Article Scrib Controls Cdc42 Localization and Activity to Promote

Current Biology 16, 2395–2405, December 19, 2006 ª2006 Elsevier Ltd All rights reserved
DOI 10.1016/j.cub.2006.10.026
Article
Scrib Controls Cdc42 Localization and
Activity to Promote Cell Polarization
during Astrocyte Migration
Naël Osmani,1,2 Nicolas Vitale,3 Jean-Paul Borg,4,5,6
and Sandrine Etienne-Manneville1,2,*
1
Cell Polarity and Migration Group
Institut Pasteur
25 rue du Dr Roux
75724 Paris cedex 15
France
2
Institut Curie
Unité mixte de recherche 144
Centre National de la Recherche Scientifique
26 rue d’Ulm
75248 Paris cedex 05
France
3
Institut des Neurosciences Cellulaires et Intégratives
Unite mixte de recherche 7168/LC2
Centre National de la Recherche Scientifique
Université Louis Pasteur
5 rue Blaise Pascal
67084 Strasbourg cedex
France
4
Inserm, U599
Centre de Recherche en Cancérologie de Marseille
Pharmacologie Moléculaire
F-13009 Marseille
France
5
Institut Paoli-Calmettes
Marseille F-13009
France
6
Université Méditerranée
F-13007 Marseille
France
Summary
Background: Mammalian Scribble (Scrib) plays a conserved role in polarization of epithelial and neuronal
cells. Polarization is essential for migration of a variety
of cell types; however, the function of Scrib in this context remains unclear. Scrib has been shown to interact
with bPIX, a guanine nucleotide exchange factor for
the small GTPases Rac and Cdc42. Cdc42 controls
cell polarity from yeast to mammals during asymmetric
cell division and epithelial cell polarization, as well as
during cell migration. Cdc42 is, in particular, required
for polarization and orientation of astrocytes in a
scratch-induced polarized migration assay. Using this
assay, we characterized Scrib function during polarized
cell migration.
Results: Depletion of Scrib by siRNA or expression of
dominant-negative constructs inhibits astrocyte polarization. Like Cdc42, Scrib controls protrusion formation,
cytoskeleton polarization, and centrosome and Golgi reorientation. Scrib interacts and colocalizes with bPIX at
the front edge of polarizing astrocytes. Perturbation of
*Correspondence: [email protected]
Scrib localization or of Scrib-bPIX interaction inhibits
bPIX polarized recruitment. We further show that bPIX
is required for astrocyte polarization and that both the
Scrib-binding motif and the GEF activity of bPIX are
essential for its function. Scrib and bPIX control Cdc42
activation and localization during astrocyte polarization.
Thereby, Scrib regulates Cdc42-dependent APC and
Dlg1 recruitment to the leading edge to promote cell
orientation.
Conclusion: We conclude that Scrib plays a key role in
the establishment of cell polarity during migration. By
interacting with bPIX, Scrib controls localization and
activation of the small GTPase Cdc42 and regulates
Cdc42-dependent polarization pathways.
Introduction
Cell polarity is an essential feature of all eukaryotic cells,
both during development and in the adult organism. A
polarized cellular organization is critical for cell division,
differentiation, and morphogenesis, as well as for a variety of cell functions. Genetic analyses have revealed
that several proteins that control cell polarity are conserved in many different contexts. The small Rho
GTPase Cdc42, for instance, is involved in the control
of polarity throughout evolution from yeast to mammals
and regulates asymmetric cell division, epithelial cell differentiation, and directed cell migration [1]. Cdc42 function in cell polarity is, at least in part, mediated by the
regulation of the Par6-Par3-atypicalPKC (aPKC) polarity
complex. The Par proteins were first identified for their
role during the C. elegans zygote first asymmetric division [2]. The Par6/Par3/aPKC complex was later shown
to regulate polarization of epithelial and neuronal cells
both in Drosophila and in mammals [3]. Other evolutionarily conserved protein complexes, including the
Crumbs and Scribble complexes, take an essential
part in the polarization process [4, 5].
In Drosophila, Scribble controls apico-basal polarity
in epithelial cells, synaptic function, and neuroblast
asymmetric divisions [6, 7]. scrib (scribble) has been
shown to interact genetically with dlg (discs large) and
lgl (lethal giant larvae) in Drosophila melanogaster [8].
Although there is no evidence for a physical interaction
between Scrib, Dlg, and Lgl, the three proteins localize
at the lateral domain of epithelial cells and form the socalled Scrib complex. The Scrib complex contributes
to the formation of the basolateral surface by repressing
both the Par and Crumbs complexes and restraining the
extension of the apical domain. Scrib encodes a multidomain scaffold protein belonging to the LAP (LRR and
PDZ) family, which includes Erbin and Densin-180. Scrib
contains 16 leucine-rich repeats (LRR domain), four PDZ
(PSD-95, Dlg, ZO-1) domains, and a carboxy-terminal
region. The functions of Scrib in epithelial-cell polarization are mediated in part by the LRR domain, which
tethers Scrib to the plasma membrane [9]. On the other
hand, Scrib PDZ domains have been shown to interact
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with ZO2, Zyxin, APC [10–12], and more recently with
bPIX/Cool-1, a guanine exchange factor (GEF) for Rac
and Cdc42 [13]. However, the role of these interactions
in Scrib function is not known yet.
In addition to their role in cell polarity, scrib, dlg, and
lgl also behave as tumor suppressors and cause massive proliferation in Drosophila imaginal discs when
they are mutated (for a review, see [5]). They cooperate
with oncogenic Ras in the transformation of Drosophila
eye-disc cells and favor metastasis in activated Ras
cells [14]. Mammalian Scrib, Lgl, and Dlg share conserved sequences and functions with their Drosophila
homologs. Scrib, Lgl, and Dlg localize to the baso-lateral
domain of epithelial cells [9, 15, 16]. Lgl and Scrib have
been shown to regulate E-cadherin-mediated epithelial
cell-cell junctions and contribute to apico-basal polarity
[17, 18]. Mammalian Dlg and Scrib may also control cell
proliferation [19, 20]. Both proteins are downregulated
during malignant progression, and their expression pattern is correlated with loss of cell polarity and tissue architecture in the colon [21]. Additionally, loss of Lgl is
correlated with the development of metastases [22].
These observations point to the participation of Scrib,
Lgl, and Dlg in a signaling pathway controlling cell polarity and migratory behavior and led us to further investigate the role of these proteins during polarization of
migrating cells.
A polarized migrating cell is characterized by a protruding front and a retracting rear. During oriented cell
migration, the cell polarity axis is oriented in a direction
defined by external factors such as a chemoattractant
concentration gradient or a physical stimulus. In socalled wound-healing assays, scratching the cell monolayer induces cell polarization and migration in a direction perpendicular to the wound. In the case of primary
astrocytes, polarization corresponds to an elongation
of the cell, which forms a protrusion in the direction of
migration. The orientation of this polarity axis can be detected by (i) the direction of cell protrusion and cell migration, (ii) the orientation of the microtubule and actin
networks, and (iii) the position of the centrosome and
the Golgi apparatus relative to that of the nucleus. Using
this assay, we previously demonstrated that, after
wounding, the Rho GTPase Cdc42 was activated and recruited to the leading edge of the cells [23]. Localized
Cdc42 activity results in a spatially confined signaling,
which controls cell polarization and cell orientation. Active Cdc42 recruits to the leading edge the polarity complex formed by Par6 and the atypical PKC PKCz and
leads to the clustering of Adenomatous Polyposis Coli
(APC) at microtubule plus ends [24]. The Cdc42-dependent Par6-aPKC pathway also controls the polarized recruitment of Dlg to the leading edge. Dlg, together with
APC, controls microtubule-network polarization and
centrosome positioning [25]. The mechanisms controlling Cdc42 recruitment and activation remain unclear.
In this article, we investigate the function of Scrib
during polarization of migrating cells by using the astrocyte wound-healing assay and show that Scrib is
essential for cell polarization and cell orientation. We
find that Scrib controls the recruitment of bPIX to the
leading edge of migrating cells and consequently regulates Cdc42 localization and activity to promote cell
polarization.
Figure 1. Scrib Plays a Critical Role in Astrocyte Polarization
Primary astrocytes were nucleofected with control siRNA (CTL) or
siRNAs directed against two different Scrib sequences (Scrib-1,
Scrib-2) and incubated for 72 hr.
(A) Sixteen hours after wounding of the cell monolayer, cells were
fixed and stained with phalloidin (upper panels) and anti-tubulin
antibodies (lower panels).
(B) Eight hours after wounding, cells were fixed and stained with
anti-pericentrin (centrosome, red), anti-GM130 (Golgi apparatus,
yellow), and Hoechst (nucleus, blue). White dotted lines indicate
the scratch orientation. The scale bar represents 10 mm.
(C) Percentage of wound-edge cells having their centrosome in the
quadrant that is in front of the nucleus and facing the wound (light
blue on the diagram). Random orientation of the centrosome with respect to the wound edge corresponds to a value of 25%. Results
shown are means 6 SEM of 3–5 independent experiments, for a
total of at least 500 cells being scored.
Results
Scrib Controls Astrocyte Polarization
and Orientation
We used two siRNAs directed against distinct Scrib sequences to reduce Scrib expression in primary astrocytes (Figure S1A). A scratch in a control astrocyte
monolayer induces the formation of long protrusions
filled with an elongated microtubule network and actin
fibers aligned along the protrusion axis (Figure 1A,
left panel), as previously reported [23]. Depletion of
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Figure 2. Role of Scrib Domains in Astrocyte
Polarization
After scratching, cells of the first row were
microinjected with the indicated DNA constructs (pEGFP as a control).
(A) Scrib constructs used in this study.
(B) Sixteen hours after wounding of the cell
monolayer, cells were fixed. Expressing cells
were scored as protruding when they formed
protrusions at least three times longer than
wide. Representative images are shown in
the right panels.
(C) Cells were fixed and stained with antipericentrin (centrosome, red) and Hoechst
(nucleus, blue). Centrosome reorientation
was assessed in expressing cells of the first
row. Microinjected cells are marked with
a star (right panels). Expression of the construct is shown in the insert. Results shown
are means 6 SEM of 3–5 independent experiments, with a total of at least 200 cells being
scored. White dotted lines show scratch positions. The scale bar represents 10 mm.
endogenous Scrib inhibits protrusion formation, and
Scrib-depleted cells show a short and disorganized microtubule network and nonpolarized actin structures
(Figure 1A, right panels). Furthermore, depletion of Scrib
significantly perturbs scratch-induced centrosome and
Golgi reorientation (Figures 1B and 1C; also Figure S1C).
These results indicate that Scrib is involved in both
polarization and orientation of migrating astrocytes.
Scrib contains an LRR domain, four PDZ domains,
and noncharacterized central and carboxy-terminal domains. The PDZ domains have been shown to mediate
Scrib interaction with several proteins, including the
Cdc42 and Rac guanine exchange factor bPIX [13]. Various Scrib constructs were microinjected in astrocytes
of the wound edge (Figure 2A). Overexpression of Scrib
WT, which localizes to multiple sites on the plasma
membrane and in the cytoplasm (Figure 2B, right panel),
evokes multiple, randomly oriented protrusions associated with a random centrosome orientation (Figures
2B and 2C). Scrib-induced multiple protrusions are inhibited by coexpression of a dominant-negative Cdc42
(+ N17 Cdc42). Similar results are obtained after expression of Scrib with a point mutation (Scrib PL) that disrupts the LRR structure and delocalizes Scrib from the
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Figure 3. Scrib Controls bPIX Recruitment to
the Leading Edge of Migrating Astrocytes
(A and B) Cells were fixed at the indicated
time after wounding. Cells were stained with
anti-Scrib (A) or anti-bPIX (B) antibodies.
The percentage of front-row cells showing
Scrib or bPIX recruitment to the leadingedge plasma membrane was scored. Results
shown are means 6 SEM of 3–5 independent
experiments, with a total of at least 500 cells
being scored. Representative images are
shown on the right. White dotted lines show
scratch positions. The scale bar represents
10 mm.
(C) Cells were fixed 4 hr after wounding and
stained with Scrib and bPIX antibodies. The
lower panel shows the merged image (Scrib
in red, bPIX in green).The scale bar represents 10 mm.
(D) Cells were lysed at the indicated time after
wounding. Immunoprecipitations with antiScrib or irrelevant (CTL) antibodies were performed and analyzed by western blotting with
Scrib (lower panels) or bPIX antibodies (upper
panels). Blots shown are representative of
three independent experiments.
(E and F) Cells were nucleofected with the
indicated siRNAs (E) or microinjected with
the indicated constructs (F). Cells were
scratched, fixed 4 hr later, and stained with
anti-bPIX. The percentage of cells in the front
row showing bPIX recruitment at the leadingedge plasma membrane was scored. Representative images are shown on the right. Microinjected cells are marked with a star (right
panels). Expression of the construct is shown
in the insert. White dotted lines show scratch
positions. The scale bar represents 10 mm.
(G) Four hours after scratching, cells were
fixed and stained with Scrib antibodies. The
percentage of front-rpw cells showing Scrib
recruitment to the leading-edge plasma
membrane was scored. Results shown are
means 6 SEM of 3–5 independent experiments, with a total of 200–500 cells being
scored.
plasma membrane without affecting its interaction with
bPIX [9]. Expression of truncated Scrib forms missing
the four PDZ domains (Scrib DPDZ, Scrib PL DPDZ) or
of constructs only encoding the four PDZ domains
(PDZ) or the carboxy-terminal domain (Cter) strongly inhibits both cell protrusion and centrosome orientation.
This phenotype is similar to that observed after expression of a dominant-negative form of Cdc42 (N17-Cdc42)
but differs from that induced by a dominant-negative
form of Rac (N17-Rac) [23]; the latter inhibits protrusion
formation but not centrosome orientation (Figures 2B
and 2C). In contrast, expression of a construct lacking
both the PDZ and the carboxy-terminal domain (Scrib
PL Nter) has no effect.
Taken together, these results suggest that mislocalization of Scrib perturbs cell polarization and that PDZ
and carboxy-terminal domains of Scrib are required for
Scrib functions. They also suggest that Scrib may act
in the same signaling pathway as Cdc42 to control
astrocyte polarization and orientation. This led us to
investigate the localization of Scrib and its interaction
with bPIX during astrocyte polarization.
Scrib Is Recruited with bPIX to the Leading Edge
of Migrating Cells
In confluent nonmigrating astrocytes, Scrib localizes at
cell-cell contacts and in the cytoplasm (data not shown).
Immediately after scratching the monolayer, Scrib cannot be detected at the wound edge. However, as soon
as 30 min after scratching, Scrib is recruited to the leading edge of front row of cells (Figure 3A). bPIX is cytoplasmic in confluent cells and between 30 min and 4 hr
after wounding progressively appears at the cell leading
edge (Figure 3B), where it colocalizes with Scrib (Figure 3C). Four hours after scratching, bPIX also localizes
in focal adhesion-like structures, as previously reported
[26]. In contrast, Scrib is not visible in these structures
(Figure 3C). Immunoprecipitation of Scrib showed that
bPIX constitutively interacts with Scrib in astrocytes
(Figure 3D). This interaction increases by a factor
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4 6 0.7 (mean 6 SEM of three independent experiments)
between 1 and 4 hr after wounding. Because Scrib and
bPIX are recruited together to the leading edge of migrating cells, we investigated the role of Scrib in bPIX localization. Depletion of Scrib, or expression of Scrib
PDZ domains, which perturbs the interaction between
Scrib and bPIX, strongly inhibits bPIX recruitment to
the leading edge (Figures 3E and 3F). Overexpression
of Scrib-WT, Scrib PL, or Scrib DPDZ, which cannot
bind bPIX, also perturbs bPIX recruitment to the leading
edge (Figure 3F). bPIX depletion does not affect Scrib recruitment (Figure 3G). These observations suggest that
Scrib is involved in a multimolecular complex required
for bPIX localization through various interaction domains. Taken together, these results show that, after
the scratching of the cell monolayer, Scrib is recruited
to the leading edge and controls the localization of its
interacting partner bPIX.
bPIX Is Required for Astrocyte Polarization
and Orientation
Two distinct siRNAs were used to deplete bPIX expression. Although one of the siRNAs (siRNA bPIX-1) almost
totally inhibits bPIX expression, the other (siRNA bPIX-2)
reduces bPIX expression to a lower extent (Figure S1B).
In both cases, bPIX depletion inhibits protrusion formation, elongation of the microtubule network, and polarization of the actin cytoskeleton (Figure 4A). bPIX is also
required for centrosome and Golgi apparatus reorientation after wounding (Figure 4B), the level of inhibition
correlating with the protein-expression level (Figure 4C;
also Figure S1C). These observations show an essential
role of bPIX during scratch-induced astrocyte polarization. We used different constructs to further investigate
the role of bPIX activity in astrocyte polarization (Figure 4D). Overexpression of bPIX (WT) has no effect on
astrocyte polarization (Figures 4E and 4F). In contrast,
expression of a truncated bPIX form lacking the three
carboxy-terminal amino acids involved in PDZ-mediated Scrib interaction (DTNL) [13] strongly inhibits protrusion formation and centrosome reorientation. A similar phenotype is obtained after expression of the last 15
amino acids including the carboxy-terminal PDZ binding
domain of bPIX (bPIX Cter) [13]. Astrocyte polarization
was not modified by expression of a control protein
(bPIX Cter DTNL) that encompasses the last 15 residues
of bPIX without the PDZ binding site and that has no affinity for Scrib. Finally, expression of an inactive form of
bPIX lacking the Dbl (diffuse B-cell lymphoma) homology domain (DDH) inhibits both protrusion formation
and centrosome orientation in migrating astrocytes to
a similar extent as dominant-negative Cdc42 (Figures
4E and 4F). Taken together, these results show that
bPIX, like Scrib, is involved in the regulation of cell protrusion and cell orientation during scratch-induced
astrocyte polarization and point to an essential role of
bPIX guanine-exchange activity.
Scrib and bPIX Control Cdc42 Recruitment to
the Leading Edge, Cdc42 Activity, and the
Cdc42-Dependent Polarity Pathway
We have previously shown that scratching an astrocyte
monolayer promotes Cdc42 recruitment and activation
to the leading edge and that both localization and
activity of Cdc42 are essential for astrocyte polarization
[23]. Cdc42 recruitment to the leading edge was visualized with a GFP-tagged construct previously shown
to localize similarly to the endogenous protein [27]
(Figure 5A). Using confocal imaging and membrane
staining with a lipophilic dye, we have verified that increased GFP signal at the leading edge is not due to
membrane ruffling (Figure S2). Cdc42 recruitment is dramatically reduced in Scrib-depleted cells and in siRNA
bPIX-1 nucleofected cells. The less efficient siRNA
bPIX-2 only partially reduces Cdc42 recruitment
(Figure 5B). As shown by a PAK-CRIB pulldown assay,
depletion of Scrib and bPIX also strongly reduces
wound-induced Cdc42 activation, whereas control
siRNA has no effect (Figures 5C and 5D).
Finally, to confirm the inhibition of Cdc42 activity, we
analyzed the effects of Scrib and bPIX depletion on
Cdc42-dependent polarity pathways. We have previously reported that Cdc42 activity is required for adenomatous polyposis coli (APC) clustering at microtubule
plus ends and for Dlg1 recruitment to the leading
edge, both events being required for centrosome reorientation [25]. Depletion of Scrib or bPIX with siRNA
strongly impaired APC clustering (Figure 6A) and Dlg1
recruitment (Figure 6B). Interestingly, bPIX depletion inhibits Dlg1 localization without perturbing Scrib recruitment to the leading edge (Figures 6B and 3G). Taken together, these results demonstrate that Scrib and bPIX
are required for Cdc42 localization, for Cdc42 activation,
and for downstream events in the Cdc42-dependent
polarity pathway.
Discussion
Using dominant-negative constructs and specific siRNAs, we demonstrate here that Scrib is essential for polarization and correct orientation of migrating cells. scrib
has been characterized as a tumor-suppressor gene,
and its expression decreases with malignant progression [21]. As reported in epithelial cells [17], the orientation more than the speed of migration was dramatically
perturbed (Figure 1 and data not shown). Similar effects
were observed after depletion of APC and Dlg in astrocytes [25], suggesting that these tumor suppressors
contribute to cell migration essentially by controlling orientation rather than speed. We might speculate that decreased expression of these genes will result in random
and uncontrolled cell migration.
Scrib plays a dual role in cell polarity by regulating
Cdc42 activation and Cdc42 recruitment to the leading
edge. Scrib PDZ and carboxy-terminal domains play
a key role in Scrib polarity function. Although we do
not know the nature of the carboxy-terminal binding
partner, it is tempting to speculate that the role of the
PDZ domain is to bind the GEF bPIX. bPIX forms a functional link between two major polarity proteins, Scrib
and Cdc42. Accordingly, the TNL carboxy-terminal residues and the exchange activity of bPIX are essential
(Figure 4).
bPIX has been described as a potential GEF for Cdc42
and Rac [28]. In astrocytes, bPIX depletion inhibits
Cdc42 activation upon wounding (Figure 5C), and expression of bPIX DDH impairs centrosome and Golgi reorientation (Figure 5C and data not shown), which are
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Figure 4. bPIX Is Involved in Astrocyte Polarization
Primary astrocytes were nucleofected with the indicated siRNAs or microinjected with the indicated DNA constructs.
(A) Cytoskeleton polarization. Sixteen hours after wounding of the cell monolayer, cells were fixed and stained with tubulin antibodies (lower
panels) and phalloidin (upper panel).
(B) Eight hours after wounding, cells were fixed and stained with anti-pericentrin (centrosome in red), anti-GM130 (Golgi in yellow), and Hoechst
(nucleus in blue). White dotted lines indicate the scratch orientation. The scale bar represents 10 mm.
(C) Centrosome orientation. Results shown are the mean 6 SEM of 3–5 independent experiments, with a total of at least 500 cells being scored.
(D) bPIX constructs used in this study.
(E) Protrusion formation. Sixteen hours after wounding of the cell monolayer, cells were fixed. Expressing cells were scored as protruding when
they formed protrusions at least three times longer than wide (labeled cells in the right panels).
(F) Centrosome reorientation was assessed in expressing cells of the first row. Cells were fixed and stained with anti-pericentrin (centrosome,
red) and Hoechst (nucleus, blue). Microinjected cells are marked with a star (right panels). Expression of the construct is shown in the insert.
Results shown are means 6 SEM of 3-5 independent experiments, with a total of at least 200 cells being scored. Representative images are
shown in the right panels. White dotted lines show scratch positions. Tje scale bar represents 10 mm.
Cdc42- but not Rac-dependent events. Moreover, we
have previously shown that Cdc42 activation is dependent on Src-like tyrosine kinase activity and integrin engagement [23]. bPIX activity toward Cdc42 has been recently shown to be regulated by Src phosphorylation
[29]. It is therefore tempting to speculate that integrins
and Src kinases are involved in Scrib and bPIX activation
or recruitment to the leading edge, or both. Taken together, our results strongly support the idea that bPIX
is directly responsible for Cdc42 activation during astrocyte polarization. Our observations differ from the previous finding that bPIX is not involved in centrosome reorientation in migrating fibroblasts [30]. Brain-specific
isoforms of bPIX, bPIX-b(L) and b2PIX, might account
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Figure 5. Scrib and bPIX Regulate Scratch-Induced Cdc42 Recruitment and Activation
(A) pEGFP or pEGFP-Cdc42 constructs were microinjected in confluent nonmigrating astrocytes or in cells just after wounding. Eight hours later,
cells were fixed, and the percentage of front-row cells showing an increased GFP fluorescence at the leading-edge plasma membrane was
scored (left panel).
(B) Primary astrocytes were nucleofected with the indicated siRNAs. Seventy-two hours later, the cell monolayer was scratched, and woundedge cells were microinjected with pEGFP-Cdc42. Cells were fixed 8 hr later, and the percentage of front-row cells showing an increased
GFP fluorescence at the leading-edge plasma membrane was scored (left panel). Results shown are means 6 SEM of 3–5 independent experiments, with a total of at least 200 cells being scored. Representative images are shown on the right. White dotted lines show scratch positions.
The scale bar represents 10 mm.
(C) Astrocytes were nucleofected with control (CTL), Scrib (Scrib-1; Scrib-2), or bPIX (bPIX-1; bPIX-2) siRNAs. Cells were lysed just after (0) or
30 min (30’) after scratching. Lysates were incubated with agarose beads coupled to GST-PAK-CRIB (GST-PAK). Affinity-purified GTP-bound
Cdc42 (top panel) and total Cdc42 (lower panel) were analyzed by western blotting. Blots shown are representative of 3–5 experiments.
(D) Quantification of wound-induced Cdc42 activation in control and siRNA-treated cells. Results are expressed as Cdc42 activity 30 min after
wounding over basal Cdc42 activity. In each condition, basal activity was defined as the GTP-Cdc42 level present in just-wounded astrocytes.
Results shown are means 6 SEM of 3–5 independent experiments.
for these differences [31, 32]. However, we could not detect these proteins by western blotting. Alternatively, the
exact nature of integrins engaged during cell polarization, the level of Scrib expression, or both may account
for cell specificity.
bPIX, but not Scrib, can also be observed at focal
adhesions, where it may contribute to their turnover
(Figure 3C and [26]). Moreover, only a minor fraction of
bPIX was found associated with Scrib (data not shown),
suggesting that bPIX is associated with different complexes that play distinct roles during cell migration. For
instance, bPIX may be involved in Rac regulation during
astrocyte migration. Rac and PAK are both involved in
astrocyte-protrusion formation [23]. Moreover, they
both directly interact with bPIX via its SH3 domain [33,
34]. In agreement with the model recently proposed by
ten Klooster and collegues, bPIX may activate Cdc42
to induce PAK phosphorylation, as well as recruitment
and activation of Rac [34].
Activation and recruitment of Cdc42 appear to be two
separate events. Scrib overexpression, which inhibits
bPIX and Cdc42 recruitment to the leading edge, does
not seem to inhibit scratch-induced activation of
Cdc42. This is supported by the fact that Scrib overexpression does not inhibit protrusion formation but rather
induces random Cdc42-dependent cell protrusions
(Figure 2B). In these conditions, APC forms clusters at
microtubule plus ends at the tip of these multiple protrusions (data not shown), suggesting that Cdc42-dependent polarity pathway is activated but not properly localized [24]. Overexpression of Scrib WT may dissociate
a multi-molecular complex by titrating a front-edge
membrane-associated protein required for the localization of bPIX and Cdc42 but not for Cdc42 activation. The
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Figure 6. Scrib and bPIX Regulate APC and
Dlg1 Recruitment to the Leading Edge
Astrocytes were nucleofected with control
(CTL), Scrib (Scrib-1; Scrib-2), or bPIX (bPIX1; bPIX-2) siRNAs.
(A) Cells were fixed 8 hr after wounding and
stained with tubulin (green) and APC (red) antibodies (right panels). The percentage of
front-row cells showing APC clustering at
the leading edge was scored. Representative
images are shown in the right panels. The
scale bar represents 10 mm. Enlargements
of boxed regions showing microtubule plus
ends at the leading edge are shown on the
right. The scale bar represents 5 mm.
(B) Cells were fixed 6 hr after wounding and
stained with Dlg1 antibodies (right panels).
The percentage of front-row cells showing
Dlg1 enrichment at the leading edge was
scored. Results shown are means 6 SEM of
3–5 independent experiments, with a total of
at least 500 cells being scored. Representative images are shown in the right panels.
The scale bar represents 5 mm. White dotted
lines show scratch positions.
LRR domain of Scrib has been shown to be essential for
Scrib localization at the plasma membrane in epithelial
cells [9]. Consistent with this result, expression of Scrib
PL in migrating astrocytes also promotes the formation
of multiple disoriented Cdc42-dependent protrusions
(Figure 2B). Determining which molecule recruits Scrib
to the plasma membrane, probably via the LRR domain,
will be the goal of further investigations.
Our results suggest that Cdc42 activation first occurs
at various cellular locations and that active Cdc42 is
then recruited to the leading edge. The kinetics of Scrib,
bPIX, and Cdc42 recruitment to the leading edge do not
exactly parallel those of Cdc42 activation, which is initiated within 5 min after wounding and reaches a maximum at 1 hr (Figure 3 and [23]). GFP-Cdc42 localizes
not only at the plasma membrane but also on the Golgi
apparatus and on numerous vesicular structures
dispersed throughout the cytosol (Figure 5A and data
not shown). In the absence of leading-edge recruitment,
active Cdc42 may localize to any of these structures.
bPIX has been involved in the recycling of integrins,
Rac, and PAK via the ARF GTPase activating protein
GIT1 [26, 35, 36]; it may also be involved in Cdc42 trafficking and delivery to plasma-membrane sites.
Together with previous observations showing the role
of Scrib in epithelial and neuronal cell polarization as
well as in planar polarity [17, 37, 38], our findings
strengthen the idea that Scrib is a major and conserved
regulator of cell polarity. Although cell polarization requires conserved proteins, how these proteins interact
seems to depend on the cellular context [1]. As in epithelial and neuronal cells [6, 37], Scrib and Dlg1 both localize in the same region of the plasma membrane. However, in contrast with Drosophila, where scrib, dlg, and
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Proligo. SiRNAs directed against the GFP sequence or corresponding to a random nucleotide sequence were used as control siRNAs.
SiRNAs were introduced into cells by nucleofection according to the
vendor’s instructions (Amaxa GmbH). Via this technique, astrocyte
transfection efficiency reached approximately 80%.
Cell Culture and Scratch-Induced Migration
Primary rat astrocytes were prepared as described previously [23,
39]. For scratch-induced assays, cells were seeded on poly-L-ornithine-coated coverslips or 90 mm diameter dishes and were grown
in serum to confluence. The medium was changed 16 hr before
scratching. Individual wounds (approximately 300 mm wide) were
made with a microinjection needle. For biochemical analysis, multiple wounds were made with an eight-channel pipette (0.1–2 ml tips).
In all cases, wounds were closed in about 24 hr. Nuclear microinjections were performed in wound-edge cells in the first 30 min after
scratching. Expression vectors were used at 100–200 mg/ml. Protein
expression was detectable about 1 hr after microinjection.
Figure 7. Diagram Showing Scrib and bPIX Functions during Astrocyte Polarization
After wounding, Scrib is recruited to the leading edge. Scrib interacts with bPIX and controls bPIX localization. In turn, bPIX promotes
Cdc42 activation and localization at the leading edge. Cdc42 controls two distinct signaling pathways, promoting (1) Rac- and PAKdependent protrusion formation and (2) centrosome and Golgi reorientation through APC clustering and Dlg1 localization at the leading
edge.
lgl produce very similar phenotypes [6], Scrib is involved
in both generation of a polarity axis (elongated cell morphology) and orientation of this polarity axis toward the
wound, whereas Dlg1 only regulates orientation [25].
Moreover, Scrib depletion inhibits Dlg1 recruitment to
the leading edge, whereas Dlg1 depletion has no effect
on Scrib localization (Figure 6B, data not shown), suggesting that Scrib lies upstream of Dlg1 in the signaling
cascade (Figure 7).
Conclusions
We have shown here that Scrib is a critical player in the
polarization of migrating cells, confirming the conserved
role of Scrib in the control of cell polarity. Scrib, through
its association with the exchange factor bPIX, controls
Cdc42, another essential polarity protein. By regulating
Cdc42 activity, Scrib acts upstream of Dlg1 and is involved in the same molecular pathway controlling cell
orientation.
Experimental Procedures
Materials
Scrib antibody was from Santa Cruz Biotechnology, anti-bPIX from
Chemicon, anti-atubulin from Sigma-Aldrich, anti-Dlg1 from Transduction Laboratories, phalloidin-rhodamine from Molecular Probes,
and anti-pericentrin from BabCO. Anti-APC was a gift from I. Näthke
(University of Dundee, Dundee, Scotland, UK). Secondary antibodies were all obtained from Jackson ImmunoResearch Laboratories. The DiI lipophilic dye was from Molecular Probes.
DNA Constructs and siRNAs
Cdc42, Rac, Scrib, and bPIX constructs have been previously described [13, 23]. Two siRNA duplexes corresponding to rat Scrib
(GenBank/EMBL/DDBJ accession no. XM_343266) starting at nucleotides 949 (siRNA Scrib-1) and 2525 (siRNA Scrib-2) and to rat bPIX
(GenBank/EMBL/DDBJ accession no. AF044673) starting at nucleotides 247 (siRNA bPIX-1) and 741 (siRNA bPIX-2) were obtained from
Immunofluorescence and Image Quantification
Cells were stained as described previously [23]. Epifluorescence images of fixed cells mounted in mowiol were obtained on a microscope (model DM6000 Leica) equipped with a 403 NA 1.25 and
a 633 NA 1.4 objective lens and were recorded on a CCD camera
(CoolSNAP HQ; Roper Scientific) with MetaMorph software (Universal Imaging Corp.).
Cell-Polarization Assays
Protrusion Formation
Protrusion formation was determined 16 hr after wounding following
fixation and microtubule staining. Images of cells transfected with
siRNAs were taken randomly along the wound. For quantification
of protrusion formation, cells of the wound edge were microinjected
with the indicated constructs just after wounding. Expressing cells,
revealed with the appropriate staining, were scored as protruding if
they displayed large membrane protrusion(s) at least three times
longer than wide.
Centrosome Positioning
Centrosome reorientation was determined as described previously
[23, 39]. Centrosomes located in front of the nucleus, within the
quadrant facing the wound, were scored as correctly oriented. In
this assay, a score of 25% represents the absolute minimum corresponding to random centrosome positioning.
Protein Localization.
Cdc42 localization. Microinjection of GFP-tagged Cdc42 WT was
used for visualization of Cdc42 because of the lack of anti-Cdc42 antibodies that work in immunofluorescence. The percentage of cells
showing an increased GFP fluorescence at the leading edge was determined 8 hr after wounding. Similar results were obtained 4 hr after
wounding (data not shown).
Scrib, bPIX, or Dlg1 localization. Cells were fixed and stained with
the appropriate antibody. For the scoring of polarized protein recruitment, cells in which the protein was specifically enriched at
the leading edge were counted as positive.
APC localization. Cells were stained with APC and tubulin antibodies. The percentage of cells showing APC clusters at microtubule tips of the leading edge was measured.
In general, only very few migrating astrocytes show membrane
ruffling at the leading edge. However, in order to avoid artifact
from increased fluorescence due to membrane ruffling, these cells
were not included in the analysis.
Immunoprecipitation and Cdc42-Activity Assay
Experiments were performed as previously described [23]. Image
quantification was performed with the ImageJ software. Intensity
of the Cdc42-GTP band was normalized by the intensity of the corresponding amount of total Cdc42. Results were expressed as
activation over basal Cdc42 activity observed in just-wounded,
nonmigrating astrocytes.
Supplemental Data
Supplemental Data include additional Experimental Procedures and
two figures and are available online at http://www.current-biology.
com/cgi/content/full/16/24/2395/DC1/.
Current Biology
2404
Acknowledgments
This work was supported by the Centre National de la Recherche
Scientifique, the Pasteur Institute, the Agence Nationale pour la Recherche, the Fondation de France, the Institut pour la Recherche sur
la Moelle épinière et l’Encéphale and the Association pour la Recherche contre le Cancer. We would like to thank Prof. Daniel Louvard and Dr. Monique Arpin for their support and the PlateForme
d’Imagerie Dynamique imaging facilitiy for technical help. N.O. is
funded by the Ministère de la Recherche.
Received: July 28, 2006
Revised: September 26, 2006
Accepted: October 12, 2006
Published online: November 2, 2006
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