|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 13 August 2008
doi: 10.1242/dev.025205
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MRC Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
* Author for correspondence (e-mail: d.strutt{at}sheffield.ac.uk)
Accepted 29 July 2008
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Planar polarity, Multiple Wing Hairs, Drosophila, Cell polarity, Frizzled, Actin
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Klein and Mlodzik,
2005; Wang and Nathans,
2007).
In many contexts, a key event in the coordinated planar polarisation of
epithelia is the asymmetric subcellular localisation of a group of `core'
planar polarity proteins to opposite cell edges. This process is best
characterised in the Drosophila pupal wing, in which the seven-pass
transmembrane protein Frizzled (Fz) and the cytoplasmic proteins Dishevelled
(Dsh) and Diego (Dgo) localise to distal apicolateral junctional regions, the
four-pass transmembrane protein Strabismus (Stbm, also known as Van Gogh) and
the cytoplasmic protein Prickle (Pk) localise proximally, and the seven-pass
transmembrane cadherin Flamingo (Fmi, also known as Starry Night) localises
both distally and proximally (reviewed by
Klein and Mlodzik, 2005). The
localisations of the core proteins define distinct distal and proximal
apicolateral membrane domains, which are thought to act as cues for subsequent
cell polarisation events mediated by downstream effector genes.
The best-characterised morphogenetic event regulated by the asymmetric
localisation of the core polarity proteins is the production of a single,
distally pointing trichome from each cell of the wing blade
(Gubb and García-Bellido,
1982; Wong and Adler,
1993). Trichome formation begins with increased actin bundling
close to the distal cell vertex at 
32 hours of pupal life, leading to
formation of a prehair that contains both F-actin and microtubules
(Mitchell et al., 1983;
Wong and Adler, 1993;
Eaton et al., 1996;
Turner and Adler, 1998).
Electron microscopy has revealed that shortly prior to prehair formation, the
apical cell surface is covered in electron-dense `pimples'
(Guild et al., 2005), which
are believed to be precursors for microvillus formation. Prehair initiation is
manifested by actin bundles sprouting from a region covering several pimples
close to the distal vertex. This suggests that it is the local activation of
pimples to polymerise actin that specifies the site of prehair formation.
Four genes have been identified that act downstream of the core polarity
proteins to specify the site of prehair initiation, namely inturned
(in), fuzzy (fy), fritz (frtz)
and multiple wing hairs (mwh)
(Gubb and García-Bellido,
1982; Wong and Adler,
1993; Lee and Adler,
2002; Collier et al.,
2005). Interestingly, whereas loss of core polarity protein
function (and thus of asymmetric localisation) causes formation of a single
prehair in the cell centre, loss of function of these downstream effectors
leads to the formation of multiple prehairs at cell edges. This led to the
suggestion that the downstream effectors repress prehair formation throughout
the cell periphery, whereas the core planar polarity proteins promote prehair
formation at the distal cell edge by locally counteracting the effectors
(Wong and Adler, 1993).
The best-studied effectors are in, fy and frtz, which all
act cell-autonomously upstream of mwh
(Gubb and García-Bellido,
1982; Wong and Adler,
1993; Park et al.,
1996; Collier and Gubb,
1997; Collier et al.,
2005) and encode, respectively, a putative two-pass transmembrane
protein, a putative four-pass transmembrane protein and a WD40-repeat
cytoplasmic protein. Interestingly, the ectopic trichome phenotype of null
alleles of all three loci is enhanced at lower temperatures, leading to the
suggestion that these proteins act in a microtubule-dependent process
(Adler et al., 1994).
An important recent observation is that the In protein localises at the
proximal apicolateral edges of wing cells under control of the core polarity
proteins, shortly prior to prehair formation
(Adler et al., 2004). This
localisation also requires the activity of fy and frtz, but
not mwh. Two alternative models have been put forward to explain the
requirement of proximally localised In for distal prehair initiation: In might
promote local formation of a repressor of prehair initiation, thus restricting
initiation to distal regions; alternatively, In could act positively to
promote polarised transport of a factor required for prehair initiation to the
distal cell edge (Adler et al.,
2004). Both models challenged the existing assumption that
distally localised Fz/Dsh act as the primary determinants for prehair
initiation, and instead suggest that proximally localised Stbm/Pk might be the
crucial cue.
The phenotype of mwh mutants is stronger than that of in,
fy or frtz, displaying a greater number of inappropriate prehair
initiations per cell (Wong and Adler,
1993). By genetic criteria, mwh acts downstream of the
other effectors and is therefore the factor most likely to interact directly
with the actin cytoskeleton, perhaps by acting as a repressor of pimple
activation. However, thus far, the molecular identity of the gene product of
mwh is unknown.
The widespread requirements of core planar polarity protein activity in
invertebrate and vertebrate morphogenesis, and the associated observation of
asymmetric core protein subcellular localisation, suggest that it will be
important to understand how the `distal' Fz/Dsh cue and/or the `proximal'
Stbm/Pk cue control cell shape and behaviour by modulating the cytoskeleton.
Significantly, homologues of the effectors In and Fy have already been found
to play crucial roles in vertebrate embryogenesis
(Park et al., 2006). In this
study, we sought to understand the mechanisms by which the core planar
polarity proteins and their effectors restrict prehair initiation to the
distal cell vertex during Drosophila wing development.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and Ubx-FLP
(Emery et al., 2005); twin
clones were generated as previously described
(Strutt and Strutt, 2007).
Overexpression was using GAL4/UAS (Brand
and Perrimon, 1993).
Immunolabelling and imaging
Pupae were aged at 25°C unless indicated otherwise and wings and cells
were processed for immunofluorescence as described previously
(Strutt, 2001), except that in
some cases, to improve labelling of actin structures, the fixative was
supplemented with 1% Triton X-100 and 1:200 Alexa568-phalloidin (Molecular
Probes). Primary antibodies were 1:400 mouse monoclonal
anti-β-galactosidase (β-gal) (Promega), 1:4000 rabbit
anti-β-gal (Cappel), 1:4000 rabbit anti-GFP (Abcam), 1:10 mouse
monoclonal anti-Fmi (#74, DSHB) (Usui et
al., 1999), 1:200 mouse monoclonal anti-Arm (DSHB), 1:1000 rat
anti-Dsh (Strutt et al.,
2006), 1:1000 rat or 1:100 rabbit anti-Mwh, 1:1000 rat or 1:100
rabbit anti-Frtz, 1:200 mouse monoclonal anti-
-tubulin (DM1A, Sigma).
Actin was visualised using 1:200 Texas Red or Alexa568-phalloidin. Confocal
z-stacks were captured on a Leica SP confocal microscope, and average
projections of several z-planes were made to provide a final image
depth of 1 µm. Fluorescent intensities were quantitated using
ImageJ.
Molecular biology and tissue culture
ESTs containing the coding sequences of fy (AT05453),
frtz (RH72421) and CG13913 (RE53394) were obtained from the
DGRC. Flies expressing EGFP-Fy and EGFP-CG13913 were generated by fusing EGFP
to the N-terminus of the coding sequence and cloning into the transformation
vector pP{w+, ActP-FRT-PolyA-FRT-PolyA}
(Strutt, 2001). Germline
transformations were performed by BestGene. EGFP-CG13913 was expressed in
cultured Drosophila S2 cells using pP{w+,
ActP-FRT-PolyA-FRT-EGFP-CG13913} transfected using Effectene (Qiagen),
with cotransfection of pActP-FLP to excise the FRT-PolyA-FRT cassette. Cells
were plated on Concanavalin A-treated coverslips.
Generation of antibodies
Frtz and Mwh antibodies were generated in rats and rabbits against
His-tagged fusion proteins corresponding to amino acids 670-951 and 440-836,
respectively; rabbit sera were affinity purified against the same fusion
protein.
Characterisation of the mwh lesion
The breakpoint in mwh1 was isolated by inverse PCR,
identifying an inversion following amino acid 367 that breaks within the
conserved FH3 domain, leading us to believe that this might result in a null
allele. In support of this, we scored mwh1 as amorphic in
the wing, with the adult trichome phenotype of mwh1
homozygotes being indistinguishable from that of hemizygotes (D.S.,
unpublished).
Immunoblotting
Protein extracts for immunoblotting were prepared by dissecting pupal wings
directly into sample buffer (141 mM Tris base, 2% lithium dodecyl sulphate,
10% glycerol, 0.51 mM EDTA, 100 mM dithiothreitol, pH 8.5) and running the
equivalent of one wing per lane. For phosphatase experiments, wings were
dissected into ice-cold lysis buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5%
Triton X-100, protease inhibitors (Roche)] supplemented with phosphatase
inhibitor (1 µM microcystin) for control samples. Experimental samples were
treated with 400 U lambda phosphatase (NEB) for 30 minutes at room
temperature. Proteins were detected using 1:500 affinity-purified rabbit
anti-Mwh, 1:5000 anti-actin (AC40, Sigma) or 1:10,000 anti--tubulin
(DM1A, Sigma).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Shimada et al.,
2001; Das et al.,
2004), but, at cell edges touching non-mutant cells, Stbm/Pk are
strongly recruited (Tree et al.,
2002; Bastock et al.,
2003), giving rise to a localised `proximal' cue in a cell lacking
a `distal' cue. Strikingly, in such cells containing just a proximal cue,
prehair initiation is still seen to occur at the opposite cell edge
(Fig. 1B). However, in
fz cells away from the clone edge, prehair initiation occurs in the
cell centre as previously reported (Wong
and Adler, 1993), but is noticeably delayed.
Patches of cells lacking stbm activity present the opposite
situation, in which no proximal cue is present, but, in cells touching
non-mutant cells, a distal cue containing Fz/Dsh assembles
(Strutt, 2001;
Bastock et al., 2003). Cells
containing only a localised distal cue were also seen to initiate a prehair at
the site of this cue, despite the lack of a proximal cue at the opposite cell
edge (Fig. 1C). Notably,
stbm mutant cells within the clone showed a shorter delay in prehair
initiation than observed with fz tissue. In addition, prehair
initiation was only consistently seen in the cell centre in the fourth row of
cells away from the clone edge. In the third row of cells, about half produced
a prehair that was positioned towards the cell edge nearest to the clone
boundary, whereas in the second row of mutant cells, almost all prehairs
initiated closer to the cell edge nearest the clone boundary.
To compare further the effects on prehair initiation of cells only having a
distal or proximal cue, we generated wings containing clones of fz
mutant cells abutting clones of stbm mutant cells (fz;
stbm twin clones) (Strutt and
Strutt, 2007). At the boundary between the fz and
stbm clones, the core polarity proteins showed normal asymmetric
localisation, but the neighbouring cells were all mutant and failed to
asymmetrically localise core polarity proteins. Hence, we can rule out any
potential influence of asymmetric core protein localisation in neighbouring
cells on the site of prehair initiation, and focus on the effects of the
asymmetric localisation straddling a single cell-cell boundary. Consistent
with the effects observed around isolated fz and stbm
clones, cells containing a distal cue initiated a prehair at this cell edge,
whereas the neighbours containing a proximal cue assembled a prehair towards
the opposite cell edge (Fig.
1D). We again saw a greater delay in prehair initiation in
fz mutant cells that were not on the clone edge, versus stbm
mutant cells, and also found that the second row of stbm mutant cells
had a site of prehair initiation that was influenced by their polarised
neighbours on the clone edge.
|
One way in which an adjacent cell might influence prehair formation in a
neighbour would be if assembly of a prehair produced a physical cue that
influences the cytoskeleton of the neighbouring cell and induces prehair
formation at the opposite edge. To test this, we generated mutant cells
containing only a proximal or distal polarity cue, juxtaposed to cells that
are unable to form a prehair by virtue of being mutant for shavenoid
(sha) (Ren et al.,
2006). Interestingly, cells on the edges of a fz clone,
adjacent to sha mutant cells, formed prehairs at the expected cell
edge despite the lack of prehairs in neighbouring cells
(Fig. 1E). Similarly, cells
containing only a distal cue also formed prehairs close to the cell edge
(Fig. 1F). Hence, prehair
formation in neighbouring cells is not necessary to enable cells with just a
proximal or distal cue to position prehairs correctly, although cell-cell
communication of another form cannot be excluded.
Additionally, we investigated the increased delay in prehair initiation in fz mutant cells versus stbm mutant cells. In theory, Fz and Stbm could be required simply to localise a prehair-promoting cue and a prehair-repressing cue, respectively, and/or could also be required for the activity of the cue. Hence, cells mutant for both fz and stbm might contain neither cue, or could contain one or both cues uniformly distributed.
Interestingly, stbm clones in a uniformly fz mutant background show no difference in the time of prehair initiation between single- and double-mutant tissue (Fig. 1G). This is consistent with Stbm acting to modulate the distribution of a cue but not altering its overall activity, such that the cue is equally active in cells with and without Stbm. Conversely, fz clones in a stbm mutant background show prehairs initiating sooner in stbm mutant tissue than in stbm; fz double-mutant tissue (Fig. 1H). This suggests that Fz is able to promote prehair initiation positively and that in the absence of Fz this promoting activity is lost.
|
Fy, Frtz and In are putative effectors of the proximal prehair initiation cue
Loss of activity of fy, frtz or in results in more than
one prehair initiating in ectopic positions in the cell, consistent with
inappropriate activation of a prehair-promoting cue or the loss of a
prehair-repressing cue (Gubb and
García-Bellido, 1982;
Wong and Adler, 1993;
Collier et al., 2005). Notably,
In localises proximally with Stbm, in a Stbm-dependent manner
(Adler et al., 2004),
suggesting that In mediates the proximal cue. Proximal localisation of In also
depends upon fy and frtz activity
(Adler et al., 2004),
consistent with the three gene products acting together to regulate prehair
initiation.
We raised an antibody against Frtz and found that Frtz also localises to
proximal junctions (Fig. 2A),
and that this localisation depends upon in and fy activity
(Fig. 2B,C), but not on the
downstream-acting gene mwh (Fig.
2D). Frtz junctional localisation also required stbm
activity (Fig. 2E), but was
only reduced in pk mutants (Fig.
2F), consistent with the effects of loss of pk on the
distribution of Stbm (Bastock et al.,
2003). Similarly, in fz mutant cells away from clone
borders, significant junctional localisation was retained
(Fig. 2G). Fluorescent
intensities of Frtz immunolabelling in the junctional regions of mutant tissue
were quantitated. Interestingly, within in and fy tissue,
levels were the same as within frtz tissue, indicating that
junctional Frtz protein is undetectable. However, in fz tissue,
fluorescent intensities were 50% of wild-type levels, and, in
stbm tissue, fluorescent intensities still achieved 25% of wild-type
levels. This suggests that although Frtz is not noticeably localised to
junctions in stbm tissue, the protein is nevertheless still present
within the cell, supporting the contention that Stbm acts to localise Frtz
activity but not necessarily to control its levels. (As our Frtz antibodies
did not work in immunoblotting, we were unable to more directly assess these
protein levels.)
These results indicate that Frtz, like In, localises proximally in a Stbm-dependent manner. We also attempted to determine the localisation of Fy using an EGFP-tagged form. We again saw preferential localisation to the apicolateral junctions, with enrichment at proximal cell edges (Fig. 2H). These results are consistent with In, Fy and Frtz all colocalising proximally and (at least in the case of In and Frtz) each requiring the activity of the other two to become localised.
mwh encodes a novel FH3-domain protein that is more strongly localised proximally in cells
The mwh locus has been mapped genetically to the cytological
position 61E-F. As the most downstream known effector of core polarity gene
function, we hypothesised that mwh might encode a protein that
interacts directly with the cytoskeleton. Searching by gene ontology in
FlyBase for `cytoskeletal protein binding' revealed a single uncharacterised
candidate gene, CG13913, in this region. A strain carrying a
transgene that expresses an inducible RNAi hairpin against the
CG13913 transcript (Dietzl et al.,
2007) closely phenocopied the mwh phenotype
(Fig. 3B). Importantly,
transgenic flies expressing the CG13913 gene product fused to EGFP
also showed complete rescue of the mwh phenotype
(Fig. 3C). In addition, we
isolated the predicted coding region of CG13913 from
mwh1 flies by PCR and determined that the locus has been
subject to a rearrangement in which an inversion breaks the coding sequence
after amino acid 367 (Fig. 3D).
Taken together, this is good evidence that mwh corresponds to
CG13913 and also suggests that the mwh1 allele is
likely to correspond to a null allele (see also Materials and methods).
Analysis of the CG13913 coding sequence for known protein domains
(Labarga et al., 2007)
revealed the presence of the Interpro domains Diaphanous formin homology 3
(FH3) and GTPase-binding/formin homology 3 (GBD/FH3)
(Fig. 3D). Formins are a class
of proteins involved in actin nucleation that generally consist of three
conserved domains known as the FH1, FH2 and GTPase-binding (GBD) domains
(Wallar and Alberts, 2003). In
addition, some formins also contain a further conserved domain known as the
FH3 domain, which partly overlaps the GBD and is thought to be involved in
subcellular localisation of the protein
(Petersen et al., 1998;
Kato et al., 2001). Homology
searches identified homologues of Mwh only in insects, with the closest
mammalian matches to the GBD/FH3 domain being found in conventional formins
also containing an FH2 domain. However, a GBD/FH3 domain is present in the
absence of FH2 domains in some Dictyostelium RasGEFs
(Rivero et al., 2005). Thus,
Mwh is a novel protein implicated in actin cytoskeleton regulation, but which
lacks the functional domains normally found in formins that mediate actin
nucleation.
|
Mwh localisation and phosphorylation are regulated by frtz
Next we asked whether Mwh levels and distribution were regulated by in,
fy or frtz. Interestingly, in all three genotypes, the apical
punctate labelling of Mwh was dramatically reduced
(Fig. 4A-C). By contrast, in
fz and stbm clones, the proximal enrichment of Mwh was lost
(consistent with loss of proximal Frtz localisation), but the levels of apical
punctate labelling were not greatly altered
(Fig. 4D,E; see Fig. S1C-F in
the supplementary material).
To further investigate the effects of frtz on Mwh, we used our Mwh
antibody for immunoblotting. In extracts from wild-type flies, the antibody
detected a number of bands in the molecular weight range expected for Mwh
(91 kDa). Only a broad band migrating at 110 kDa was lost in
mwh mutants or CG13913-RNAi extracts, consistent with this
representing the Mwh protein (Fig.
5A). Expression of EGFP-Mwh gave rise to bands 20-30 kDa
larger, reflecting the expected shift in molecular weight due to the tag.
Extracts from fz and stbm flies showed negligible changes in
Mwh levels, consistent with the immunolabelling results
(Fig. 5A). However, loss of
frtz activity resulted in a slight increase in Mwh gel-mobility, with
the loss of a higher-molecular-weight form
(Fig. 5A,C). We surmise that
the higher-molecular-weight form constitutes the apical and proximally
enriched punctate populations of Mwh seen by immunolabelling, as both were
reduced in frtz cells.
A plausible explanation for the presence of the higher-molecular-weight form of Mwh is that Frtz promotes post-translational modification of Mwh. When wild-type protein extracts were treated with phosphatase (Fig. 5B), the slower-migrating form of Mwh was lost and an increase in gel-mobility similar to that seen upon loss of frtz was observed, consistent with Frtz promoting Mwh phosphorylation.
|
;
Collier et al., 2005). This
could be explained if Mwh stability was temperature dependent. On immunoblots,
we observed a dramatic reduction in Mwh levels in wings from animals raised at
18°C, as compared with 29°C (Fig.
5C). This was additive to the effects of frtz on Mwh
gel-mobility, consistent with the cold-sensitivity of in, fy and
frtz nulls being a result of reduced stability of Mwh.
mwh mutant cells show ectopic actin bundles across their apical surface
The earliest reported manifestation of the mwh phenotype is
ectopic prehair initiation at the cell edge
(Wong and Adler, 1993). Using
fixation conditions optimised for preservation of F-actin structures, we
re-examined the earliest stages of the mwh phenotype, looking in
wings from animals raised at 18°C, 25°C and 29°C. A similar
phenotype was seen at each temperature
(Fig. 6A-D, and data not
shown). We observed that prior to and during the appearance of prehair
structures at the cell periphery, cells showed excess actin bundling across
the apical surface of the cells, sometimes in a `starburst' pattern, with
actin bundles radiating from the cell centre
(Fig. 6A,B). Subsequently,
prehair structures were seen at cell edges, as previously reported.
Interesting, the excess actin bundles often extended at least 1 µm basally
into the cytoplasm from the apical surface, particularly once prehair
initiation was underway (Fig.
6C,D). These results suggest that excess actin polymerisation
across the apical surface of the cell is the primary defect in mwh,
and that ectopic prehairs forming at cell edges might be a secondary
consequence.
Mwh affects actin structures in cultured cells
From the molecular homology of Mwh with other proteins of known function,
one can suggest that it could directly interact with the cytoskeleton or
cytoskeletal modulators via its GBD/FH3 domain. However, the lack of other
functional domains seen in formins might indicate that Mwh negatively
influences actin filament formation, which would explain the unrestricted
actin bundling seen across the apical surface of cells in its absence.
To gather more evidence for Mwh repressing actin filament formation, we
transfected Mwh into cultured Drosophila S2 cells and assayed the
effect on their actin cytoskeleton and behaviour. Intriguingly, cells
expressing high levels of EGFP-Mwh showed an altered morphology compared with
their contacting neighbours, characterised by a less rounded shape, the
appearance of slender projections at the cell periphery and a reduction in
F-actin bundles visible at the cell periphery
(Fig. 6E). When we quantitated
the reduction in fluorescent labelling intensity at the edges of transfected
cells we found, on average, a 63% reduction. Furthermore, we observed that
isolated transfected cells progressively developed a more dramatically altered
morphology, with radially projecting slender extensions
(Fig. 6F, present in 84% of
transfected cells, compared with only 6% of control cells). This phenotype is
reminiscent of the effects of reducing the activity of a number of proteins
involved in actin dynamics, including the Arp2/3 complex, which is required
for nucleation of actin filaments (Kiger
et al., 2003; Kunda et al.,
2003). Together, these results suggest that EGFP-Mwh
overexpression can inhibit actin filament formation.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
Here we present evidence that core polarity protein localisation at both
proximal and distal cell edges provides redundant cues for specifying distal
prehair initiation.
Regarding the mechanistic basis of the proximal cue, this and previous work
provide evidence for a plausible model
(Fig. 7). The downstream
effectors In, Fy and Frtz all colocalise at the proximal cell edge with Stbm
and in a Stbm-dependent manner. Activity of In, Fy and Frtz is required for
Mwh phosphorylation and its sub-apical subcellular localisation, which is thus
concentrated towards the proximal side of the cell. Genetic studies have shown
that loss of fy, in, frtz or mwh activity leads to excess
prehair initiation (Wong and Adler,
1993; Collier et al.,
2005), and we find that the initial defect in mwh mutant
cells is excess actin bundling across the entire apical face of cells. Thus,
proximal restriction of Mwh activity in the cell results in actin bundling and
prehair initiation specifically in distal regions.
Additional evidence for the sufficiency of a Stbm-dependent cue for prehair
initiation at opposite cell edges comes from experiments in the
Drosophila abdomen (Lawrence et
al., 2004). Here, it was reported that cells lacking fz
activity, but juxtaposed to cells with fz activity, were able to
produce polarised trichomes, as we also observed in the first row of cells
within a fz clone in the wing.
We have less information regarding the distal cue. Its existence is based upon two pieces of evidence. First, if prehair initiation were entirely dependent on Stbm-mediated localisation of Mwh activity, then prehairs should show no bias in their site of initiation in cells lacking stbm activity. In fact, stbm mutant cells with Fz localised at one cell edge show a strong bias towards initiating prehairs at that edge. Second, if prehair initiation were controlled only by a Stbm-dependent repressive cue, then in the absence of stbm activity, Fz would have no influence over prehair initiation. Instead, in a stbm background, fz activity still weakly promotes prehair formation. Taken together, these data support the view that distally localised Fz acts as a prehair-promoting cue.
|
|
;
Winter et al., 2001).
Alternatively, Fz might promote prehair initiation in a Mwh-independent
fashion, via RhoA/Drok or other effectors. It is notable that the absence of fz activity results in a delay in prehair formation and in a greater tendency, compared with loss of stbm, for prehairs to form in the cell centre rather than towards a cell edge. We surmise that in fz mutant cells, there is no Fz-dependent prehair-promoting cue, and the Stbm-dependent repressive cue is evenly distributed around the cell edge, resulting in delayed prehair initiation in the cell centre. Conversely, in stbm mutant cells, there is no change in the activity of the repressive cue, but the Fz-dependent prehair-promoting cue is localised to cell edges, albeit more thinly spread than in the wild-type situation. This results in approximately normally timed prehair initiation near the cell edges.
An unexplained observation is that within stbm mutant tissue, the
site of prehair initiation appears to be biased towards that seen in
neighbouring cells. Thus, in the first rows of cells within a clone, prehairs
tend to point towards the adjacent wild-type tissue. This phenomenon is
presumably independent of core protein asymmetric localisation and might
depend upon some mechanical linkage between cells. In this context, there is
already evidence to suggest that the microtubule cytoskeletons of adjacent
cells are linked and that this could coordinate cell polarity
(Turner and Adler, 1998). An
alternative, core-protein-independent mechanism to align wing hairs that
relies on the activities of Gliotactin and Coracle, has also been reported
(Venema et al., 2004).
|
). As In,
Fy and Frtz are all required for the apical punctate distribution of Mwh
within cells, and also appear to stabilise each other (this work)
(Adler et al., 2004), this
suggests that In, Fy and Frtz act together to activate Mwh and promote its
apical localisation. Conversely, although Stbm plays a role in localising Mwh
within the cell, it is not required for its activity, as loss of stbm
does not phenocopy mwh mutants in which increased apical actin
bundling is observed. This role of Stbm in localising but not regulating Mwh
activity is most simply explained by Stbm acting to localise, but not
regulate, the activity of In, Fy and Frtz. This is supported by the
observation that whereas loss of fz or stbm has a strong
effect on the distribution of Frtz to the apicolateral junctions, it has a
negligible effect on the apparent phosphorylation state of Mwh. The regulation of Mwh activity appears to be largely post-translational, as although the subcellular distribution of Mwh changes dramatically in frtz mutant cells, total levels of Mwh are not similarly altered. Further evidence that In, Fy and Frtz regulate Mwh activity by a mechanism largely independent of Mwh protein levels comes from the observation that Mwh overexpression in the wing has no effect on trichome formation (D.S., unpublished), rather than repressing trichome formation as might be predicted if Mwh protein levels were the main determinant of activity.
Our data strongly suggest that Mwh activity is regulated by phosphorylation. Treatment of cell extracts with phosphatase results in increased gel-mobility of Mwh. A similar increase in mobility is observed when frtz activity is removed, but not when stbm or fz activities are removed. Thus, at the least, Mwh phosphorylation correlates with Mwh activity and apical punctate localisation. Hence, we propose that the roles of In, Fy and Frtz might be to activate, or bring into proximity with Mwh, a kinase or kinases responsible for activating Mwh. Similarly, Fz could locally promote the dephosphorylation of Mwh to induce prehair initiation, although any such effect would have to be small, as Mwh phosphorylation is not obviously altered in the absence of Fz.
Definitive proof that phosphorylation of Mwh is important for its activity would require the identification of particular phosphorylation sites required for specific molecular functions and/or the identification of a kinase essential for Mwh activity.
An alternative regulatory mechanism for Mwh, by analogy with Diaphanous
family formins, would be via RhoA GTPase activity
(Wallar and Alberts, 2003).
The FH2 domain of such formins promotes actin nucleation, an activity that is
autoinhibited by interaction with the GBD. Upon interaction of the GBD with
GTPase-bound Rho GTPases, this autoinhibition is released. Notably, genetic
interaction data suggest that Fz/Dsh can activate RhoA activity
(Strutt et al., 1997;
Winter et al., 2001). This is
consistent with a model whereby in the proximal cell, Rho GTPase activity is
low and Mwh inhibits prehair initiation, whereas in the distal cell, activated
RhoA alleviates the inhibitory activity of Mwh.
Notwithstanding our evidence for post-translational regulation of Mwh activity in the normal context of the pupal wing, in cultured cells we do see an effect of Mwh overexpression on the actin cytoskeleton. This seems likely to be due to the much higher levels of expression that can be achieved in transfected cells as opposed to cells in vivo, and hence the result should be treated with caution, but might nonetheless suggest that S2 cells express a factor able to constitutively activate Mwh.
Our results also indicate that Mwh levels are influenced by temperature,
which provides a plausible explanation for why in, fy and
frtz phenotypes are stronger at 18°C than at higher temperatures
(Adler et al., 1994;
Collier and Gubb, 1997;
Collier et al., 2005). We
suggest that loss of in, fy and frtz reduces Mwh activity
and that lower temperatures additively reduce Mwh levels, resulting in lower
overall Mwh activity.
What is the molecular function of Mwh?
As already noted, the FH3 domain of conventional formins is thought to be
involved in targeting the protein to particular cellular sites, whereas the
GBD domain is involved in inhibition of the actin-nucleating function of the
FH2 domain (Wallar and Alberts,
2003). A plausible model is that Mwh acts as a dominant-negative
by binding via its GBD domain to other FH2-domain-containing formins involved
in the nucleation of actin filaments, thereby inhibiting their activity.
Notably, this dominant-negative activity of Mwh could then be inhibited
distally in the cell by Fz-mediated activation of RhoA GTPase activity.
Electron microscopy studies suggest that prior to prehair initiation, the
apical cell surface is covered in electron-dense pimples that are normally
only activated at the distal cell edge and serve as foci for actin filament
formation (Guild et al.,
2005). We propose that at around 32 hours of pupal development,
cells receive a general signal for pimple activation that results in actin
nucleation, and that Mwh activity is required to inhibit this activation at
locations away from the distal cell edge.
The lack of direct vertebrate homologues of Mwh might indicate that in
insects, the GBD/FH3 domain of a conventional formin has become separated from
the rest of the molecule, but retained its function in inhibiting
formin-mediated actin nucleation. Nevertheless, it is also plausible that the
core polarity proteins would use similar regulatory mechanisms to promote
local changes in cytoskeletal structure in vertebrate cells as those employed
in the Drosophila wing. Importantly, vertebrate homologues of both
Fuzzy and Inturned have been shown to be involved in regulating apical actin
assembly and, thus, in specifying the orientation of cilia
(Park et al., 2006). By
analogy with our findings, we suggest that core polarity proteins in
vertebrates are likely to localise Fuzzy/Inturned activity within cells and to
regulate formin activity via phosphorylation and/or Rho GTPase activation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/18/3103/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adler, P. N., Charlton, J. and Park, W. J. (1994). The Drosophila tissue polarity gene inturned functions prior to wing hair morphogenesis in the regulation of hair polarity and number. Genetics 137,829 -836.[Abstract]
Adler, P. N., Zhu, C. and Stone, D. (2004). Inturned localizes to the proximal side of wing cells under the instruction of upstream planar polarity proteins. Curr. Biol. 14,2046 -2051.[CrossRef][Medline]
Axelrod, J. D. (2001). Unipolar membrane
association of Dishevelled mediates Frizzled planar cell polarity signalling.
Genes Dev. 15,1182
-1187.
Bastock, R., Strutt, H. and Strutt, D. (2003).
Strabismus is asymmetrically localised and binds to Prickle and Dishevelled
during Drosophila planar polarity patterning.
Development 130,3007
-3014.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118,401 -415.[Abstract]
Collier, S. and Gubb, D. (1997). Drosophila tissue polarity requires the cell-autonomous activity of the fuzzy gene, which encodes a novel transmembrane protein. Development 124,4029 -4037.[Abstract]
Collier, S., Lee, H., Burgess, R. and Adler, P. (2005). The WD40 repeat protein fritz links cytoskeletal planar polarity to frizzled subcellular localization in the Drosophila epidermis. Genetics 169,2035 -2045.
Das, G., Jenny, A., Klein, T. J., Eaton, S. and Mlodzik, M.
(2004). Diego interacts with Prickle and Strabismus/Van Gogh to
localize planar cell polarity complexes. Development
131,4467
-4476.
Dietzl, G., Chen, D., Schnorrer, F., Su, K. C., Barinova, Y., Fellner, M., Gasser, B., Kinsey, K., Oppel, S., Scheiblauer, S. et al. (2007). A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448,151 -156.[CrossRef][Medline]
Eaton, S., Wepf, R. and Simons, K. (1996).
Roles for Rac1 and Cdc42 in planar polarisation and hair outgrowths in the
wing of Drosophila. J. Cell Biol.
135,1277
-1289.
Emery, G., Hutterer, A., Berdnik, D., Mayer, B., Wirtz-Peitz, F., Gaitan, M. G. and Knoblich, J. A. (2005). Asymmetric Rab 11 endosomes regulate delta recycling and specify cell fate in the Drosophila nervous system. Cell 122,763 -773.[CrossRef][Medline]
Gubb, D. and García-Bellido, A. (1982). A genetic analysis of the determination of cuticular polarity during development in Drosophila melanogaster. J. Embryol. Exp. Morphol. 68,37 -57.[Medline]
Guild, G. M., Connelly, P. S., Ruggiero, L., Vranich, K. A. and
Tilney, L. G. (2005). Actin filament bundles in Drosophila
wing hairs: hairs and bristles use different strategies for assembly.
Mol. Biol. Cell 16,3620
-3631.
Kato, T., Watanabe, N., Morishima, Y., Fujita, A., Ishizaki, T. and Narumiya, S. (2001). Localization of a mammalian homolog of diaphanous, mDia1, to the mitotic spindle in HeLa cells. J. Cell Sci. 114,775 -784.[Abstract]
Kiger, A. A., Baum, B., Jones, S., Jones, M. R., Coulson, A., Echeverri, C. and Perrimon, N. (2003). A functional genomic analysis of cell morphology using RNA interference. J. Biol. 2,27 .[CrossRef][Medline]
Klein, T. J. and Mlodzik, M. (2005). Planar cell polarization: an emerging model points in the right direction. Annu. Rev. Cell Dev. Biol. 21,155 -176.[CrossRef][Medline]
Kunda, P., Craig, G., Dominguez, V. and Baum, B. (2003). Abi, Sra1, and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions. Curr. Biol. 13,1867 -1875.[CrossRef][Medline]
Labarga, A., Valentin, F., Anderson, M. and Lopez, R.
(2007). Web services at the European bioinformatics institute.
Nucleic Acids Res. 35,W6
-W11.
Lawrence, P. A., Casal, J. and Struhl, G.
(2004). Cell interactions and planar polarity in the abdominal
epidermis of Drosophila. Development
131,4651
-4664.
Lee, H. and Adler, P. N. (2002). The function
of the frizzled pathway in the Drosophila wing is dependent
on inturned and fuzzy. Genetics
160,1535
-1547.
Mitchell, H. K., Roach, J. and Petersen, N. S. (1983). The morphogenesis of cell hairs on Drosophila wings. Dev. Biol. 95,387 -398.[CrossRef][Medline]
Park, T. J., Haigo, S. L. and Wallingford, J. B. (2006). Ciliogenesis defects in embryos lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling. Nat. Genet. 38,303 -311.[CrossRef][Medline]
Park, W. J., Liu, J., Sharp, E. J. and Adler, P. N. (1996). The Drosophila tissue polarity gene inturned acts cell autonomously and encodes a novel protein. Development 122,961 -969.[Abstract]
Petersen, J., Nielsen, O., Egel, R. and Hagan, I. M.
(1998). FH3, a domain found in formins, targets the fission yeast
formin Fus1 to the projection tip during conjugation. J. Cell
Biol. 141,1217
-1228.
Ren, N., He, B., Stone, D., Kirakodu, S. and Adler, P. N. (2006). The shavenoid gene of Drosophila encodes a novel actin cytoskeleton interacting protein that promotes wing hair morphogenesis. Genetics 172,1643 -1653.
Rivero, F., Muramoto, T., Meyer, A. K., Urushihara, H., Uyeda, T. Q. and Kitayama, C. (2005). A comparative sequence analysis reveals a common GBD/FH3-FH1-FH2-DAD architecture in formins from Dictyostelium, fungi and metazoa. BMC Genomics 6,28 .[CrossRef][Medline]
Shimada, Y., Usui, T., Yanagawa, S., Takeichi, M. and Uemura, T. (2001). Asymmetric co-localisation of Flamingo, a seven-pass transmembrane cadherin, and Dishevelled in planar cell polarisation. Curr. Biol. 11,859 -863.[CrossRef][Medline]
Strutt, D. I. (2001). Asymmetric localisation of Frizzled and the establishment of cell polarity in the Drosophila wing. Mol. Cell 7,367 -375.[CrossRef][Medline]
Strutt, D. (2003). Frizzled signalling and cell
polarisation in Drosophila and vertebrates.
Development 130,4501
-4513.
Strutt, D. and Strutt, H. (2007). Differential activities of the core planar polarity proteins during Drosophila wing patterning. Dev. Biol. 302,181 -194.[CrossRef][Medline]
Strutt, D. I., Weber, U. and Mlodzik, M. (1997). The role of RhoA in tissue polarity and Frizzled signalling. Nature 387,292 -295.[CrossRef][Medline]
Strutt, H., Price, M. A. and Strutt, D. (2006).
Planar polarity is positively regulated by casein kinase I
in
Drosophila. Curr. Biol.
16,1329
-1336.[CrossRef][Medline]
Tree, D. R. P., Shulman, J. M., Rousset, R., Scott, M. P., Gubb, D. and Axelrod, J. D. (2002). Prickle mediates feedback amplification to generate asymmetric planar cell polarity signalling. Cell 109,371 -381.[CrossRef][Medline]
Turner, C. M. and Adler, P. N. (1998). Distinct roles for the actin and microtubule cytoskeletons in the morphogenesis of epidermal hairs during wing development in Drosophila. Mech. Dev. 70,181 -192.[CrossRef][Medline]
Usui, T., Shima, Y., Shimada, Y., Hirano, S., Burgess, R. W., Schwarz, T. L., Takeichi, M. and Uemura, T. (1999). Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity under the control of Frizzled. Cell 98,585 -595.[CrossRef][Medline]
Venema, D. R., Zeev-Ben-Mordehai, T. and Auld, V. J. (2004). Transient apical polarization of Gliotactin and Coracle is required for parallel alignment of wing hairs in Drosophila. Dev. Biol. 275,301 -314.[CrossRef][Medline]
Vinson, C. R. and Adler, P. N. (1987). Directional non-cell autonomy and the transmission of polarity information by the frizzled gene of Drosophila. Nature 329,549 -551.[CrossRef][Medline]
Wallar, B. J. and Alberts, A. S. (2003). The formins: active scaffolds that remodel the cytoskeleton. Trends Cell Biol. 13,435 -446.[CrossRef][Medline]
Wang, Y. and Nathans, J. (2007). Tissue/planar
cell polarity in vertebrates: new insights and new questions.
Development 134,647
-658.
Winter, C. G., Wang, B., Ballew, A., Royou, A., Karess, R., Axelrod, J. D. and Luo, L. (2001). Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar polarity signalling to the actin cytoskeleton. Cell 105, 81-91.[CrossRef][Medline]
Wong, L. L. and Adler, P. N. (1993). Tissue
polarity genes of Drosophila regulate the subcellular location for
prehair initiation in pupal wing cells. J. Cell Biol.
123,209
-221.
Xu, T. and Rubin, G. M. (1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117,1223 -1237.[Abstract]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  D. Strutt and S. J. Warrington Planar polarity genes in the Drosophila wing regulate the localisation of the FH3-domain protein Multiple Wing Hairs to control the site of hair production J. Cell Sci., September 15, 2008; 121(18): e1807 - e1807. [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
