1 Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2
3EJ, UK
2 Faculty of Life Sciences, University of Manchester, Michael Smith Building,
Oxford Road, Manchester M13 9PT, UK
3 School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG,
UK
4 Facultad de Ciencias, Centro de Biologia Molecular, `Severo Ochoa', CSIC-UAM,
Cantoblanco, Madrid 28049, Spain
5 Centro Andaluz de Biología del Desarrollo,Universidad Pablo de Olavide,
Carretera de Utrera, Km 1, Sevilla 41013, Spain
* Author for correspondence (e-mail: hs17{at}cam.ac.uk)
Accepted 18 March 2005
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SUMMARY |
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Key words: crossveinless-c, Drosophila, Morphogenesis, RhoGTPase, RhoGAP, Actin cytoskeleton, Convergent extension
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Introduction |
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The Rho GTPase family of proteins act as `molecular switches' that cycle
between two conformational states, a GTP-bound state in which they are active
and a GDP-bound inactive state. The balance between these states is controlled
principally by two classes of regulatory proteins: the guanine nucleotide
exchange factors (GEFs) that promote the active state by facilitating the
release of GDP and subsequent rebinding of GTP; and the GTPase activating
proteins (GAPs) that promote the inactive state by catalysing the weak
intrinsic GTP hydrolysing capacity of the GTPase, thereby converting it to the
inactive form. In many cases, it is crucial that the correct balance between
these two states is properly regulated. This is evident in cases in which they
are not, as deregulated GTPases can have catastrophic developmental
consequences and can lead to cellular pathologies such as cancer
(Boettner and Van Aelst, 2002;
Jaffe and Hall, 2002
).
In all animal genomes sequenced to date, there are considerably more genes
encoding for GAPs and GEFs than the GTPases they regulate. For example, the
human genome encodes 20 Rho-family GTPases, but in excess of 50 different
GAPs and GEFs (Bernards, 2003
;
Peck et al., 2002
); similarly,
the Drosophila genome encodes seven Rho-family GTPases but 21 GAPs
and 20 GEFs (Bernards, 2003
).
The preponderance of GAPs and GEFs probably reflects the importance of
controlling the activity of RhoGTPase family members. The 21 GAP proteins in
Drosophila show considerable diversity in their domain architecture
and this may provide context specificity for the multitude of functions and
outcomes of GTPase signalling. Although much is known about the catalytic
function of the GAPs and GEFs, relatively little is known about how they
function in a cellular or developmental context, e.g. their spatial and
temporal regulation and the factors that control their specificity. Of the 21
Drosophila RhoGAPs, five have been studied to date
(Billuart et al., 2001
;
Guichard et al., 1997
;
Lundström et al., 2004
;
Raymond et al., 2001
;
Sagnier et al., 2000
;
Sotillos and Campuzano, 2000
).
In most cases, these studies have used either gain-of-function analyses or
dominant-negative approaches; therefore, the phenotypic defects in mutants
have not been analysed.
We present evidence that viable alleles in the crossveinless-c
(cv-c) gene are hypomorphic alleles of the RhoGAP88C gene. Three
cv-c alleles have been described previously. These alleles are
characterised by partial or complete loss of the posterior crossvein (PCV) and
variable detachment of the anterior crossvein (ACV) in the wing
(Fig. 1A)
(Diaz-Benjumea, 1990;
Edmondson, 1952
;
Stern, 1934
). Despite having
been first described over 70 years ago
(Stern, 1934
) and used as a
marker for recombination studies, the molecular nature of the locus has until
now remained a mystery.
|
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Materials and methods |
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Mutagenesis and screens
MpT screen
The cv-cM62 and cv-cC524
EMS-induced alleles were generated on a multiply marked third chromosome
(rucuca) following standard techniques. The primary screen used
luminal uric acid as a marker for MpT morphology
(Fig. 4O,P).
|
Immunocytochemistry and histochemistry
Immunostaining was performed using standard techniques with antibodies
against the following proteins: 22C10 (1:200), Baz (1:1500),
ß-galactosidase (1:10,000), Cno (1:200), Cut (1:200), E-cad (1:20),
FasIII (1:20), GFP (1:1500, Abcam), p-Mad (1:2000) and Stranded-at-Second
(1:500). Appropriate biotinylated secondaries were used in conjunction with
the Vector Elite ABC Kit (Vector Laboratories, CA) for DAB staining. For
fluorescent labelling, we used appropriate secondary antibodies conjugated
either to FITC or Cy3. When required, we performed an additional amplification
step using streptavidin-conjugated Cy3. cv-c RNA localisation was
performed by in situ hybridisation using a digoxigenin-labelled antisense
probe directed against the entire coding sequence of cv-c. GFP was
visualised by antibody staining using a GFP antibody
(Fig. 5E-H,
Fig. 6G) or by observing GFP
fluorescence in living embryos (Fig.
5K-P; Fig.
6E,F).
|
|
SNP mapping
SNP mapping of cv-cM62 and cv-cC524
was performed as previously described
(Martin et al., 2001). Novel
SNP loci were found by sequencing PCR-amplified intergenic DNA fragments from
rucuca/FRT82B flies. SNP loci were scored using restriction fragment
length polymorphisms (RFLPs), or allele-specific primers. The following SNP
loci were used (FRT/rucuca alleles are given for novel SNPs):
SNP-87A; SNP-88C (Martin et al.,
2001
) (contrary to the previous report, we find that the
rucuca allele is A and FRT allele is G at SNP-88C); SNP-Abi
[nucleotide 81776 in AE003703 (C/T, RFLP for XhoI)]; and SNP-NK7.1
[nucleotide 75924 in AE003704 (T/A)].
Cuticle preparations
Embryonic cuticle preparations were performed as previously described
(Hu and Castelli-Gair, 1999).
Wings were dissected from adult flies in 70% ethanol and mounted in
Euparal.
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Results |
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No mutants have been reported for any of the nine predicted genes flanked
by our SNPs, precluding complementation analysis. However, a UAS-RNAi
construct directed to RhoGAP88C (CG31319) has previously been generated
(Billuart et al., 2001). We
tested whether expression of this RNAi construct could recapitulate the
cv-c phenotypes we observe. To do this, we used Gal4 driver lines
that express in the developing wing throughout pupal development and are
active during the period of PCV specification at 24-26 hours APF
(Conley et al., 2000
). The
four Gal4 driver lines we tested produce a phenotype indistinguishable from
the cv-c1 phenotype
(Fig. 1D). No other wing
structures, including the longitudinal veins, are affected
(Fig. 1D). This result
contrasts with previous data reporting that expression of UAS-RhoGAP88C in the
developing wing pouch (using T80-Gal4, a driver we also used) results in a
reduction or absence of wing vein L2
(Billuart et al., 2001
). These
results suggest that cv-c encodes RhoGAP88C, and encouraged us to
search for molecular lesions in the coding sequence of RhoGAP88C in our EMS
alleles.
Molecular characterisation of cv-c mutations
The Drosophila RhoGAP88C gene spans a region of 14 kb and contains
eight exons. The BDGP database contains a single RhoGAP88C cDNA (RE02250) of
4.4 kb in length. Conceptual translation of this cDNA reveals an open reading
frame of 1017 amino acids (Fig.
2B). In addition to the GAP domain, RhoGAP88C contains two other
previously described domains: a sterile motif (SAM), originally
defined as a protein-protein interaction domain
(Schultz et al., 1997
), but
more recently implicated in RNA (Aviv et
al., 2003
; Green et al.,
2003
; Kim and Bowie,
2003
) and lipid membrane-binding
(Barrera et al., 2003
); and a
lipid transfer START domain so called because it was initially isolated in the
steroidogenic acute regulatory (StAR) protein
(Clark et al., 1994
;
Sugawara et al., 1995
).
Database searches identify several closely related RhoGAP proteins containing identical domain architecture to Drosophila RhoGAP88C (Fig. 2C). The closest related vertebrate proteins are the human deleted in liver cancer 1 and 2 [DLC1 and DLC2 (STARD13 Human Gene Nomenclature Database)], the mouse serologically defined colon cancer 13 antigen and rat p122-RhoGAP (Fig. 2C). A C. elegans protein, gut on exterior-interacting protein (GEI) also shares a high level of identity with Drosophila RhoGAP88C in the GAP and START domains, but lacks the SAM motif (Fig. 2C).
We identified molecular lesions in the coding region of three of our
cv-c EMS alleles. Two of these, cv-cC524 and
cv-cM62, contain nonsense mutations at amino acid
positions 369 and 666, respectively, resulting in protein products truncated
either before the GAP domain for cv-cC524, or within it
for cv-cM62 (Fig.
2C). The absence of a complete GAP or START domain in either
mutant product suggests they are likely to be amorphic mutations. In
cv-c7 (Table
1), arginine 601 is substituted for glutamine
(Fig. 2C). This arginine is
highly conserved at the corresponding position in RhoGAP88C homologues in
other species and is also conserved in GAP proteins more distantly related to
the RhoGAP family, underlining the importance of this residue for GAP
function. A combination of biochemical and structural studies have shown that
the conserved arginine residue projects into the active site of the GTPase and
stabilises the transition state of the hydrolytic reaction
(Barrett et al., 1997;
Nassar et al., 1998
;
Rittinger et al., 1997a
;
Rittinger et al., 1997b
;
Scheffzek et al., 1997
).
Mutational analysis reveals that substitution of this so-called `arginine
finger' with another residue dramatically reduces the catalytic activity of
the GAP protein (Leonard et al.,
1998
). Our finding that the arginine finger is specifically
mutated in cv-c7 indicates that GAP activity is central to
the function of cv-c. Surprisingly, however, we find
cv-c7 homozygous embryos have consistently stronger
phenotypes than either cv-cC524 or
cv-cM62 homozygotes (see below), suggesting that
cv-c7 is an antimorphic allele. Our interpretation of
these findings is that substitution of arginine 601 to glutamine not only
abolishes the catalytic activity of cv-c, but also impedes the weak,
intrinsic GTPase activity of its substrate. Therefore, the balance between
GTP-bound and GDP-bound states of the substrate GTPase will be shifted even
further towards the active, GTP-bound form in cv-c7
homozygous embryos.
The P-element insertion in cv-cl(3)06951
(Table 1) is 60 kb
upstream to the start of transcription
(Fig. 2; see Materials and
methods). The reporter activity faithfully recapitulates the endogenous
expression of cv-c (Fig.
3; data not shown). Precise excision of the P-element reverts the
lethality and cv-c phenotype. Both cv-cl(3)06951
and the new imprecise excision alleles we have generated are homozygous lethal
but are weaker than those of the amorphic mutations
(Table 1; see below). In one of
these excision alleles, cv-cJ17, cv-c transcripts are
absent from most, but not all tissues (Fig.
3F). These results indicate that sequences controlling
cv-c transcription reside at a considerable genomic distance from the
transcription unit.
|
As previously discussed, cv-cl(3)06951 is likely to disrupt regulatory sequences. To test this hypothesis, we examined the expression of cv-c in cv-cl(3)06951 and the jump-out allele cv-cJ17. Although we were unable to detect any major differences in cv-c expression in cv-cl(3)06951, cv-c expression is reduced in all tissues apart from the LVM in cv-cJ17 embryos (compare Fig. 3D with 3F). This confirms that the cv-cJ17 lesion removes cv-c regulatory elements.
Mutations of cv-c produce defects in morphogenesis
Here, we report embryonic phenotypes associated with mutations in
cv-c; further details of the wing phenotypes will be described
elsewhere. Unless specified otherwise, we describe the mutant phenotypes of
cv-cM62 homozygotes, as we believe this to be an amorphic
cv-c allele. Analysis of cv-c germline clones (data not
shown) confirms that cv-c is not supplied maternally and for this
reason, we used zygotic mutants for the phenotypic analysis.
The cells that contribute to the larval mouth skeleton of Drosophila are internalised during development in a process known as head involution (Fig. 4A). In cv-c mutants, cells fail to move into the embryo so that structures such as the medial tooth, the H piece and dorsal bridge remain on the surface of the embryo (Fig. 4B). cv-c7 consistently shows stronger defects, in which the head does not involute normally so that the remnants of the whole head skeleton lie on the surface (Fig. 4F).
The posterior spiracle forms by invagination of surface ectodermal cells
into the interior to form the spiracular chamber
(Hu and Castelli-Gair, 1999)
(Fig. 4C). In cv-c
mutants, invagination of these cells is aberrant, causing defects ranging from
complete failure of invagination, so that the entire internal cuticular
structure (the Filzkörper) is later found on the surface, to branching of
the spiracular chamber (Fig.
4D).
During dorsal closure sheets of epithelial cells on either side of the embryo move dorsally over the amnioserosa, so that their epithelial fronts meet and fuse at the dorsal midline, enclosing the embryo (Fig. 4E,G). Although embryos mutant for strong cv-c alleles complete dorsal closure, staining with an antibody against the apical membrane marker Stranded at second (Sas) shows that closure is delayed (compare equivalently aged embryos in Fig. 4G,H), and we frequently observe puckering of the dorsal cuticle (Fig. 4F). The phenotypes we observe do not result from abrogation of Dpp signalling, that underlies dorsal closure (see Fig. S1 in the supplementary material), suggesting defects occur in the morphogenetic movements themselves.
In wild-type embryos, the midgut becomes subdivided into four compartments
by three constrictions that result from interactions between the visceral
mesoderm and the underlying endoderm (Fig.
4I). The formation of these constrictions involves changes in cell
shape and movement that occur without cell division (reviewed by
Bienz, 1994). In cv-c
mutant embryos, the anterior (and occasionally also the posterior) midgut
constriction fails to develop (Fig.
4J). Failure of the gut constrictions is not due to defects in
visceral mesoderm specification or signalling (see Fig. S1 in the
supplementary material) and is therefore likely to result from defects in the
normal cell movements.
In cv-c embryos, the MpTs form a single large cyst- or ball-like
structure instead of four elongated tubules
(Fig. 4K-N). This phenotype
results from the failure of tubule remodelling, which normally occurs by a
series of convergent extension movements, such that each tubule extends along
its proximodistal axis and narrows around its circumference (compare
Fig. 4K,L with 4M,N)
(Denholm and Skaer, 2004). The
cyst-like structure in mutants sometimes, but not always, exhibits a central
lumen, which is revealed by the accumulation of secreted urates
(Fig. 4O,P). In a small
proportion of embryos, the distal regions of one and, occasionally, two
tubules undergo normal convergent extension movements, with the distal tips
finding their correct location within the body cavity (an example of this is
shown in Fig. 5P).
As part of our analysis to confirm that cv-c encodes RhoGAP88C, we generated a UAS-RhoGAP88C construct and asked whether this could rescue the cv-c phenotype. Expression of the construct in cv-cM62 mutant embryos using the CtBGal4 driver, which is specific for the MpTs, rescues the tubule phenotype (Fig. 4Q), confirming that cv-c encodes RhoGAP88C. Furthermore, rescue of the MpT phenotype in an embryo otherwise mutant for cv-c demonstrates that the requirement for Cv-c activity is cell autonomous in this tissue at least. In summary, our phenotypic analysis shows that the tissues most sensitive to loss of Cv-c function are those actively undergoing morphogenetic movement or remodelling. We propose that the cv-c gene product functions autonomously to perform a fundamental role in the control of morphogenesis of multiple tissues.
cv-c is required to organise the actin cytoskeleton
The morphogenetic defects we observe could result from alterations in cell
polarity or cell shape through cytoskeletal reorganisation. In order to
distinguish between these, we first analysed both planar and apicobasal
polarity of cells in cv-c embryos. We chose to examine planar
polarity in epidermal cells during dorsal closure because it is not known
whether the MpTs exhibit planar polarity. To do this, we visualised the
redistribution of Canoe (Cno). In wild-type embryos, Cno is expressed at the
cell cortex of leading edge cells, but as dorsal closure proceeds it clears
from sites of apposition with the amnioserosa and refines to distinct puncta
where adjacent epidermal cells meet
(Kaltschmidt et al., 2002)
(Fig. 5A,C). This
redistribution depends on the planar cell polarity pathway
(Kaltschmidt et al., 2002
). We
find that this redistribution of Cno occurs normally in cv-c mutant
embryos (Fig. 5B,D), indicating
that planar polarity is not disrupted in the absence of Cv-c activity. We next
examined whether cv-c tubule cells have normal apicobasal polarity.
Using CD8-GFP to label the entire membrane of MpT cells and staining for the
apical membrane marker Bazooka (Baz) reveals that apicobasal polarity is
established normally and maintained in cv-c `tubules' until the end
of embryogenesis (Fig. 5E-H).
In addition, the presence and position of the adherens junction marker
dE-Cadherin relative to apical markers such as Sas are normal in mutant tubule
cells (Fig. 5I,J).
We then focussed our attention on the actin cytoskeleton. To do this, we
made use of the UAS-GMA construct, which encodes the actin-binding region of
moesin fused to GFP and provides a faithful readout of filamentous actin
localisation (Dutta et al.,
2002). Using the CtB-Gal4 driver, we expressed UAS-GMA in the MpTs
throughout embryonic development. In wild-type embryos at stage 13, F-actin is
found localised to the cell cortex of all tubule cells and is particularly
enriched at the apical (luminal surface)
(Fig. 5K). A similar
distribution of F-actin is maintained throughout the remainder of
embryogenesis (Fig. 5L,M). F-actin distribution is more diffuse in stage 13 cv-c mutant embryos,
and although it is present around the cell cortex, its levels here are
significantly lower than in wild-type embryos
(Fig. 5N). Similarly, apical
accumulation of actin is either absent or, at best, very weak
(Fig. 5N). Although F-actin
becomes localised to the cell cortex in later stages of development, its
distribution is less compact than in wild type and diffuse staining throughout
the cytoplasm suggests that attachment of the subcortical actin network to the
membrane is disrupted (Fig.
5O,P; compare inset in L with inset in O). Although apical
accumulation of F-actin is never observed in cv-c cystic MpTs, those
in which the distal end does become tubular possess weak apical F-actin
accumulation at the luminal membrane (Fig.
5P, arrowhead). These data show that Cv-c is required to regulate
the spatial organisation of F-actin in tubule cells during morphogenesis.
Furthermore, apical accumulation of F-actin, which is largely absent in
cv-c tubules, is crucial for the maintenance of tubular integrity; in
its absence the MpTs collapse to form cyst-like sack structures.
Gain of function analyses suggest that Cv-c stabilises membrane/actin associations
In order to analyse the GOF phenotype, we overexpressed Cv-c in the MpTs
throughout embryogenesis. Tubules in which Cv-c is ectopically expressed are
significantly shorter in length and fatter around their circumference,
suggesting that elevated Cv-c activity also causes defects in convergent
extension movements (data not shown). The most obvious phenotype we observe,
however, affects the tip cell, a specialised cell that protrudes from the
distal end of each of the four tubules
(Fig. 6A)
(Skaer, 1989). The length of
the tip cell stalk is
7 µm in both wild-type tubules and in the
tubules of cv-c loss-of-function embryos
(Fig. 6B,C). However, when Cv-c
is overexpressed, the stalk length can increase to over 50 µm
(Fig. 6D) [average length=24
µm (n=56)]. Analysis using 22C10, which labels the tip cell
membrane, as well as observations of F-actin, reveal that the extended stalk
is associated with underlying F-actin (Fig.
6D,F). Staining for Sas reveals that the stalk is an extension of
the basolateral, rather than apical membrane
(Fig. 6G). We reasoned that if
the normal function of Cv-c is to downregulate the activity of RhoGTPases,
then overexpression of a dominant-negative RhoGTPase might be expected to
produce a similar phenotype. This is indeed the case: ectopic expression of
the dominant-negative Rho1 construct UAS-Rho.N19 (but not the
dominant-negative Rac1 construct UAS-Rac1.N17 see below) resulted in
an increase in tip cell stalk similar to the phenotype in cv-c
overexpression (Fig. 6H)
[average length, 15 µm (n=22)]. These data, taken together with
the loss-of-function analysis described above, are consistent with a role for
Cv-c in regulating or stabilising links between the cell membrane and the
cortical actin cytoskeleton. Furthermore, in agreement with the genetic
interaction experiments described below, these data further suggest that this
function for Cv-c, in the MpTs at least, is mediated at the level of Rho1 and
not Rac1 activity (see below).
Genetic interactions between cv-c and RhoGTPases
RhoGAPs normally function to stimulate the intrinsic GTP hydrolysing
capacity of the GTPases, thereby converting them to the inactive GDP-bound
form. Thus, the phenotypes we observe in the absence of Cv-c function are
likely to be caused by elevated activities of the cognate GTPase substrate(s)
of Cv-c. Such relationships can be tested by genetic interactions; a reduction
in the activity of a substrate GTPase would rescue the cv-c
phenotype, while an increase in GTPase activity would enhance it. To identify
the substrate(s) of Cv-c, we analysed genetic interactions between
cv-c and candidate GTPases for which mutants are available. Embryos
homozygous for cv-cM62 and homozygous or hemizygous for
mutations in the GTPases Rho1, Rac1, Rac2, Mtl and Cdc42
were analysed for both the MpT and embryonic cuticle phenotypes.
Removal of any of the Rac candidates, Rac1, Rac2 or Mtl, and reduction or removal of Cdc42 failed to modify the cv-cM62 phenotype in the MpTs. By contrast, 50% of cv-cM62 mutant embryos additionally homozygous for Rho172R (n=48) and 20% of cv-cM62 mutant embryos heterozygous for Rho172R (n=56) had a phenotype significantly less severe than that of the cv-cM62 homozygote alone. The MpTs of doubly mutant embryos undergo convergent extension movements to some extent (compare Fig. 7A,B and 7A',B'), such that they resemble weaker alleles of cv-c (data not shown). These data strongly suggest that Rho1 is a substrate for Cv-c in the MpTs.
|
The posterior spiracle phenotype in cv-c embryos was not
suppressed by any of the RhoGTPase mutants, possibly because maternal
contribution of the substrate GTPase is sufficient to provide full activity in
this tissue. However, we unexpectedly find that embryos mutant for both
Rac1 and Rac2 do not decrease, but enhance the
cv-cM62 posterior spiracle phenotype (compare
Fig. 7E with 7F). One possible
explanation for this interaction comes from observations in cell culture,
where it has been shown that Rac activity downregulates Rho activation
(Nimnual et al., 2003;
Sander et al., 1999
). If Rac
normally functions to inhibit Rho in the posterior spiracle, then loss of Rac
and Cv-c together, would lead to hyperactivity in Rho1 and this would lead to
the enhancement of the cv-c phenotype. In support of this, we see
cv-c-like posterior spiracle phenotypes with low penetrance in
Rac1 Rac2 embryos (data not shown).
In summary, our data from these genetic analyses in the embryo indicate that cv-c interacts with Rho1, Rac1 and Rac2. Furthermore, these data suggest that interactions between Cv-c and its substrates might be regulated in a tissue-specific manner.
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Discussion |
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The common feature linking the morphogenetic processes in which cv-c activity is required is the coordinated reorganisation of large groups of cells during tissue remodelling. Such processes involve choreographed cell movements as well as alterations in cell shape. We have focused our attention on the role of Cv-c in the morphogenesis of the MpTs as they undergo convergent extension movements. In contrast to cell polarity, which is relatively normal in cv-c mutant embryos, we find that the organisation of the actin cytoskeleton is aberrant. We show that the normal strong subcortical accumulation of F-actin is lost in cv-c mutant MpTs. In particular, the striking concentration of F-actin on the luminal membrane at the onset of tubule elongation fails.
How do these defects in the actin cytoskeleton relate to the MpT
morphogenetic phenotype? By a process of convergent extension, starting at
stage 13 and continuing until stage 16, the MpT cells remodel from short fat
tubes with 10-12 cells surrounding the lumen to long thin tubes with only two
cells encircling the lumen. The convergent extension movements in the MpTs are
likely to occur in a manner similar to those described in the epidermis during
germband extension, whereby a coordinated reorganisation of cell partners
drives a change in tissue dimension (Bertet
et al., 2004). Central to this reorganisation is the remodelling
of zonula adherens junctions (ZAs), which are progressively disassembled as
ZAs between new cell partners are formed
(Bertet et al., 2004
).
Remodelling of the actin cytoskeleton is thought to drive these changes, both
to facilitate alterations in cell shape and finally to stabilise the new
junctional configuration. We suggest that the defects in the actin
cytoskeleton observed in cv-c tubules perturbs the cell
rearrangements that underlie convergent extension movements required during
tubule elongation. As the MpT phenotype becomes progressively more severe
during the period when cell rearrangement normally occurs, we suggest that it
is the coordination of cell reorganisation rather than cell intercalation per
se that is defective. In addition, morphogenesis of the MpTs requires the
maintenance of a tubular structure and this also fails in cv-c
mutants. It is likely that the accumulation of actin at the luminal membrane
is required both to remodel cell-cell associations and to retain the integrity
of the lumen. We therefore propose that Cv-c plays an important regulatory
role to ensure that the actin cytoskeleton is properly remodelled during
tubule morphogenesis. Furthermore, as cv-c is required in multiple
tissues, we predict that cv-c is reiteratively used during
development in the regulation of actin cytoskeletal remodelling during
morphogenesis.
It seems likely that Cv-c acts to orchestrate the actin cytoskeleton via the direct regulation of RhoGTPase activity; our finding that cv-c7 is defective in a specific crucial residue required for GAP enzymatic activity provides strong support for this hypothesis. Although our genetic interaction experiments highlight potential substrates and furthermore suggest the possibility that substrates might be regulated in a tissue-specific manner, additional biochemical analyses will be necessary to confirm this.
Cv-c function appears to be evolutionarily conserved. The rat and human
cv-c homologues, p122 and DLC2 respectively, have been shown to
inhibit Rho-mediated assembly of actin stress fibres when overexpressed in
cell culture (Ching et al.,
2003; Nagaraja and Kandpal,
2004
; Sekimata et al.,
1999
). Furthermore, the C. elegans cv-c homologue, GEI-1,
is thought to provide a molecular link with the actin assembly machinery.
GEI-1, was isolated as a binding partner of GEX-2, one of a group of
interacting proteins that localise to the plasma membrane and are required for
the process of ventral enclosure, a morphogenetic event similar to dorsal
closure in the fly (Soto et al.,
2002
; Tsuboi et al.,
2002
). The recent molecular characterisation of GEX-1 as a
WAVE-family protein (M. Soto, personal communication) sheds light on the
function of these proteins in morphogenesis. Proteins of the WAVE-family are
known to relay signals from Rho-family GTPases to the actin cytoskeleton,
possibly by permitting the assembly of multi-protein complexes, including
components that act to nucleate actin [such as Arp2/3- and actin-binding
proteins, GTPase proteins, GAPs and GEFs
(Hussain et al., 2001
;
Scita et al., 1999
;
Soderling et al., 2002
;
Welch and Mullins, 2002
)].
Thus, a molecular link exists between the Gex proteins, GTPases, their
regulators and the actin cytoskeleton. Cv-c and its homologues may provide GAP
activity within this large multi-protein complex, contributing to dynamic
regulation of the actin cytoskeleton. For these reasons, it will be
interesting to determine the relationship between Cv-c and the Gex homologues
in the fly, Sra1/Gex2 and Kette/Gex3, and the fly SCAR/WAVE
(Hummel et al., 2000
;
Kitamura et al., 1996
;
Kunda et al., 2003
;
Schenck et al., 2003
;
Zallen et al., 2002
).
Our demonstration that overexpression of cv-c is sufficient to
induce stable membrane extensions supported by F-actin suggests that Cv-c acts
to coordinate or stabilise interactions that occur between the plasma membrane
and the actin cytoskeleton. The Cv-c protein contains two domains implicated
in lipid membrane binding, the START and SAM domains
(Barrera et al., 2003;
Hanada et al., 2003
;
Romanowski et al., 2002
;
Tsujishita and Hurley, 2000
),
suggesting that Cv-c localises to a membrane domain. This would place Cv-c in
an ideal position to regulate an interaction between the plasma membrane and
the actin cytoskeleton. There is compelling evidence that moesin is required
to organise the cortical actin network which it does, at least in part, by
linking the actin cytoskeleton to the plasma membrane (for reviews, see
Bretscher et al., 2002
;
Gautreau et al., 2002
;
Polesello and Payre, 2004
).
Given that moesin has been shown to antagonise Rho1 by altering its activation
state (Speck et al., 2003
), it
will also be interesting to examine the requirement of Cv-c for moesin
function.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/10/2389/DC1
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