with an accessory involvement of FAK in integrin-mediated
adhesion, we show here that the Fak56-induced wing blister phenotype is
sensitized in an integrin mutant background, thus indicating a genetic
interaction between Fak56 and integrins in this context. Employing the
UAS-GAL4 system to drive Fak56 specifically in muscles it is clear from our
results that overexpression of Fak56 disrupts muscle attachment, thus
overexpression of Fak56 causes much more serious defects than its absence.
This is consistent with Fak56 either functioning as an adaptor by displacing
more critical proteins from the integrin cytoplasmic tail required for the
extracellular binding to ECM ligands, or causing a dissociation of the
integrin-containing complex by excessive phosphorylation. The evidence in
mammalian systems suggests that FAK plays a critical role in cell migration,
which is a complex, highly regulated process that involves the continuous
formation and disassembly of adhesions
(Gelman, 2003;
Parsons, 2003
;
Schlaepfer and Mitra, 2004
).
Indeed, recently it has elegantly been shown that FAK is important for
adhesion turnover at the cell front, a process central to migration
(Webb et al., 2004
). We have
examined the migration of (1) the endoderm, (2) border cells during oogenesis,
(3) germ cells and (4) the trachea, in Fak56 mutant animals, and have
found no defects in either process in the absence of Fak56. However, such a
role of FAK in the disassembly, rather than in the actual assembly of these
structures, is in agreement with our findings that Fak56 overexpression
results in detached muscles where the
PS2 integrin is still localized
at the muscle ends, as though dissociated from the ECM. This then raises the
question of why Fak56 is normally found in the developing muscles. We
speculate that it contributes in keeping the strong adhesive junctions dynamic
so that they can be remodeled during the formation of the attachments and the
subsequent growth of the muscles. Perhaps a defect in this process would be
more apparent under more strenuous growth conditions than those found in the
laboratory.
One important point to consider is the wealth of data from mammalian cell
experiments indicating a critical role for FAK family PTKs in integrin
function (reviewed by Gelman,
2003; Parsons,
2003
). Such a function is corroborated by the phenotype of the FAK
knockout mouse, which dies early in embryogenesis
(Ilic et al., 1995
). Indeed,
Ptk2/ fibroblasts derived from FAK mutant
embryos, exhibit a rounded morphology, and have an increased number of focal
contact sites and decreased rates of cell migration
(Ilic et al., 1995
). A recent
RNAi-based screen of the Drosophila genome to find novel genes
affecting cell morphology identified Fak56 as a molecule affecting cell
spreading (Kiger et al.,
2003
). In addition, overexpression of Fak56 in the fly using the
GAL4-UAS system also produces a wealth of integrin-related phenotypes (C.G.
and R.H.P., unpublished). We have noted that overexpressed Fak56 does localize
to muscle attachment sites under such conditions, and it is entirely possible
that this creates a neomorphic phenotype, i.e. such that the presence of
excess Fak56 protein or indeed Fak56 activity binds up
multiple factors and acts in a dominant negative fashion to produce these
defects. Thus, experiments in mammalian cells employing overexpression of FAK
to analyze integrin function may not be an ideal model for the in vivo
scenario. Furthermore, a radical explanation for the strong defects observed
in Ptk2/ fibroblasts, which would be
consistent with our findings, is if the primary defect of removing FAK is the
observed elevated expression and activity of the related kinase Pyk2, and the
Src family PTKs (Sieg et al.,
1998
). If overexpressed Pyk2 causes similar dominant negative
effects on other components of integrin adhesion, as we have seen when Fak56
is overexpressed, than this could contribute to the severe phenotype observed
in the FAK mutant mice. Finally, it is also possible that the functions of the
FAK family PTKs are not totally conserved between Drosophila and
mice, and that FAK has assumed a more critical role during evolution of
vertebrates.
Disruption of the single Drosophila Fak56 gene is the first example of an animal completely lacking FAK PTK function. However, it is surprising that a genetic null for a protein such as FAK, that has always been thought to be a critical link between integrins and the actin cytoskeleton, exhibits a viable phenotype. Since this also appears to be true for the C. elegans FAK family PTK, we must assume that while FAK may play an accessory role in modulating integrin functions in vivo, it is by no means essential for integrin-mediated adhesion or signaling in the fruitfly. It is obvious that further experiments will be required to define and understand the exact role of the Fak56 PTK in such processes in vivo.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/23/5795/DC1
Present address: Academy of Athens, Foundation of Biomedical Research,
Genetics Laboratory, Soranou Efesiou 4, 11527, Athens, Greece
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