1 ZMBH, Universität Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg,
Germany
2 Max-Planck-Institut für Entwicklungsbiologie, Spemannstraße 35,
72076 Tübingen, Germany
3 Institut für Genetik, Heinrich-Heine-Universität Düsseldorf,
Universitätsstrasse 1 Geb. 26.02., 40225 Düsseldorf, Germany
Author for correspondence (e-mail:
j.grosshans{at}zmbh.uni-heidelberg.de)
Accepted 24 December 2004
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SUMMARY |
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Key words: formin, blastoderm, furrow canal, morphogenesis, Drosophila melanogaster
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Introduction |
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The enclosure of the nuclei into cells during the cellular blastoderm of
Drosophila embryos is achieved by a specialised process of membrane
invagination and reorganisation of the actin cytoskeleton
(Foe et al., 1993;
Schejter and Wieschaus, 1993
;
Mazumdar and Mazumdar, 2002
).
Following the exit from the last mitosis of the cleavage stage, the plasma
membrane folds in between adjacent nuclei to form the furrow canals that are
visible by light microscopy as the cellularisation front after about 10 to 20
minutes when the shape of the nuclei is already ellipsoid. The furrow canals
have a diameter of about 0.2 µm, are coated with actin filaments and remain
connected with the surface plasma membrane. Apical to the furrow canal the
so-called basal junction tethers the two adjacent membranes and thus
stabilises the furrow (Hunter and
Wieschaus, 2000
). The mechanism of the spatially restricted
assembly of F-actin at the furrow canal, as well as the factors that nucleate
F-actin at this site, have not yet been identified. Furthermore, it is unclear
whether actin filaments have an instructive function for the initial formation
and shape of the furrow canal.
A few genes are known to be involved in furrow canal formation. Embryos
mutant for nullo, sry-, nuf, Rab11, Abl, dah or
dia lack furrow canals between adjacent nuclei to a variable extent,
which leads to the formation of multinuclear cells
(Schweisguth et al., 1990
;
Postner and Wieschaus, 1994
;
Zhang et al., 1996
;
Rothwell et al., 1998
;
Afshar et al., 2000
;
Riggs et al., 2003
;
Grevengoed et al., 2003
).
Among this group of genes, nullo and sry-
are
particularly interesting because they are early markers for the furrow canal
and affect the formation of the basal junction
(Hunter and Wieschaus, 2000
).
Another early marker for the furrow canal is the novel protein Slam, which is
required for timed invagination of the furrow. Localised to the furrow canal
and the basal junction, Slam recruits MyoII to the furrow canal and affects
the accumulation of Arm at the basal junction (Lecuit et al., 2001;
Stein et al., 2002
). However,
a direct link to furrow canal formation and F-actin polymerisation has not
been established for any of the genes in this group.
To identify additional components required for proper cell morphology in
the blastoderm we have screened a large collection of female-sterile mutants
derived from germline clones (Luschnig et
al., 2004) (our unpublished data). We found two allelic mutations
that affect the cellularisation front. Mapping and complementation analysis
identified RhoGEF2 as the mutated gene.
RhoGEF2 is required during gastrulation for apical constriction of
the cells undergoing mesoderm invagination
(Barrett et al., 1997;
Häcker and Perrimon,
1998
). Although there is evidence that RhoGEF2 genetically
interacts with Rho1 and is controlled by Folded gastrulation during
Drosophila gastrulation, the mechanism of how RhoGEF2
controls cell shape at this stage and whether this involves spatially
restricted control of F-actin is not understood.
Potential effectors of RhoGEF2 and Rho1 are Rho kinase/sqh/myoII
(Royou et al., 2004), citron
kinase (Shandala et al., 2004
;
Naim et al., 2004
;
D'Avino et al., 2004
), protein
kinase N (Lu and Settleman, 1999b) and Diaphanous (Dia). As there are no
indications that Rho kinase/sqh/myoII and citron kinase would have a similar
function for furrow canal formation as RhoGEF2, we concentrated in our
analysis on dia, which is a member of the protein family with
formin-homology domains (FH) that control formation of actin filaments
(Wallar and Alberts, 2003
).
Biochemical and structural studies of the yeast (BNI1) and mouse (mDia; also
known as Diap1 - Mouse Genome Informatics) homologues have shown how actin
filaments are nucleated (Pruyne et al.,
2002
; Sagot et al.,
2002
; Li and Higgs,
2003
; Xu et al.,
2004
; Shimada et al.,
2004
; Higashida et al.,
2004
; Romero et al.,
2004
). mDia1 is assumed to be activated by binding of Rho1 that
releases an inhibitory intramolecular interaction of the C-and N-terminal
domains of mDia1 (Alberts,
2001
; Watanabe et al.,
1997
; Watanabe et al.,
1999
). However, this activation mechanism could only partially be
reconstituted in vitro (Li and Higgs,
2003
). Besides controlling actin fibres, Dia may also regulate
microtubules (Ishizaki et al.,
2001
; Palazzo et al.,
2001
; Wen et al.,
2004
; Yasuda et al.,
2004
). On a physiological level, the function of Dia and formins
are less defined. The M-formin1 may link actin filaments to adherence
junctions by an interaction with
-catenin
(Kobielak et al., 2004
), and
mDia3 may regulate attachment of microtubules to kinetochores during mitosis
(Yasuda et al., 2004
). In
Drosophila dia is required for cytokinesis in the male germline,
formation of pole cells and pseudo cleavage furrows during embryonic cleavage
stage (Castrillon and Wasserman,
1994
; Afshar et al.,
2000
). Furthermore cellularising embryos from dia
germline clones have defects in the arrangement and cortical connection of the
nuclei (Afshar et al., 2000
).
During this stage Dia protein localises to the furrow
(Afshar et al., 2000
), which
makes it a good candidate for controlling spatially restricted actin
polymerisation at the furrow canal. However, the site of Dia protein
localisation has neither been defined in detail nor correlated to the
morphological defects of the mutant.
We describe here a new function of RhoGEF2 and dia in the formation of the furrow canal. Mutant embryos have strongly enlarged furrow canals containing cytoplasmic blebs. Double immunolabelling studies show that RhoGEF2 and Dia are specifically concentrated between adjacent nuclei before the cellularisation front becomes visible and thus may serve as a template for the hexagonal pattern of membrane invagination. The amount of F-actin at the furrow canal is reduced in RhoGEF2 and dia mutants, suggesting that RhoGEF2 and Dia assemble actin filaments at the site of invagination and thus control the location, size and stability of the furrow canal. This further implies that spatially restricted F-actin polymerisation plays an important role for the initial infolding of the plasma membrane. Since many furrow canals still form in the absence of RhoGEF2 or dia, we tested whether nullo has a redundant function. Embryos lacking both RhoGEF2 and nullo, as well as embryos lacking both dia and nullo, do not form any furrow canals. This additive effect indicates that nullo functions in a genetic pathway separate from RhoGEF2 and dia. We propose that these pathways work in parallel to control two distinct but complementary aspects of furrow canal formation: actin polymerisation at the site of the infolding membrane and adherens junction formation.
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Materials and methods |
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The lethality on chromosome fs(2R)201 was fine-mapped to 53EF by
meiotic recombination with the w+ of P-element insertions
(l(2)k03609, l(2)k04222b, l(2)k07805b). The
lethality of the two mutations from our screen, fs(2R)201 and
fs(2R)350 was not complemented by the previously identified
RhoGEF21.1 (Barrett et
al., 1997). As the transcript is missing in the allele
RhoGEF204291, the described defects most probably
represent a complete loss-of-function phenotype (Häcker and Perrimon,
1988). The RhoGEF2 phenotype (allele fs(2R)201), as well as the
dia phenotype depend only on the maternal genotype, because
zygotically homozygous and heterozygous embryos, as marked with a
hb-lacZ reporter gene showed the same range of defects (data not
shown). Molecular characterisation of the dia5 locus
showed that about 3 kb of the original P element remained at the original
insertion site (data not shown).
Histology
Embryos were heat-fixed in 0.4% NaCl, 0.03% Triton X-100 or fixed with 4%
formaldehyde in PBS (except for phalloidin staining when 8% formaldehyde was
used) and stored in methanol. Fixed embryos were stained in PBS with 0.2%
Triton X-100 consecutively with solutions of primary antibody, fluorescent
secondary antibodies (4 µg/ml, Alexa488, Alexa546, Alexa647; Molecular
Probes), DNA dyes (DAPI, Hoechst, Oligreen or propidium iodide) and mounted in
either Mowiol/DABCO or Aquapolymount (Polyscience). Antibodies to the
following proteins were used: RhoGEF2 (0.1 µg/ml), Dia (1:4000; J. G. and
S. Wasserman), Slam [1:5000 (Stein et al.,
2002)], MyoII (B. Mechler), Arm (1:50), Sry-
(1:10), Dlg
(1:20; Hybridoma Center), HA (12CA5, 1 µg/ml; Roche), ß-gal (0.1
µg/ml; Roche),
-tubulin (0.2 µg/ml; Sigma), phalloidin coupled to
Alexa dyes (6 nM; Molecular Probes). To compare Dia and F-actin distribution
in wild-type, RhoGEF2 and dia embryos, embryos of the two
genotypes to be compared were mixed prior to fixation and processed as a
mixture. Wild-type embryos were marked with a nullo-HA transgene, by the
presence of RhoGEF2 staining or recognised by a proper F-actin array. For
quantification of the RhoGEF2 or dia mutant phenotype nuclei
in fields of 238x238 µm were counted in embryos stained with either
Arm or Dlg antibodies or phalloidin.
Microscopy
Fluorescent images were recorded with a Leica confocal microscope (DMIRE2,
20x NA 0.7 water, HCX PL APO 63x NA1.2 corr, HCX PL APO 63x
NA1.4-0.6 oil, laser at 405, 488, 543, 633 nm). Development of live embryos
was recorded using an inverted microscope with differential interference
contrast optics and a computer controlled stage (Leica DMIRE2, PL APO
63x NA1.4-0.6 oil; Hamamatsu ORCA-ER, Openlab software, Improvision).
Digital photographs were processed with Photoshop (Adobe). For the analysis of
the ultrastructure, embryos were staged in halocarbon oil (27S, Sigma) with a
dissecting microscope. After removing the chorion with hypochlorite and
rinsing, embryos were transferred to hexadecen, mounted in 100 µm deep
aluminium plates and fixed by rapid high pressure freezing using a Balzer HPFM
10 machine. Fixed embryos were collected in liquid nitrogen and
freeze-substitution was carried out with acetone containing 2% OsO4
at -90°C for 24 hours, -60°C for 6 hours and -40°C for 3 to 9
hours. Following embedding in Epon, sectioning and staining with Pb(II)citrate
and U(II)acetate, specimens were examined on a Zeiss 109 or Philips CM10
transmission electron microscope.
Microinjection, drug treatment
Eggs were dechorionated in 50% bleach, dried in a desiccation chamber,
covered with halocarbon oil and subsequently injected posteriorly with 50-100
pl of aqueous dsRNA at 1 µg/µl except for sry- dsRNA,
which was injected into wild-type embryos at 6 µg/µl. Embryos were fixed
with 4% formaldehyde for 30 minutes. The vitelline membrane was removed
manually. For the latrunculin A treatment, dechorionated embryos were
incubated for 2.5 minutes in n-heptane to permeabilise the vitelline
membrane, briefly rinsed in PBT (PBS plus 0.1% Tween 20), incubated for 6
minutes at room temperature in PBS containing 50 µg/ml latrunculin A (P.
Crews, Santa Cruz, USA) and subsequently fixed in formaldehyde.
Molecular genetics
DNA encoding indicated fragments were amplified by PCR, cut by appropriate
restriction enzymes and cloned into the indicated vectors: RhoGEF2
(aa1-687)-His6, as a NcoI-BglII into pQE80N60
(Görlich), GST-RhoGEF2 (aa1512-1897), EcoRI-SalI into
pGEX-4T-1 (Pharmacia). Dia aa 1-464 (pCS-diaC464) as
SalI-XbaI into pCS2 (R. Rupp, Munich). Dia aa 318-1091
(pCS-dia
N318) as SalI-XbaI into pCS2.
ZZ-dia-His6 fusions: Dia aa 1-518 (pZZ-dia
C518) or Dia aa
519-1091 (pZZ-dia
N519), KpnI-SalI into pQE80ZZ
(Görlich). The point mutations T1544A (codon1544 mutated to GCT) in the
GEF domain of RhoGEF2 and the Rho1Q63L (codon 63 mutated to CTG) were
introduced by inverse PCR with Pfu polymerase (Stratagene). GST fusion
constructs of RhoA, RhoL, Cdc42, Rac1, Rac2, Mtl1 and the TrioGEF-D1 are
described previously (Newsome et al.,
2000
). DNA templates for the synthesis of dsRNA were amplified
with a T7 promoter site at their ends (nullo 586 bp,
sry-
568 bp, Bsg25D 561 bp). Respective dsRNA fragments were
synthesised with T7 RNA polymerase (Ambion MEGAscript). Details are available
upon request.
Biochemistry
Purification of GST fusion proteins
E. coli BL21DE expressing the fusion proteins from pGEX plasmids
were lysed in 50 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM MgCl2, 1 mM
DTT, 1 mM PMSF in a French press. GST fusion proteins were purified from the
soluble fraction by GSH affinity chromatography (GSTrapFF, Amersham; wash
buffer 50 mM Tris-HCl pH 8, 500 mM NaCl, 10 mM MgCl2, 1 mM DTT,
elution buffer 50 mM Tris-HCl pH 8, 50 mM NaCl, 10 mM glutathione, 1 mM DTT),
dialysed against 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM MgCl, 1 mM DTT, 10%
glycerol and stored in aliquots at -80°C with an additional 250 mM
saccharose.
Purification of His-tagged proteins
Native or denatured extracts from E. coli BL21DE were purified by
nickel chelate chromatography (Ni NTA agarose, Qiagen).
Immunisation
Rabbits or guinea pigs were immunised with a denatured N-terminal fragment
of RhoGEF2 (aa 1-687) or native ZZ-diaN519. Antibodies were purified by
affinity chromatography with Sepharose (BrCN activated Sepharose; Pharmacia,
2.5 ml) with coupled RhoGEF2 (aa 1-687+H6, 10 mg, native).
Antibodies were eluted with 50 mM glycine pH 3.5, dialysed against PBS and
concentrated.
Guanyl-nucleotide exchange assay
GTPase (0.2 µM) loaded with [8-3H]GDP (426 GBq/mmol;
Amersham) and 0.1 µM of the corresponding GEF were incubated at 25°C
for 20 minutes, or as indicated. After nitrocellulose filtration, the
radioactivity on the filter was determined in a liquid scintillation counter.
The assay was performed in duplicate
(Debant et al., 1996) (S.
Schmidt, Montpellier, France).
For western blot analysis, extracts of approximately 50 embryos were separated on SDS-PAGE and transferred by semi-dry blotting to a nitrocellulose membrane (Schleicher and Schuell). Antibodies were diluted as follows: Dia 1:5000, Dlg 1:100, RhoGEF2 1:20000, ß-tubulin 1:10000. The blots were developed with IgG coupled with peroxidase and chemiluminescence (ECLplus, Amersham, Kodak XOMAT).
For radioactive labelling, proteins were expressed from template plasmids carrying a modified ß-globin leader sequence (pCS2 derivatives) with SP6 RNA polymerase in a coupled in vitro transcription-translation system (TNT, Amersham) supplemented with [35S]methionine (37 TBq/mmol; Amersham).
Binding assay
10 µl of GSH-Sepharose (Pharmacia) loaded with approximately 10 µg
GST fusion proteins were incubated with 2 µl of labelled protein in 400
µl binding buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM DTT, 10 mM
MgCl2, 0.2% Tween) for 1 hour at 4°C. After washing six times
with binding buffer, proteins were eluted with 2x50 µl of 10 mM
glutathione, 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 10 mM
MgCl2, 0.2% Tween and precipitated with 5% TCA. Following SDS-PAGE
the label was visualised with a phosphoimager (Fuji BAS1000).
Actin polymerisation assay
The indicated protein solution (60 µl) in G-buffer (20 µM
CaCl2, 20 µM ATP, 0.5 mM Tris-HCl pH 8) and 10 µl of
polymerisation buffer (12.5 mM KCl, 0.5 mM MgCl2, 25 µM ATP) were added to
30 µl of G-actin (10% labelled with pyrene, final concentration 3 µM;
Cytoskeleton, USA). Fluorescence was excited at 365 nm and recorded at 407
nm.
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Results |
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To investigate possible morphological defects, we analysed the ultrastructure of the furrow canals in embryos during mid-cellularisation by transmission electron microscopy. In wild-type embryos, the furrow canal can be seen as a hairpin loop with a diameter of about 0.2 µm in specimens fixed by high pressure freezing (Fig. 1A,B). Sometimes the diameter of furrow canals appears enlarged because of the plane of the section, in particular at the intersections of furrow canals. In six different embryos from two preparations, we found that 90% of furrow canals had the typical compact loop structure. In contrast, in embryos from RhoGEF2 germline clones, the furrow canal diameters were enlarged up to three fold and in most cases (78% in 6 embryos examined) did not show the typical hairpin loop (Fig. 1C,D). Instead, cytoplasmic blebs of variable size were present in the furrow canal, which we did not observe in wild-type embryos, suggesting that the enlarged mutant furrow canals are less stable, possibly because of their larger size or changed properties of the membrane. Defects in the morphology of the furrow canals were also observed in embryos from dia germline clones. The furrow canals in these embryos were more variable than in RhoGEF2 mutants ranging from about threefold dilated to diameters of more than 1 µm (Fig. 1E,F). Based on the analysis of the ultrastructure we conclude that RhoGEF2 and dia are required for compact and stable furrow canals during cellularisation.
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|
Distinct functions of RhoGEF2 and nullo in furrow canal formation
nullo and sry- are also required for correct
furrow canal formation (Postner and
Wieschaus, 1994
; Schweisguth
et al., 1990
). nullo and sry-
mutant
embryos contain multinucleate cells because furrow canals are frequently
absent (Fig. 2C).
sry-
probably acts downstream of nullo, because
Sry-
protein localisation depends on nullo
(Postner and Wieschaus, 1994
).
To test whether they act in the same genetic pathway as RhoGEF2 or
dia, we analysed the formation of the furrow canals, marked by
F-actin in embryos lacking both gene functions by producing RNAi-induced
phenocopies of nullo or sry-
in embryos from
RhoGEF2 or dia germline clones
(Fig. 3; see Fig. S2 in the
supplementary material). In embryos from RhoGEF2 germline clones
treated with RNAi for nullo (73%, n=33,
Fig. 3B,E) or
sry-
(63%, n=16,
Fig. 3C,D), F-actin at the
furrow canal was almost absent and only a few singular rings and irregular
patches remained. The strength of the `double mutant' phenotype directly
correlated with the amount of Sry-
protein as seen in embryos with
locally deposited sry-
dsRNA at the posterior pole
(Fig. 3C). In transversal
sections, F-actin staining at the furrow canal was observed only in anterior
regions. This staining was gradually lost towards posterior regions in the
`double mutant' situation, which suggests that furrow canals are completely
absent (Fig. 3D,E). Following
nullo or sry-
RNAi injection into embryos from
dia germline clones we observed, in both cases, an almost complete
absence of furrow canals (data not shown).
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RhoGEF2, Dia and F-actin colocalise at the furrow canal
Dia protein is localised at the furrow
(Afshar et al., 2000), whereas
the distribution of RhoGEF2 protein has not yet been determined. We raised
antibodies against an N-terminal RhoGEF2 fragment to investigate the
intracellular distribution of RhoGEF2. The antibody specifically detects
RhoGEF2 because in western blots a band migrating at more than 200 kDa was
detected in extracts of wild-type but not of mutant embryos
(Fig. 4G). In whole-mount
staining of germline clone embryos from four alleles of RhoGEF2
(RhoGEF2201, RhoGEF21.1, RhoGEF24.1,
RhoGEF204291) only a low and uniform signal was detected
(Fig. 4D), whereas a locally
restricted staining was observed at the tip of the furrow throughout
cellularisation in wild-type embryos (Fig.
4A-C). The specific staining was visible when the nuclei were
still spherical, that is before the cellularisation front appeared. In surface
view RhoGEF2 formed a hexagonal array enclosing the nuclei
(Fig. 4E). This distribution
changed at the onset of gastrulation when RhoGEF2 staining was lost basally in
the invaginating ventral cells and simultaneously appeared apically
(Fig. 4F). These data show that
RhoGEF2 localisation precedes furrow canal formation at the site of
invagination, suggesting that it may determine the hexagonal pattern of
membrane invagination.
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|
Does the small GTPase Rho1 provide a link among RhoGEF2, Dia and F-actin?
The proposed biochemical activities of the guanyl nucleotide exchange
factor RhoGEF2 and the formin Dia suggest that RhoGEF2 activates a Rho type
GTPase and that actin polymerisation by Dia is activated by a Rho type GTPase.
To critically test such a model we reconstituted the interactions in vitro
with purified components (see Fig. S5 in the supplementary material). First,
to identify the substrates of the RhoGEF2 guanyl-nucleotide exchange activity
we sampled all six Drosophila Rho GTPases
(Newsome et al., 2000) in an
in vitro GDP-GTP exchange assay (Fig.
8A,B). We loaded GST fusion proteins of Rho GTPases with
3H-labelled GDP and measured the release of GDP catalysed by the
GEF domain of RhoGEF2. We found that GDP was significantly released only from
the Rho1 protein, but not from the RhoL, Rac or Cdc42 GTPases, whereas the GEF
domain of Trio was specific for the three Rac GTPases
(Newsome et al., 2000
). The
GEF domain of RhoGEF2 with a single point mutation (T1544A)
(Zhu et al., 2000
) and GST had
no activity. Consistent with the reported genetic interaction of
RhoGEF2 and Rho1 (Barrett
et al., 1997
), these data show that Rho1 is the only GEF substrate
of RhoGEF2.
|
In a third step, we tested whether Dia can induce actin polymerisation in a
Rho1-dependent manner (Li and Higgs,
2003). We purified two fragments of Dia: an N-terminal fragment
(Dia
C518, containing the Rho-binding site) and a C-terminal fragment
(Dia
N519, containing the FH1, FH2 and autoinhibitory domains) and
tested their activity on actin polymerisation in vitro. The C-terminal part
alone induced actin polymerisation at submicromolar concentrations (see Fig.
S6 in the supplementary material) similar to BNI1 fragments from yeast
(Pruyne et al., 2002
). This
activity was inhibited by equimolar amounts of the N-terminal part, but was
partially restored by a 10-fold molar excess of Rho1
(Fig. 8D). However, no
difference in activity was observed between activated forms of Rho1 (loaded
with GTP
S, GMP-PNP or Rho1Q63L) and Rho1 loaded with GDP (data not
shown). Our data show that the C-terminal part of Dia is sufficient for
efficiently polymerising actin filaments. The polymerisation activity is
inhibited by the N-terminal part, suggesting that Dia activity is controlled
by an intramolecular inhibition. However, since it was not possible to fully
reconstitute the release of Dia autoinhibition by Rho1, a behaviour also
described for mDia1 (Li and Higgs,
2003
), the mechanisms for Dia activation remains elusive. Together
with the weak binding of Rho and Dia that only slightly depended on the
activation state of Rho1 and consistent with the properties of mDia1, these
data indicate, that Rho1-independent mechanisms appear to be involved
(Wallar and Alberts,
2003
).
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Discussion |
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Our morphological analysis of the mutant phenotypes reveals a new function
of RhoGEF2 and dia in the formation of the furrow canal.
This function is consistent with the co-localisation of both proteins with
F-actin at the furrow canal and the reduced amounts of F-actin in
RhoGEF2 and dia mutants. Biochemical analysis demonstrates
actin polymerisation by Dia and thus supports the model that RhoGEF2 and Dia
organise actin filaments to control the formation of the furrow canals.
Furthermore, we provide evidence that the previously characterised genes
nullo and sry- act in a genetic pathway in parallel
to RhoGEF2 and dia, suggesting that they control two
distinct aspects of furrow canal formation. This conclusion is based on the
assumption that we used amorphic situtations in our experiment. We cannot
exclude that RhoGEF2 and dia stabilise the furrow canal
rather than control its initial formation. A function in the formation is
supported by our observation that the proportion of nuclei in multinuclear
cells does not increase in the course of cellularisation.
The following arguments support the hypothesis that RhoGEF2 and
dia act in the same genetic pathway that controls spatially
restricted assembly of actin filaments. In both dia and
RhoGEF2 mutants the morphology of the furrow canal is disrupted. The
furrow canals are much larger than normal and filled with cytoplasmic blebs
(Fig. 1). Both proteins are
localised at the furrow canal and both precede the appearance of the
cellularisation front (Fig. 5).
The localisation of both proteins does not depend on F-actin
(Fig. 6). However, they are
directly or indirectly involved in the assembly of F-actin since the amount of
F-actin is reduced at the furrow canal of the mutant embryos
(Fig. 7). The strongest
argument for a functional connection is that Dia localisation at the furrow
canal depends on RhoGEF2 during the early phase of cellularisation
(Fig. 7). Rho1 may mediate this
functional link by direct interactions with RhoGEF2 and Dia
(Fig. 8). However, our findings
do not show that RhoGEF2 exclusively functions via dia.
Other targets of Rho1-GTP, like citron kinase, protein kinase N or Rho kinase
(Lu and Settleman, 1999b; Shandala et al.,
2004; Naim et al.,
2004
; D'Avino et al.,
2004
; Royou et al.,
2004
) may be activated in parallel to Dia. Although we observe a
reduction of MyoII at the furrow canal during the first half of
cellularisation in embryos from RhoGEF2 germline clones,
correspondingly lower MyoII levels are also observed in embryos from
dia germline clones, which indicates that the reduction of MyoII may
be a consequence of reduced F-actin levels. Consistent with the reduction of
F-actin at the furrow canal, levels of MyoII were also reduced in the mutant
embryos (see Fig. S3 in the supplementary material). In contrast to the
reduction at the furrow canal, cortical F-actin appeared to be increased in
some embryos from dia germline clones. This increase was variable and
not observed in all of the experiments, however.
The difference in the RhoGEF2 and dia mutant phenotypes
clearly shows that dia has additional functions and may be controlled
by other not yet identified factors besides RhoGEF2. Whereas RhoGEF2
mutants pass through the cleavage cycles without obvious defects (data not
shown), dia is involved in formation of pole cells and pseudo
cleavage furrows (Afshar et al.,
2000). As a possible consequence of these additional functions,
dia mutants in contrast to RhoGEF2 mutants often have a more
disrupted F-actin array, larger furrow canals and a more disturbed
cellularisation than RhoGEF2 mutants (Figs
1,
2). Furthermore in the early
phase of cellularisation Dia localisation depends on RhoGEF2, whereas
later, after the furrow has formed, Dia becomes enriched to a certain degree
at the cellularisation front independently of RhoGEF2. One gene that
may act in parallel to RhoGEF2 to control Dia localisation is Abl.
Embryos from Abl germline clones have reduced amounts of Dia at the
furrow canal and show a disrupted F-actin array similar to that observed in
dia and RhoGEF2 mutants
(Grevengoed et al., 2003
).
However, the molecular link between Abl and Dia is elusive and no
abnormalities in the morphology of the furrow canal in Abl mutants
have been described. Thus Dia may be controlled and activated by multiple
pathways including RhoGEF2 among others.
It is not known how the position of the invaginating plasma membrane is determined. RhoGEF2 and Dia are not likely to be part of a pattern formation process, but their localisation reflects an early readout of this pattern, since the nuclei and centrosomes are properly arranged in RhoGEF2 and dia mutants (Fig. 2). RhoGEF2 and Dia proteins are early markers for these sites and precede furrow canal formation because we detected specific staining for both Dia and RhoGEF2 when the nuclei were still spherical and when the cellularisation front was not yet visible (Fig. 4A, Fig. 5B). Other factors beside RhoGEF2 and Dia are also involved in furrow canal formation, because many furrow canals still form in RhoGEF2 and dia mutants, which indicates that there is genetic redundancy.
At present we can only speculate about which factors and mechanisms are
responsible for RhoGEF2 localisation. Candidates may be among the group of
genes involved in furrow canal formation. However, for all of these mutations
no ultrastructural analysis has been reported that would allow us to define
the morphological defect and compare their function for furrow canal formation
with the function of RhoGEF2 and dia. Among this group are
Rab11 and nuf, which encode a GTPase of the recycling
endosome and its putative effector (Riggs
et al., 2003). Considering the assumed biochemical activities, it
is conceivable that vesicle targeting is important for transporting factors to
the site of membrane invagination (Riggs
et al., 2003
). This raises the possibility that RhoGEF2 is
transported by such vesicles to the sites of membrane infolding. Analysis of
RhoGEF2 protein distribution in nuf and Rab11 mutants and
the phenotype of double mutants may address this hypothesis. Alternatively,
RhoGEF2 may be transported to the site of the future furrow canal along
microtubules that form open baskets around the nuclei, or other recruiting
factors may precede at the site of membrane invagination.
Furthermore, slam is required for timed formation of the furrow
and invagination of the membrane in the first half of cellularisation. Like
Dia and RhoGEF2 Slam protein localises to the furrow canal and localisation
precedes furrow canal formation. Slam may act by recruiting MyoII to the
furrow canal, but the biochemical activities of Slam have not been defined
(Lecuit et al., 2002;
Stein et al., 2002
). Although
the membrane does not invaginate initially in slam mutants, a
complete F-actin array is visible (Fig.
2). Thus despite the overlapping localisation of Slam, RhoGEF2 and
Dia their functions are clearly distinguishable.
How do RhoGEF2 and Dia act in furrow canal formation? If we consider the
biochemical activity of Dia to nucleate actin filaments
(Fig. 8) and the enlarged and
labile furrow canals in the dia mutants
(Fig. 1), it is conceivable
that Dia organises and assembles a coat of F-actin at the site of membrane
invagination and furrow canal formation. The coat of F-actin may be important
for the compactness and stability of the furrow canal to prevent infoldings of
the cytoplasm. Such a function may be related to the function of F-actin in
endocytic events (Engqvist-Goldstein and
Drubin, 2003). The subset of actin filaments controlled by
RhoGEF2 would not significantly contribute to pulling in the plasma
membrane, since membrane invagination proceeds with normal speed in
RhoGEF2 mutants. Alternatively, RhoGEF2 and Dia may perform their
function independently of actin polymerisation. Although we have shown that
the amount of F-actin is reduced in the mutants
(Fig. 7), we do not exclude the
possibility that the polymerisation activity of Dia is not required for all or
part of its function. Dia may also influence the organisation of microtubules,
as interactions of mDia1 with microtubules and EB1, a microtubule-associated
protein, have been described (Ishizaki et
al., 2001
; Palazzo et al.,
2001
; Wen et al.,
2004
).
The differences in protein localisation and mutant phenotypes of
RhoGEF2 and nullo suggest that they have distinct
activities. In contrast to the frequently missing furrow canals in single
mutants, their complete absence in embryos lacking both gene functions
(Fig. 3) clearly implies,
however, that their functions are redundant from a genetic point of view. As
proposed by Hunter and Wieschaus (Hunter
and Wieschaus, 2000) the basal junction that tethers the two
membranes of the furrow and that is located apically to the furrow canal is
controlled by nullo, whereas our results show that RhoGEF2
and dia are required for the formation of a compact and stable furrow
canal. If one of the two pathways is disturbed, the furrow canal can still
form, albeit with a lower and variable efficiency that depends on the
conditions. For example the nullo phenotype is strongly temperature
sensitive (Hunter et al.,
2002
). However, if both pathways are affected, furrow canals do
not form at all. Future studies will resolve how the actin filaments are
involved in bending the plasma membrane that leads to the furrow canal and
will further demonstrate how RhoGEF2 protein is expressed in the hexagonal
array to serve as a template for local actin polymerisation.
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Supplementary material |
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ACKNOWLEDGMENTS |
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Footnotes |
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