Institut für Neurobiologie, Universität Münster, Badestr. 9, D-48149 Münster, Germany
* Author for correspondence (e-mail: klaembt{at}uni-muenster.de)
Accepted 12 June 2003
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SUMMARY |
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Key words: F-actin, Drosophila, NAP1/Kette
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INTRODUCTION |
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Actin dynamics crucially depend on the ability of the protein to switch
from a monomeric (G-actin) to a filamentous form (F-actin). Polymerization of
F-actin starts with the de novo nucleation of an actin trimer, a process that
occurs relatively slowly and requires the action of the Arp2/3 complex
(Machesky et al., 1994;
May, 2001
;
Robinson et al., 2001
;
Welch et al., 1997
).
Subsequent elongation is fast and cells have to prevent spontaneous actin
polymerization by expressing a variety of actin-binding proteins such as
profilin (Schafer and Cooper,
1995
). The nucleation activity of the Arp2/3 complex in turn is
regulated by a set of activators, such as the members of the Wasp
(Wiskott-Aldrich syndrome protein) and Wave (Scar - FlyBase) families
(Higgs and Pollard, 2001
;
Suetsugu et al., 2002b
;
Takenawa and Miki, 2001
). Wasp
proteins are usually self-inhibiting and require small G proteins of the Rho
family for activation (Kim et al.,
2000
; Miki et al.,
1998a
). In its activated GTP-bound form, Cdc42 can bind to the
Crib (Cdc42/Rac Interactive Binding) domain of Wasp, releasing the
auto-inhibition and thereby leading to the activation of the Arp2/3 complex
(Higgs and Pollard, 2001
;
Rohatgi et al., 1999
). By
contrast, Wave, which does not bind Cdc42, is trans-inhibited through its
association with members of the Kette (Hem - FlyBase) family, Sra1
(specifically Rac associated 1) and Abi (Abelson-interactor). Rac1 binding,
presumably to Sra1, relieves the inhibitory function of this complex
(Eden et al., 2002
;
Kobayashi et al., 1998
;
Miki et al., 2000
).
Both, Cdc42 and Rac1 exert distinct functions in rearranging the F-actin
cytoskeleton, GTP-bound Rac1 promoting lamellipodia and activated Cdc42
promoting filopodia formation (Hall,
1998). This matches the finding that Wasp enhances the generation
of filopodia, whereas Wave activation near the cell membrane results in an
increase of lamellipodia (Miki et al.,
1998a
; Takenawa and Miki,
2001
).
Drosophila possesses only two genes, wasp and
scar, that encode a Wasp and Wave homolog, respectively
(Zallen et al., 2002;
Ben-Yaacov et al., 2001
). Both
genes act largely independently of each other and have a strong maternal
component. Only embryos lacking both the zygotic and the maternal gene
functions display severe embryonic nervous system phenotypes. In the adult,
wasp is required for formation of external mechanosensory organs,
whereas scar/wave is - unlike wasp - required for
normal formation of the compound eye. Recent structure function analyses
demonstrated that Drosophila Wasp can perform at least part of its
function independent of Cdc42 (Tal et al.,
2002
). Alternative modes of Wasp activation such as
phosphorylation or SH3 (Src homology 3) domain binding have been described
(Cory et al., 2002
;
Scott et al., 2002
;
Suetsugu et al., 2002a
).
Obviously, regulation of the dynamic F-actin cytoskeleton is pivotal
especially during cell movement and thus requires a close link to the plasma
membrane and guidance receptors involved in the perception of extracellular
signals. The latter generally induce conformational changes in the cytoplasmic
domain of membrane-anchored receptors, which recruits other proteins to the
ligand-receptor complex. In the case of receptor tyrosine kinases (Rtk),
autophosphorylation of tyrosine residues leads to the recruitment of SH2
domain containing adapter proteins. One of these is the SH2 SH3 adapter
protein Nck, which is able to link several Rtks as well as guidance receptors
like the Netrin-receptor DCC to the actin cytoskeleton
(Li et al., 2001;
Li et al., 2002
). Nck can
recruit additional proteins to the cell membrane via its three SH3 domains.
Among the highly conserved Nck-interacting proteins are Wasp
(Rivero-Lezcano et al., 1995
),
the non-receptor tyrosine kinase Abl (Adler
et al., 2000
) and Nap1 (Nck-associated protein 1), which is a
member of the evolutionary conserved Kette family
(Baumgartner et al., 1995
;
Hummel et al., 2000
;
Kitamura et al., 1996
;
Soto et al., 2002
).
Based on its requirement for axonal growth, we have previously identified
mutations in the Drosophila kette gene
(Hummel et al., 2000). Loss of
kette function primarily affects neurite growth and subsequently
causes glial migration defects leading to a characteristic commissure
phenotype in the embryonic CNS similar to the one observed in
waspmat/zyg mutants
(Zallen et al., 2002
) and
Drosophila Nck (dock) mutants
(Desai et al., 1999
;
Garrity et al., 1996
).
Furthermore, kette genetically interacts with mutations in the Nck
homolog dock as well as with the small GTPase Rac1, supporting the
notion that Kette may provide a novel mechanism linking extracellular signals
to the actin cytoskeleton (Hummel et al.,
2000
). Recently, the Kette homolog Nap1 was found in a large 500
kDa protein complex comprising PIR121/Sra1, Abi, HSPC300 and Wave that keeps
Wave in an inactive state in vitro (Eden
et al., 2002
).
However, to date it is still unclear how Kette could regulate the
organization of the actin cytoskeleton in vivo. As Kette has been predicted to
be an integral membrane protein with six transmembrane domains
(Baumgartner et al., 1995), it
might serve as a receptor recruiting Nck/Dock or Rho-GTPases to the membrane.
We present biochemical and genetic evidence revealing that Kette is found
predominantly in the cytosol. Only a small amount of Kette is recruited to the
plasma membrane. In vivo as well as in tissue culture models, Kette protein
colocalizes with F-actin and co-sedimentation assays revealed a direct
interaction with F-actin. Within the membrane, Kette accumulates at the
insertion sites of large F-actin bundles, suggesting that targeted
localization of Kette may be required for its function. Loss of Kette protein
leads to a Scar/Wave-dependent accumulation of F-actin within the cell.
Ectopic expression of wild-type or different truncated Kette proteins in
tissue culture cells or during Drosophila development does not affect
F-actin formation or viability. However, expression of membrane-tethered Kette
efficiently induces ectopic bundles of F-actin in a process depending on Wasp
but not on Scar/Wave. These data indicate that Kette fulfils a novel role in
regulating F-actin organization by antagonizing Wave and activating
Wasp-dependent actin polymerization.
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MATERIALS AND METHODS |
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Two hybrid assay
To isolate proteins interacting with Kette protein, we inserted the
kette as well as the wasp ORF in frame with the GAL4 DNA
binding domain into the GBK-T7 vector (Clontech). With pGBK-Kette we screened
a matchmaker Drosophila embryo cDNA library (1x106
clones tested) using the GAL4-based Two-hybrid System 3 from Clontech.
pGAD-Abi contains the entire Abi-ORF.
Antibody production
The rabbit anti-Kette antibody (97/82), directed against a peptide derived
from the middle region of Kette (652-666), KHFDDIRKPGDESYR, was made by
Eurogentec (Belgium). In addition, polyclonal antibodies were generated
against parts of Kette fused with a His6-tag (Qiagen). pQE plasmids
express amino acid regions 1-374, 375-906 and 907-1126 of Kette, respectively.
Using these expression constructs, His6-Kette fusion proteins were
expressed in E. coli and purified with Ni-NTA resin (Qiagen) under
denaturing conditions. Rabbits were immunized with purified proteins by Davids
Biotechnologie (Germany).
Cell culture, transfection and immunofluorescence
Drosophila S2 cells were propagated in 1x Schneider's
Drosophila media (Gibco) supplemented with 10% FBS, 50 units/ml
penicillin and 50 µg/ml streptomycin in 75 cm3 T-flasks
(Sarstedt) at 25°C. For transfections, 5x105 cells were
plated on glass cover slips (pretreated with fibronectin) in 24-well plates,
cultured for 24 hours and transfected with Fugene 6 (Roche) at a transfection
reagent:DNA ratio of 3:1. Cells were incubated 24 hours after transfection,
fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, stained
with Drosophila anti-Kette antibody 97/82 (1:2000 dilution) followed
by Alexa-fluorophore-conjugated goat anti-rabbit IgG antibody (Molecular
Probes, 1:1000 dilution). For filamentous actin and nucleic acid staining,
cells were prepared as described above and incubated in 1-2 units
Alexa-fluorophore-conjugated phalloidin (Molecular Probes) and in 300 nM DAPI
(Molecular Probes), respectively. Tyrosine phosphorylated proteins were
detected using the PT-66 anti-phosphotyrosine antibody (Sigma). The samples
were mounted in a 25% (w/v) Mowiol (Sigma) solution containing DABCO (Sigma),
and visualized on a Leica LSM.
dsRNA production and RNAi treatment
570 bp of the kette ORF and 750 bp of the wasp and
scar/wave ORF were inserted into the vector pLITMUS-28i (BioLabs)
flanked by T7 promoters. After linearization, dsRNA was generated using a
HiScribe RNAi Transcription Kit (BioLabs). dsRNA products were resuspended in
water and annealed by incubation at 65°C for 30 minutes followed by slow
cooling to room temperature and stored at -20°C. For RNAi treatment,
5x105 Drosophila S2 cells in 0.5 ml serum-free
medium were plated on fibronectin coated cover slips in 24-well plates
(Sarstedt). dsRNA (5 µg) were added directly to the media followed by
vigorous agitation. The cells were incubated for 30 minutes at 25°C
followed by addition of 1 ml medium containing 15% FCS. dsRNA treated cells
were incubated for additional 2-3 days and analyzed by immunofluorescence and
western blot analysis.
Actin binding assay
Actin was purified from rabbit skeletal muscle following the methods of
Spudich and Watt (Spudich and Watt,
1971) and stored as G-actin at -172°C
(Spudich and Watt, 1971
).
Actin was polymerized in a buffer of 100 mM KCl, 2 mM MgCl2, 20 mM
HEPES, pH 7.4, 0.5 mM ß-mercaptoethanol and 2 mM NaN3. Prior
to use, MBP, MBPKette and actin were centrifuged at 200,000 g
for 30 minutes in a tabletop ultracentrifuge (Beckmann Instruments) to pellet
any aggregated protein. For binding assays, increasing amounts of full-length
or truncated Kette proteins were incubated with 3 µM F-actin for 30 minutes
at room temperature in polymerization buffer. The mixtures were centrifuged at
200,000 g for 30 minutes at 4°C. Equal amounts of
supernatant and pellet were separated by SDS-PAGE followed by Coomassie blue
staining.
Fractionation experiments
Fractionation of Drosophila embryonic and S2 cell extracts were
performed as described (Zhang and Hsieh,
2000). The pellets were solubilized in SDS sample buffer and
supernatants were precipitated in trichloroacetic acid, followed by
solubilization in SDS sample buffer. Western blot analysis was carried out as
described previously (Bogdan et al.,
2001
).
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RESULTS |
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|
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The recovery of Kette protein in the 1% NP-40 insoluble fraction containing
F-actin and other cytoskeletal proteins suggest an association between Kette
and F-actin. To test this directly, we determined a possible interaction of
Kette with F-actin in a co-sedimentation assay. Soluble, maltose-binding
protein (MBP) fusion proteins containing full-length Kette or its N-terminal
third (Kette1-374) were generated. Both fusion proteins, and
unaltered MBP as a control, were subjected to ultracentrifugation either alone
or in a mixture with F-actin. Only the fusion proteins pelleted with F-actin
(Fig. 2A,B), indicating that
actin binding is mediated by the N terminus of Kette, which, however, lacks
known F-actin binding sequence motifs. Furthermore, we identified three
different actin isoforms (Act42A, Act5C and Act57B) as putative interaction
partners of Kette in a yeast two-hybrid screen using full-length Kette as bait
(Fig. 2C). Based on sequence
analyses of these functionally redundant actin isoforms
(Wagner et al., 2002), Kette
is able to bind to the C-terminal 110 amino acids of G-actin.
|
|
Loss of kette function leads to an excess of F-actin in the
cytosol
The above data showed that Kette can be detected at the membrane and it is
able to bind to F-actin. First evidence for a regulatory function of Kette in
F-actin organization stems from our phenotypic analysis of kette
mutants (Hummel et al., 2000).
Loss of kette function affects the organization of the F-actin
cytoskeleton and is characterized by an excess of disorganized F-actin
bundles. To better analyze the function of Kette in F-actin dynamics we
reduced kette expression in Drosophila S2 cells by RNA
interference (see Materials and Methods). As judged by western blot and
immunofluorescence analyses, treatment of S2 cells with a 570 bp dsRNA
fragment generated from the kette-coding region resulted in a marked
reduction of Kette protein expression (compare
Fig. 4A with
Fig. 5A,B). No interference was
seen with sense and antisense RNAs (data not shown). With increasing time
after dsRNA treatment, cells with reduced levels of Kette show dramatic
alterations in cell morphology and concomitant changes of the F-actin
cytoskeleton. Within 3 days, cells appear to collapse and accumulate large
amounts of F-actin. Two days after RNAi treatment, intense granular F-actin
structures are observed which are similar to those in kette-null
mutants (Hummel et al., 2000
)
(Fig. 5B, arrowhead).
|
Further support for a Kette mediated repression of Scar/Wave activity stems
from genetic analyses. Wild-type embryos are characterized by clearly
separated anterior and posterior commissures, which is due to the migrating
midline glia (Fig. 6A). Embryos
that lack zygotic kette function display a characteristic CNS
phenotype and commissures appeared fused
(Fig. 6B). By contrast, loss of
zygotic Scar/Wave expression does not affect embryonic nervous system
development (Zallen et al.,
2002) (Fig. 6D). To
test whether the kette mutant phenotype might due to an upregulation
of scar/wave activity we removed one copy of the scar/wave
locus in a kette background, which indeed suppressed the homozygous
kette phenotype and distinct commissures are now recognizable
(Fig. 6C). Taken together,
these data suggest that in vivo Kette represses scar/wave
activity.
|
To test this possibility directly, we generated a membrane-tethered form of Kette by fusing a myristyolation signal derived from the Drosophila Src1 protein to the N terminus of Kette (KetteMyr). Overexpression of comparable amounts of KetteMyr in S2 cells also led to a dramatic rearrangement of the cytoskeleton and F-actin accumulates close to the membrane at sites of Kette expression (Fig. 4B, Fig. 5F). Thus, membrane tethered Kette protein is able to induce local actin polymerization.
In order to assess whether the membrane-tethered Kette protein performs similar to the wild-type protein we established UAS-ketteMyr transgenic flies and conducted genetic rescue experiments. Expression of KetteMyr in the rhomboid pattern in all CNS midline cells of mutant kette embryos suppresses the mutant phenotype in such that the segmental commissures can be clearly identified again which is not possible in mutant kette embryos (Fig. 6F). This indicates that the KetteMyr protein performs wild-type functions.
Increased expression of membrane-tethered Kette reorganizes the actin
cytoskeleton
Elevated expression of the activated, membrane-tethered, Kette protein is
able to induce a dominant phenotype in a dose-dependent manner. Flies carrying
a scabrous-GAL4 driver (Klaes et
al., 1994) and three copies of the UAS-KetteMyr
transgene are characterized by pronounced alterations in bristle morphology
(Fig. 7). Whereas in wild-type
flies macrochaete and microchaete form as thin and relatively straight
cuticular structures, expression of KetteMyr leads to thicker and
often forked bristles (Fig.
7A,B,E,G). In addition to the bristle phenotypes we observed
defects in the epidermal hairs. In the wild-type, each epithelial cell will
develop one fine hair, whereas KetteMyr expression results in
multiple hairs emerging from one cell (Fig.
7B,C, arrows). When we expressed only two copies of the
KetteMyr transgene, bristle development and epidermal hair
formation was affected more moderately
(Fig. 7C,F).
|
Membrane recruitment of Kette induces F-actin formation via Wasp
Treatment of S2 cells expressing the membrane-bound KetteMyr
protein with wave dsRNA did not suppress the KetteMyr
induced phenotype, suggesting the Kette may induce F-actin formation
independent of Wave (data not shown). Another important regulator of F-actin
formation is Wasp, which has a single Drosophila homolog. Removal of
one copy of the wasp gene significantly restores the bristle
phenotype evoked by overexpression of KetteMyr
(Fig. 7D). If Kette can act via
activating Wasp, similar phenotypes might be expected following disruption of
either gene. This is indeed the case, and loss of zygotic and maternal Wasp
function results in a kette-like embryonic CNS phenotype
(Tal et al., 2002;
Zallen et al., 2002
). To test
whether a reduction in the gene dose of wasp may also affect the
mutant kette phenotype, we generated a ketteC3-20
wasp3 double mutant. However, the kette wasp
phenotype appeared identical to the kette mutant phenotype
(Fig. 6E), which is in
agreement with the notion that Kette and Wasp act positively in the same
pathway.
In summary, Kette appears to repress Wave and, after membrane association, is able to activate Wasp. In the cytosol Kette is found in a complex with Wave but to date no interaction between Kette and Wasp has been reported. To elucidate further how Kette may regulate Wasp activity, we determined possible protein-protein interactions between Kette and Wasp in a yeast two hybrid assay (Fig. 8). Although no direct interaction was found between Kette and Wasp, it could be demonstrated between Kette and Abi as well as between Abi and Wasp, which thus is able to link Kette and Wasp.
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DISCUSSION |
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Regulation of Scar/Wave activity
Recent work from the Kirschner laboratory showed that a large 500 kDa
complex comprising Nap1/Kette, PIR121/Sra1, Abi, HSPC300 and Wave silences the
otherwise constitutive activity of Wave in stimulating actin polymerization
(Eden et al., 2002). The 500
kDa complex is stabilized by direct protein-protein interactions that have
been demonstrated between Nap1/Kette and Abi
(Tsuboi et al., 2002
;
Yamamoto et al., 2001
), and
Nap1/Kette and PIR121/Sra1 (also called Gex2)
(Soto et al., 2002
) (see
Fig. 9 for a model). The exact
binding partners of Wave are presently unknown.
|
In agreement with the work of Eden et al.
(Eden et al., 2002) disruption
of Kette function leads to an excess formation of cytoplasmic F-actin.
However, expression of even very high levels of wild-type Kette protein do not
evoke any mutant phenotype. Thus, in wild type cells the inactive Wave
complexes are already formed and Kette overexpression does not result in an
additional sequestering of Wave into the silencing complexes and/or an
incorporation of additional Kette into these complexes. In addition, as Kette
requires Sra1 function to bind to membrane-associated adapters such as Nck,
excess cytosolic Kette will not be able to reorganize subcellular Wave
distribution.
Further support for the notion that Kette mediates repression of Scar/Wave
activity stems from genetic analyses. Embryos lacking zygotic kette
function display a characteristic CNS phenotype, whereas loss of zygotic
Scar/Wave expression does not affect embryonic nervous system development
(Zallen et al., 2002). The
kette mutant phenotype, which is due to defects in neurite outgrowth
(Hummel et al., 2000
), could
significantly be suppressed by reducing the dose of Scar/Wave expression. This
demonstrates that in wild-type embryos, Kette acts as a negative regulator of
Scar/Wave. Similar results were obtained when we reduced Kette and or
Scar/Wave expression in Drosophila S2 cells. These experiments also
revealed that Scar/Wave is required for the normal subcellular distribution of
Kette, which may, however, be an indirect effect caused the disruption of the
F-actin actin cytoskeleton.
Kette function at the membrane
Kette protein localizes to the plasma membrane where it accumulates in
focal adhesion contacts. A prime candidate that may mediate recruitment of
Kette to the membrane is the SH2 SH3 adapter protein Nck which, besides
binding of the Sra1/Kette/Abi complex, also recruits numerous other proteins
to focal contact sites (Li et al.,
2001). Among these is Wasp, which binds to the third SH3 domain of
Nck (Quilliam et al., 1996
;
Rivero-Lezcano et al., 1995
)
(Fig. 9).
The genetic interaction of kette and dock which encodes
the Drosophila Nck homolog has recently been shown
(Hummel et al., 2000). We have
demonstrated that membrane recruitment of Kette is sufficient to activate
actin polymerization in the cell cortex mediated by Wasp. How is this brought
about? One explanation might be that recruitment of Kette to the membrane
disintegrates the inhibitory Wave complex - independent of the Nck/Sra1
association. This would then lead to an excess of Wave activity and
subsequently to an excess of actin polymerization. However, the genetic data
clearly show that membrane bound Kette functions independent of Scar/Wave but
depends on Wasp.
Genetic interaction between kette and wasp
Wasp usually adopts an auto-inhibited conformation and is activated after
Cdc42, Nck binding or phosphorylation (Kim
et al., 2000; Miki et al.,
1998a
). A structure-function analysis of the Drosophila
Wasp demonstrated that the Cdc42-binding domain is not necessary for function,
suggesting that alternative pathways, such as phosphorylation can activate
Wasp (Tal et al., 2002
). Kette
might be a part of such an alternative pathway, as we could demonstrate a
genetic interaction between kette and wasp in the regulation
of actin dynamics. Regulation of Wasp by Kette is not mediated by direct
protein-protein interaction, but probably involves Abi that is able to link
Kette and Wasp. The Abl interactor (Abi) protein localizes to sites of actin
polymerization at the tips of lamellipodia and filopodia and has been
implicated in the cytoskeletal reorganization in response to growth factor
stimulation (Stradal et al.,
2001
). As a positive regulator of the non-receptor tyrosine-kinase
Abelson (Abl) Abi may bring Abl into position to phosphorylate and thus
activate Wasp. Abl is known to phosphorylate many proteins regulating focal
adhesion and F-actin dynamics and overexpression of activated Abl induces
F-Actin formation in Cdc42-independent manner
(Woodring et al., 2002
). Some
tyrosine kinases activate by phosphorylation of Wasp
(Cory et al., 2002
;
Scott et al., 2002
;
Suetsugu et al., 2002a
);
however, direct phosphorylation of Wasp by Abl remains to be demonstrated.
Further support of the model (Fig.
9) that in vivo Kette activates Wasp but suppresses Wave are the
phenotypic analyses of Drosophila kette, wasp and scar/wave
mutants. Mutations in kette have been isolated due to defects in
commissure formation in the embryonic CNS
(Hummel et al., 1999). If
Kette acts via activating Wasp, similar phenotypes are expected following
disruption of either gene. This is indeed the case and loss of zygotic and
maternal Wasp function results in a kette-like embryonic CNS
phenotype (Hummel et al.,
2000
; Zallen et al.,
2002
).
In agreement with the proposed function of Kette in regulating both, Wasp
and Wave, is its subcellular distribution. Whereas the majority of Kette is
present in the cytoplasm to keep Wave in its inactive state
(Eden et al., 2002) some is
present leading edge of lamellipodia-like structures. However, highest amounts
of Kette are present at the insertion points of large F-actin bundles where
N-Wasp is also present (Nakagawa et al.,
2001
). Kette might be recruited to these focal adhesion sites via
Sra1/Nck (Goicoechea et al.,
2002
) and via Wasp could enhance the formation of F-actin
bundles.
How Wasp activity results in straight F-actin bundles, whereas Wave
stimulates the formation of a meshed F-actin network is presently unclear
(Takenawa and Miki, 2001). In
the cytosol, Kette may act as a scaffold protein that keeps Wave close to
F-actin and recruits additional factors to F-actin such as Profilin, which not
only binds to Kette but also enhances actin nucleation
(Tsuboi et al., 2002
;
Witke et al., 1998
;
Yang et al., 2000
). Thus,
Kette could promote the formation of a meshed F-actin network characteristic
for lamellipodia. At the membrane other proteins may interact with Kette and
in this respect it is interesting to note that the F-actin crosslinking
protein Filamin, which plays an important role in filopodia formation, also
binds to Kette (S.B., unpublished). This suggests that Kette, in addition to
regulating Wasp and Wave, may also contribute to the decision whether
filopodia or lamellipodia are formed.
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ACKNOWLEDGMENTS |
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