The Netherlands Cancer Institute, Antoni van Leeuwenhoekhuis, * Division of Cell Biology, Division of Cellular Biochemistry,
1066 CX Amsterdam, The Netherlands
The invasion-inducing T-lymphoma invasion
and metastasis 1 (Tiam1) protein functions as a guanine
nucleotide exchange factor (GEF) for the small GTPase
Rac1. Differentiation-dependent expression of Tiam1
in the developing brain suggests a role for this GEF and
its effector Rac1 in the control of neuronal morphology.
Here we show that overexpression of Tiam1 induces
cell spreading and affects neurite outgrowth in N1E-115
neuroblastoma cells. These effects are Rac-dependent
and strongly promoted by laminin. Overexpression of
Tiam1 recruits the 6
1 integrin, a laminin receptor, to
specific adhesive contacts at the cell periphery, which
are different from focal contacts. Cells overexpressing
Tiam1 no longer respond to lysophosphatidic acid-
induced neurite retraction and cell rounding, processes
mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively
active RhoA, which may indicate that regulation occurs
at the level of Rho or upstream. Conversely, neurite
formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate
that Rac- and Rho-mediated pathways oppose each
other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential
role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process.
MEMBERS of the Ras superfamily of small GTPases
control a wide variety of cellular responses,
which include proliferation, cell cycle regulation, and intracellular transport (Bogusky and McCormick, 1993 Activation of both Ras and Rho proteins in response to
growth factors involves the recruitment of guanine nucleotide exchange factors (GEFs),1 which activate these GTPases by catalyzing the exchange of GDP for GTP. A hallmark
of GEFs specific for Rho family members is the presence
of a Dbl homology domain, flanked by a pleckstrin homology
domain. More than 20 proteins with a Dbl homology/
pleckstrin homology domain combination have been identified, and they are considered to be the primary targets of
signaling pathways that control the activity of Rho-like
GTPases (Cerione and Zheng, 1996 Rho-like GTPases have recently been put forward as potential regulators of neuronal outgrowth (Mackay et al.,
1995 In earlier work, we have identified the invasion-inducing
T-lymphoma invasion and metastasis 1 (Tiam1) gene (Habets et al., 1994 Expression Constructs
The C1199 Tiam1 cDNA was cloned as a BssHII\\ScaI fragment into pRc/
CMV (Invitrogen Corp., Carlsbad, CA) as described earlier (Habets et al.,
1994 Transient Transfection Assays
N1E-115 cells were seeded in 6-cm dishes at a density of 5 x 105 cells in
DME, supplemented with 10% FCS and antibiotics. The next day, plasmids containing the various cDNAs were mixed in a 5:1 ratio with pCMVLacz (Stratagene, La Jolla, CA), and transfections were carried out using
the calcium phosphate precipitation method as described earlier (van
Leeuwen et al., 1995 Immunoprecipitation and Western Blotting
Transfected cells were lysed in a buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 5 mM EDTA supplemented with 20 µg leupeptin, 100 µg aprotinin, and 180 µg PMSF per milliliter. Extracts were clarified by centrifugation. An equal volume of Laemmli loading buffer containing 10% Attachment Assays
Adhesion of cells to laminin was performed in 24-well dishes that were
coated in triplicate with increasing concentrations of laminin-1 (Collaborative Biomedical Products, Bedford, MA). After overnight incubation at
4°C, unbound substrate was washed away with PBS and the wells were
blocked with PBS containing 1% BSA for 1 h at 37°C. Cells were harvested, washed in DME containing 1% BSA and seeded at a density of 105
cells per well. For antibody-blocking experiments, cells were pretreated in
suspension with DME/1%BSA in the presence of 10 µg/ml of affinity-
purified antibody GoH3, a rat monoclonal antibody which specifically recognizes the extracellular domain of the Immunofluorescence
Cells grown on (laminin-coated) coverslips were fixed with 3.7% formaldehyde in PBS for 20 min, washed, and permeabilized with 0.2% Triton
X-100 in PBS for 5 min. After a brief washing step, fixed cells were
blocked with PBS containing 2% BSA (PBS/BSA) for 1 h. All antibody
incubations were done in PBS/BSA and coverslips were washed three
times between each incubation step. Tiam1 was detected using a rabbit
polyclonal anti-Tiam1 antibody (Habets, 1994). Myc-tagged GTPases
were detected using antibody 9E10. FITC-labeled secondary antibodies
were from Zymed Labs (San Francisco, CA). In the last incubation step,
TRITC-labeled phallodin (Molecular Probes, Eugene, OR) was included
to allow detection of F-actin. Antibody NM6, directed against B-50/GAP-43,
was obtained from Dr. L Schrama (University Utrecht, The Netherlands).
Anti-neurofilament 200 (NE14) was from Sigma Chemical Co. (St. Louis,
MO). Phosphotyrosine-containing proteins were detected using antibody
PY20 (Transduction Laboratories, Lexington, KY). Antipaxillin antibodies (mAb3060) were obtained from Chemicon Int. Inc. (Temecula, CA).
Antivinculin antibody VII F9 was obtained from Dr. Marina Glukhova
(CNRS Institut Curie, Paris, France), and the Tiam1 Expression in Neural Cell Lines
We analyzed Tiam1 protein levels in a number of adherent
cell lines by immunoprecipitation in combination with
Western blotting (Fig. 1). Whereas no detectable expression was found in NIH 3T3, Rat-1, or COS-7 cells, relatively high levels of expression were observed in N1E-115
cells and PC12 cells, cell lines commonly used to study aspects of neuronal differentiation in vitro. Another neuroblastoma cell line, SK-N-MC, which cannot be induced to
produce neurites in vitro, but rather shows growth factor-
dependent scattering (van Puijenbroek et al., 1997), did
not express Tiam1. In view of the proposed role of Tiam1
in regulating neuronal differentiation in the brain (Ehler
et al., 1997
Effects of C1199 Tiam1, V12Rac1, or V12Cdc42 on
N1E-115 Morphology
We studied the consequences of overexpression of Tiam1,
constitutively active V12Rac1, or V12Cdc42 on N1E-115
morphology after transient transfection. Rather than using
a full-length Tiam1 cDNA, we used an NH2-terminally truncated variant, known as C1199 Tiam1. This variant, which
can efficiently activate Rac1 and induces morphological
transformation and membrane ruffling in NIH 3T3 cells, is
more stably expressed and appears to be more active than
the full-length protein (Michiels et al., 1995 In the presence of serum, cells expressing C1199 Tiam1
showed a characteristic spreading morphology, accompanied by a redistribution of F-actin to the cell periphery
(Fig. 2, A and B). Expression of V12Rac1 produced a phenotype very similar to that of Tiam1 (Fig. 2, C and D),
which is consistent with earlier observations that Tiam1
activates Rac1. The phenotype induced by Cdc42 resembled that of Rac1 and Tiam1 (Fig. 2, E and F), although
most cells expressing Cdc42 were less well spread. Transfection of constitutively active V12Cdc42 in combination
with dominant-negative N17Rac1 abolished this phenotype
(not shown), indicating that the V12Cdc42-induced phenotype is Rac-dependent. This is in agreement with experiments in Swiss 3T3 cells which show that injection of
V12Cdc42 activates Rac (Nobes et al., 1995). Apparently a
similar regulation of Rac by Cdc42 occurs in N1E-115 cells.
We next determined the effect of serum withdrawal on
these transfected populations. Whereas after 48 h in low
serum ~50% of the non transfected cells had formed neurites, cells expressing C1199 Tiam1, V12Rac1, or V12Cdc42
retained their flat lamellar appearance, similar to what is
shown in Fig. 2. We conclude that expression of C1199
Tiam1 or the respective GTPases does not allow neurite
formation when grown on plastic.
Tiam1 Promotes the Rapid Formation
of Neurite-like Outgrowths and Lamellar
Spreading on a Laminin-1 Substrate
Neurite formation in vitro, in response to differentiative
signals, is strongly promoted by extracellular matrix components such as laminin, fibronectin, or collagen (Hynes
and Lander, 1992 N1E-115 cells grown on fibronectin-coated dishes adhered slowly, and the C1199 Tiam1-expressing cells within
this population were mostly flat, a phenotype very similar
to that produced on plastic (see Figs. 2 and 5 B). In contrast, on laminin-coated dishes, cells adhered rapidly and
the C1199 Tiam1-expressing cells went through a number
of dramatic changes in cell morphology and behavior.
During the first hours after plating, cells expressing Tiam1
either spread along their entire surface (Fig. 3 A) or became polarized, retracting from the substrate at one end of
the cell and carrying a leading lamella at the other end,
reminiscent of migrating fibroblasts (Fig. 3 B). After some
time, polarized cells began to form neurite-like processes
that carried prominent lamellipodia (Fig. 3 C). After overnight growth on laminin, many of these processes had lost
their lamellar appearance (Fig. 3 D). The highest expressing cells produced extreme phenotypes overnight, either
as a result of uncontrolled branching (Fig. 3 E) or extreme
spreading (Fig. 3 F). Surprisingly, these changes took place
in the presence of serum, conditions which normally prevent neurite formation.
To establish the nature of these neurite-like extensions,
two different neuronal markers were used. Neurofilament,
a marker for mature neurites, could not be detected in
these overnight cultures (results not shown). However, the
"growth-associated" protein B-50/GAP-43, an early marker
for developing neurites and growth cones (Skene et al.,
1986
We found that expression C1199 Tiam1, V12Rac1, or
V12Cdc42 produced very similar effects, although in cells
expressing V12Cdc42, the morphological changes occurred more slowly and fewer cells developed neuronal
processes. Since these experiments were performed in the
presence of serum, no neuronal processes were induced in the vector control cells. Fig. 5 A shows representative
phase-contrast images of cells carrying neurite-like processes within each transfected population, 4 h after plating
and after overnight growth on laminin.
To obtain a more quantitative analysis of the effects of
C1199 Tiam1, Rac1, or Cdc42 on morphology, phenotypes
of individual lacZ-positive cells were scored as either
round, flat, or neurite-bearing after overnight growth on
laminin (Fig. 5 B). For comparison, C1199 Tiam1-transfected cells and vector-transfected control cells were
grown overnight on fibronectin. Whereas on laminin ~50%
of the Tiam1-transfected cells developed neurite-like extensions, on fibronectin most of these cells showed a
spreading morphology, and the number of cells that carried neurite-like outgrowths was not increased relative to
the control cells (Fig. 5 B). Correct expression of each construct was confirmed by Western blotting (Fig. 5 C).
In summary, expression of C1199 Tiam1 prevents neurite formation on fibronection or plastic, even under conditions that normally favor neuritogenesis, i.e., serum
withdrawal. In contrast, when grown on a laminin substrate, neurite-like processes are induced even in the presence of serum. We conclude that, rather than inducing neurite formation per se, overexpression of Tiam1 and
consequently activation of Rac1 modulate the ability of
these cells to respond to differentiative signals, which is to
a large extent determined by specific cell-substrate interactions.
Tiam1 Promotes Adhesion and Spreading on Laminin-1
via the Integrin a6 To further explore the role of laminin in Tiam1-induced
spreading and the formation of neurite-like outgrowths,
we compared adhesion of N1E-115 cells stably expressing
C1199 Tiam1 after retroviral transduction and G418 selection with empty vector control cells. The C1199 Tiam1-
expressing cells showed a significant increase in adhesion
relative to the control cells (Fig. 6). Adhesion was inhibited ~75% by preincubating cells with a rat monoclonal antibody (GoH3), directed against the
Integrins are known to be involved in regulating neurite
formation (for review see Hynes and Lander, 1992 Tiam1/Rac1 Overexpression Leads to a Distribution of
Since we did not find any changes in the expression levels
of
In Swiss 3T3 fibroblasts, activation of Rac leads to the
formation of similar integrin-containing complexes at the
leading edge of cells (Nobes and Hall, 1995 Relative Levels of Rac and Rho Activity Determine
Neurite Formation in N1E-115 Cells
Readdition of serum to serum-starved N1E-115 cells leads
to rapid neurite retraction and (transient) cell rounding
(Jalink and Moolenaar, 1992 To substantiate this observation, cells transiently expressing the C1199 Tiam1 protein were plated on laminin-1
and allowed to adhere in the absence of serum for 6 h. Under these conditions, not only did the Tiam1-expressing
cells produce neuronal processes, but also the nonexpressing cells in the population began to spread and to produce
short extensions (Fig. 8 A). However, 5 min after adding 1 µM LPA to these cultures, ~80% of the cells that did not
express C1199 Tiam1 showed complete cell rounding (Fig.
8 B). In contrast, cells expressing C1199 Tiam1, identified
by LacZ staining, did not show any cell rounding or neurite retraction (Fig. 8 B). These results demonstrate that
Tiam1-induced activation of Rac prevents Rho-dependent
neurite retraction and cell rounding. Also, in view of the
apparent loss of contractility accompanied by extreme
spreading that occurs in cells highly expressing C1199 Tiam1 (Fig. 3 F), we suspect that activation of Rac1 leads
to inhibition of the Rho pathway. Neurite retraction and
cell rounding was restored by coexpression of constitutively active V14RhoA (see below), suggesting that regulation of the Rho pathway by Rac occurs at the level of
Rho or upstream, rather than downstream.
To gain more insight into the contributions of Rac and
Rho activity on Tiam1-induced neurite formation, we examined the effects of constitutively active V14RhoA and
dominant-negative variants of Rac1, RhoA, and Cdc42 on
cells stably expressing Tiam1 after retroviral transduction.
These cells expressed moderate levels of the C1199 Tiam1
protein (Fig. 8 D) and showed a less extreme phenotype
than cells transiently expressing Tiam1 after transfection. When plated on a laminin substrate and in the presence of
serum, ~50% of these cells showed spreading, whereas
another 20% produced neuronal processes. The empty-vector control cells were mostly round and barely produced processes.
Transient transfection of a construct containing either
dominant-negative N17 Rac1 or constitutively active V14
RhoA reduced the amount of flat and neurite-bearing
cells to levels comparable with the control cells (Fig. 8 C).
Conversely, expression of dominant-negative N19RhoA
or p190, which has been shown to inactivate RhoA in fibroblasts (Ridley et al., 1993 Tiam1 Determines N1E-115 Morphology
by Activating Rac
Modifications of the actin cytoskeleton and cell-matrix interactions are essential determinants of neurite formation.
Rho family members are likely to play an important role
in these events (Mackay et al., 1995 The Effects of Tiam1 on Neurite Formation Are
Substrate Dependent
On a laminin substrate, N1E-115 cells expressing C1199
Tiam1, V12Rac1, or V12Cdc42 are rapidly induced to produce neurite-like processes, which at an early stage carry
prominent lamellipodia. In contrast, on fibronectin or
plastic, expression of C1199 Tiam1 prevents rather than
promotes neurite formation, even under conditions that
normally induce neurite formation. These differences illustrate the importance of reciprocal interactions between
the extracellular matrix and the cytoskeleton during neurite formation. Adhesive interactions between developing
neurites and matrix components such as laminin or collagen not only provide the traction that is required to allow
directional movement but also induce elaborate changes in
the organization of the cytoskeleton (Hynes and Lander, 1992 Although the signals responsible for activating Tiam1
and consequently Rac1 in neuronal cells are not known,
they may also involve receptor tyrosine kinases. Soluble
trophic factors such as NGF and FGF stimulate neurite
formation in rat PC12 cells and primary neurons (Chao,
1992 Involvement of the Integrin C1199 Tiam1 expression in N1E-115 neuroblastoma cells
promotes adhesion and spreading on laminin, and this adhesion is mediated by the integrin Opposing Roles for Rac and Rho Regulating
Neuronal Morphology
Activation of Rho, induced by the lipid LPA, triggers a
rapid contraction of cortical actin cytoskeleton to induce
cell rounding and neurite retraction in differentiated N1E-115 cells (Jalink et al., 1994 We find that one of these proteins, P190, promotes the
Tiam1-induced phenotype, presumably by inactivating
Rho. In Swiss 3T3 cells, this protein was shown to preferentially inactivate Rho, affecting both cell adhesion and
stress fiber formation (McGlade et al., 1993 Role of Tiam1 in Neuronal Development
Expression of Tiam1 in neurons in the brain is developmentally regulated and increases during neuronal migration and differentiation. In the adult, expression remains
high in regions of the brain undergoing synaptic remodeling (Ehler et al., 1997). A subgroup of this family, the Rho-like GTPases, affect the organization of the actin cytoskeleton. In
fibroblasts, Cdc42 is involved in the induction of filopodia,
Rac induces the formation of lamellipodia, and activation
of Rho leads to the formation of actin stress fibers (Ridley
and Hall, 1992
; Ridley et al., 1992
; Kozma et al., 1995
;
Nobes and Hall, 1995
). In addition, these GTPases promote the assembly of adhesive complexes connecting the
actin cytoskeleton with the extracellular matrix (Ridley et al., 1992
; Nobes and Hall, 1995
). Integrins, cytoskeletal proteins, and signaling molecules converge at these sites, which
are known as focal contacts when present at the end of actin stress fibers (Jockusch et al., 1995
). Studies using microinjection of activated Rho-like proteins in Swiss 3T3
cells suggest that there is a hierarchy in which these GTPases regulate each others activities. Expression of activated Cdc42 leads to activation of Rac, which subsequently can induce Rho activity (Nobes and Hall, 1995
). There are
also links between signaling of Ras and Rho family members. In fibroblasts, Ras-induced membrane ruffling is dependent on Rac activity (Ridley et al., 1992
) and focus formation induced by Ras involves both Rac and Rho mediated
signals (Prendergast et al., 1995
; Qiu et al., 1995a
,b). Little
is known however, about the mechanisms responsible for
this regulation.
; Collard, 1996
). Inactivation of Rho proteins is regulated by GTPase activating
proteins, (Rho-GAPs), which elevate the intrinsic GTPase activity of Rho proteins, returning them to an inactive
state. Several Rho GEFs and Rho GAPs show restricted
expression patterns and specificity towards particular Rho
family members (Collard, 1996
; Lamarche and Hall, 1994
),
and it remains a challenge to understand how the activity of
such proteins directs complex cellular behaviors such as cell
migration or differentiation during development and in the
adult.
; Tanaka and Sabry, 1995
). Thrombin and the lysophospholipid LPA induce cell rounding and neurite retraction
of differentiated mouse neuroblastoma cells (Jalink and
Moolenaar, 1992
; Jalink et al., 1993
). LPA-induced cell
rounding can be inhibited by the C3 endotoxin (Jalink et al., 1994
) or dominant-negative N19RhoA (Gebbink et al.,
1997
), establishing a key role for Rho in this process. In
addition, ectopic expression of constitutively active or
dominant-negative variants of Rac1 and Cdc42 affect neurite formation during Drosophila and mouse development
(Luo et al., 1994
, 1996
).
). More recently, we showed that the
Tiam1 protein functions as GEF for Rac both in vitro and
in vivo (Michiels et al., 1995
). Tiam1 is expressed at low
levels in most tissues but at markedly higher levels in brain
and testis (Habets et al., 1995
). Expression in the brain is
restricted to a subset of neurons, and the onset of expression during development correlates well with neuronal differentiation and/or migration, suggesting a role for Tiam1 in these processes (Ehler et al., 1997
). Therefore, we have
investigated the effects of Tiam1 on neuronal morphology
in mouse N1E-115 neuroblastoma cells. We show that
overexpression of Tiam1, by activating Rac1, affects
lamellar spreading and neurite formation, and that these effects are strongly promoted by laminin. Overexpression
of Tiam1 increases adhesion to laminin and directs the integrin
6
1, a laminin receptor, to specific contacts at the
cell periphery. We also find that Tiam1-promoted neurite
outgrowth is determined by a balance between Rac- and
Rho-mediated signals and that activation of Rac antagonizes the Rho pathway. We propose that interactions between Rac- and Rho-mediated signaling pathways coordinate neurite formation, and that GEFs and GAPs are key
regulators in this process.
Materials and Methods
). cDNA's for Rac, Rho, and Cdc42 contained an NH2-terminal myc
tag and were cloned into the eukaryotic expression vector pCDNA3 (Invitrogen Corp.). Rac and Rho cDNAs were originally made available by
Dr. A. Hall (MRC, London, UK) and cloned as EcoRI fragments into
pCDNA3 (Invitrogen Corp.). The pCDNA3 construct containing
N19RhoA was obtained from M. Gebbink and W.H. Moolenaar (The
Netherlands Cancer Institute, Amsterdam, The Netherlands). V12Cdc42
and N17Cdc42 were derived by PCR from a wild-type Cdc42 cDNA,
which was made available by Dr. R. Cerione (Cornell University, Ithaca,
NY). A p190 cDNA was obtained from Dr. M. Symons (Onyx Pharmaceuticals, Richmond, CA) and cloned into pcDNA3. Construction, packaging, and infection of recombinant retroviruses containing the C1199
Tiam1 cDNA fused to a IRES-neo sequence will be described in detail
elsewhere.
). The next day, transfected cells were detached from
the dish and replated into six-well dishes or onto glass coverslips that were
coated overnight with 20 µg/ml laminin-1. Cells were fixed in PBS containing 2% formaldehyde/0.2% glutaraldehyde and assayed for
-galactosidase activity. The morphology of single lacZ-positive cells was scored as either round, flat, or neurite bearing. Cells with at least one process
greater than one cell diameter were considered neurite bearing. An average of 250 cells were counted per well and the values presented are the
mean percentages (± SEM) of at least two independent transfections.
-mercaptoethanol was added. For immunoprecipitations,
clarified lysates were incubated with anti-Tiam1 antibody C16 (Santa
Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h. Immunocomplexes
were removed using a 10% protein A-sepharose slurry (Pharmacia Biotech, Inc., Piscataway, NJ) and washed three times before adding 25 µl of
Laemmli sample buffer. Samples were boiled, separated by SDS-PAGE,
and subjected to Western blot analysis as described earlier (van Leeuwen
et al., 1995
). For detection of Tiam1, an affinity-purified rabbit polyclonal antibody (Habets et al., 1994
) was used. Myc-tagged GTPases were detected using monoclonal antibody 9E10.
6 integrin (Sonnenberg et al.,
1988
). Cells were allowed to adhere for 15 min, after which unattached
cells were removed by washing with PBS. After fixation in PBS/3% formaldehyde (10 min), cells were stained for 30 min with a 2% crystal violet
solution in PBS. Excess stain was washed away with PBS and cells were
lysed in PBS containing 1% NP-40. Attachment was quantitated by measuring the absorbance of these lysates at 570 nm. Maximal adhesion was determined on poly-L-lysine-coated dishes and set at 100%. Values depict the average of two experiments. Each experiment consisted of four independent determinations.
6 integrin subunit was detected with antibody GoH3 (Sonnenberg et al., 1988
), which was also used
in the adhesion assays. To facilitate detection of these proteins, the signal
was enhanced by including a biotinylated secondary antibody (Amersham
Corp., Arlington Heights, IL), followed by a third incubation with streptavidin-FITC (Zymed Labs). Coverslips were mounted in vectamount (Vector Laboratories, Burlingame, CA) and viewed under a confocal microscope (model MRC600; Bio-Rad Labs, Hercules, CA).
Results
), the prominent expression of Tiam1 in neural
cell lines may relate to the ability of these cells to form
neurites. Mouse N1E-115 neuroblastoma cells can be
transfected with high efficiency (Kranenburg et al., 1995
) and are rapidly induced to spread and produce neurites after serum withdrawal. Therefore, we decided to use N1E-115
cells as an in vitro model for Tiam1 function in neuronal cells.
Fig. 1.
Tiam1 expression in various cell lines. Detection of the
Tiam1 protein by immunoprecipitation and Western blotting.
The Tiam1 protein was precipitated using antibody C16. Samples
were separated on a 7.5% polyacrylamide gel and analyzed by
Western blotting using a rabbit polyclonal antibody raised against
a COOH-terminal fragment of the Tiam1 protein (Habets et al.,
1994).
[View Larger Version of this Image (38K GIF file)]
; van Leeuwen et al., 1995
). Cells expressing recombinant proteins were
identified 48 h after transfection using immunofluorescence detection and confocal microscopy as described in Materials and Methods.
Fig. 2.
Morphological characteristics of N1E-115 cells expressing Tiam1, Rac1, or Cdc42. Transiently transfected cells were
grown on glass coverslips for two days, fixed, permeabilized, and
stained with anti-Tiam1 antibodies (A) or anti-myc antibody
9E10 to detect V12Rac1 (C) and V12Cdc42 (E). TRITC-labeled
phalloidin was used to detect F-actin (B, D, and F). Cells were
viewed by confocal microscopy. The untransfected control cells
are only visible in the panels stained for F-actin (B, D, and F).
Bar, 25 µm.
[View Larger Version of this Image (159K GIF file)]
). We therefore determined if and how
different substrates would affect the phenotype of N1E-115 cells expressing C1199 Tiam1, V12Rac1, or V12Cdc42.
Fig. 5.
C1199 Tiam1, V12Rac1, and V12Cdc42 promote neurite formation on a laminin-1 substrate. (A) Phase contrast photographs
of neurite bearing N1E-115 cells transiently transfected with either C1199 Tiam1, V12Rac1, V12Cdc42, or a control plasmid, and
cotransfected with plasmid pCMV-lacZ. 20 h after transfection, cells were seeded onto laminin-coated six-well plates, grown for 4 or 24 h,
and subsequently fixed and stained for -galactosidase activity to identify transfected cells in the population. (B) Quantification of morphological changes. The morphology of transfected (lacZ-positive) cells on either laminin (LN) or fibronectin (FN) was scored as round,
flat, or neurite bearing, as described in Materials and Methods. About 250 cells were counted per well. Numbers represent the mean
percentage of three independent experiments. Error bars indicate SEM (n = 3). (C) Expression of myc-tagged V12Rac1 or V12Cdc42
(left) or C1199 Tiam1 (right) in transiently transfected N1E-115 cells, as analyzed in B. Total cell lysates were separated on 7.5 or 6%
polyacrylamide gels and immunoblotted using anti-Myc or anti-Tiam1 antibodies. In the anti-Tiam1 blot (right), the signal present at
200 kD represents endogenous Tiam1, whereas the smaller 170-kD product is C1199 Tiam1. Bar, 25 µm.
[View Larger Versions of these Images (102 + 33 + 31K GIF file)]
Fig. 3.
Morphological transitions observed in Tiam1 cells grown on laminin. C1199 Tiam1-transfected cells were seeded onto laminin-coated coverslips in the presence of serum and allowed to adhere for various lengths of time before fixation and staining. Representative examples of each timepoint are given. (A) Spreading cell, 2 h after seeding. (B) Polarized cell 2 h after seeding, carrying a large
lamellipodium at the leading edge. (C) Cell developing neurite-like outgrowths, 4 h after seeding. Note the presence of lamellipodia on
both ends of the cell, moving in opposite directions. (D) Neurite-bearing cell after overnight growth on laminin. (E) Extreme branching
induced by high expression of C1199 Tiam1 after overnight growth on laminin. (F) Some cells show extreme spreading and an apparent
loss of contractility induced by high expression of C1199 Tiam1 after overnight growth on laminin. Tiam1 is shown in green, F-actin in
red. Note that D and F represent a 400x magnification, whereas the other images were magnified 600x. The red cells represent untransfected controls. Bars, 25 µm.
[View Larger Version of this Image (29K GIF file)]
), was highly enriched in these processes (Fig. 4). We
therefore conclude that these processes have at least neurite-like properties. The expressed C1199 Tiam1, identified by immunofluoresence detection, appeared to be
mainly cytosolic and was not particularly enriched at the
plasma membrane, neither in spreading cells nor in developing neuronal processes (Fig. 3). This contrasts with our
earlier studies in NIH 3T3 cells, where overexpressed
Tiam1 colocalized with cortical F-actin (Michiels et al.,
1997
). In spite of the seemingly cytosolic distribution of
C1199 Tiam1 in these cells, biochemical fractionation experiments in N1E-115 cells have demonstrated that part of
the endogenously expressed Tiam1 protein is present in a
Triton X-100 insoluble fraction, suggesting association
with the actin cytoskeleton (Stam et al., 1997
).
Fig. 4.
The neurite-specific marker, B-50/GAP-43, is present
in neurite-like extensions of C1199 Tiam1-expressing N1E-115
cells. (A) F-actin; (B) B-50/GAP-43. Note how expression of B-50/
GAP-43 is enriched in neuronal processes in comparison to the
cell showing a spreading morphology. Bar, 25 µm.
[View Larger Version of this Image (82K GIF file)]
1
6 integrin subunit
(Fig. 6), which is part of the laminin receptor
6
1 (Sonnenberg et al., 1988
). Although we cannot exclude the possibility that other laminin-binding integrins may be present
on N1E-115 cells, these results implicate
6
1 in adhesion
and spreading of N1E-115 cells to laminin.
Fig. 6.
Tiam1 overexpression results in increased adhesion to
laminin. N1E-115 cells stably expressing C1199 Tiam1 or a control cell line were plated on 24-well plates coated with increasing
concentrations of laminin. A small portion of the cells was pretreated in suspension with DME/1% BSA in the presence of 10 µg/ml of affinity-purified anti-6 antibody (GoH3), or mock
treated, and plated in wells coated with 20 µg/ml laminin. Cells
were allowed to attach for 15 min, washed, and adhesion was
quantitated by crystal violet staining and measuring absorbance
at 570 nm (for details see Materials and Methods). Maximal adhesion was determined on poly-L-lysine-coated dishes and set at
100%. Values represent the average of quadruplicate samples
from two representative experiments. Error bars indicate SEM (n = 2). The observed differences at laminin concentrations of 20 and
10 µg/ml were found statistically significant by students t test (P < 0.01).
[View Larger Version of this Image (27K GIF file)]
).
Therefore, the effects of Tiam1 on cell adhesion, spreading, and neurite formation might be explained by changes
in expression, activation, or distribution of integrins. Since
it was shown in PC12 cells that expression of the integrin
1
1, a laminin/collagen receptor, is upregulated in response to differentiative signals, (Zhang et al., 1993
), we
determined if expression of
6
1 was altered in the
Tiam1-expressing cells. Flow cytometry performed on
control cells and C1199 Tiam1-expressing cells, however,
did not reveal any differences in cell surface expression of
either the
6 or the
1 subunit (not shown). We conclude
that the effects of Tiam1 on adhesion and spreading on laminin are not due to increased expression, but may reflect activation and/or redistribution of the laminin receptor
6
1.
6
1 Integrin to Adhesive Contacts at the Leading Edge
6
1, we examined the distribution of this integrin in
cells expressing Tiam1. For these experiments, we used a
population of virally transduced N1E-115 cells that stably
expressed C1199 Tiam1. Cells were allowed to adhere on
laminin overnight, and the localization of the
6
1 integrin was analyzed by immunofluorescence detection using
an antibody against the
6 subunit. In the control cells, we
did not see any specific distribution of the
6
1 integrin (Fig. 7 A). In the C1199 Tiam1-expressing cells, however,
6
1 was found in small adhesive complexes along the distal end of advancing lamellae. These complexes were most
clearly seen in round spreading cells (Fig. 7 B), but also
present in lamellar endings of neurite-like outgrowths
(Fig. 7 F). A similar distribution was found using an antibody against
1 (not shown). Neuronal processes produced by control cells after serum withdrawal did not
carry prominent lamellipodia. As a consequence, a specific
distribution of
6
1 was not found in these cells (not
shown).
Fig. 7.
The integrin 6
1
but not focal adhesion components are enriched in adhesive complexes at the cell
periphery of cells expressing C1199 Tiam1. Confocal images of C1199 Tiam1-
expressing cells grown for
24 h on laminin-coated coverslips before fixation. F-actin was detected using TRITC-labeled phalloidin. (A) Distribution of the
6 integrin
subunit in control N1E-115
cells. B-F represent examples of N1E-115 cells expressing C1199 Tiam1. (B)
Presence of the
6 integrin
subunit in adhesive complexes at the cell border of
spreading cells overexpressing C1199 Tiam1. (C-E) Paxillin, phosphotyrosine, and
vinculin are only detected in
structures that resemble focal contacts. (F) Distribution
of the
6 integrin subunit in
neurite-bearing cells. For antibodies used, see Materials and Methods. Merge: F-actin
in red;
6, paxillin, phosphotyrosine, and vinculin in green.
Bars, 25 µm.
[View Larger Version of this Image (41K GIF file)]
). These structures contain a number of cytoskeletal proteins like vinculin and paxillin, components of focal contacts that form at
the end of stress fibers. Therefore, we looked at the distribution of paxillin, vinculin, and tyrosine-phosphorylated
proteins. Although these proteins were clearly present in
focal contacts (Fig. 7, C, E, and D) we did not find paxillin,
vinculin or phosphotyrosine-containing proteins in
6
1-containing complexes at the cell periphery. Conversely, we
did not find
6
1 in focal contacts (Fig. 7 B). Since the antibody that was used to detect
6 was raised against the
ligand binding domain of this integrin, it could be argued
that
6 integrins present in these focal contacts may have
escaped detection because of antigenic site blocking. However, in other cell types this antibody readily detects focal
adhesions (Hogervorst et al., 1993
). Therefore, our results
suggest that other integrins are present in these structures.
The fact that focal contacts were only seen after overnight
growth on laminin and could also be detected on fibronectin (not shown) suggests that the formation of these structures is promoted by extracellular matrix components
other than laminin. We propose that the Tiam1/Rac1-
induced adhesive complexes, as they occur at the cell periphery, are involved in lamellipodia formation and/or
may act to stabilize advancing lamellae in developing growth cones. We further conclude that in N1E-115 cells,
these
6
1-containing complexes are different from focal
contacts.
; Jalink et al., 1993
). Responsible for this phenomenon is the platelet-derived phospholipid LPA (Jalink et al., 1993
), which is present in an albumin-bound form in serum (Tigyi and Miledi, 1992
). LPA
signals through a G protein-coupled receptor to induce
contraction of the cortical actin cytoskeleton in a Rho-
dependent manner (Jalink et al., 1994
). The fact that C1199
Tiam1- or V12Rac1-expressing cells rapidly produced
neurite-like outgrowths, even in the presence of serum,
suggested that these cells were no longer responsive to LPA.
Fig. 8.
Effects of Rac and Rho activity on Tiam1-induced morphology. (A and B) N1E-115 cells expressing C1199 Tiam1 no longer
respond to LPA. Phase contrast micrographs of serum-deprived N1E-115 cells on a laminin substrate. Transfected cells were identified
after fixation by LacZ staining. (A) Untreated serum-deprived cells. (B) Serum-deprived cells exposed to 1 µM LPA for 5 min before fixation and lacZ staining. Note that C1199 Tiam1-expressing cells do not respond to LPA in contrast to control cells. (C) Quantification of morphological changes induced by Cdc42, Rac and Rho activity. NIE-115 cells stably expressing C1199 Tiam1 were transiently
transfected with myc-tagged constitutively active V14RhoA, dominant-negative N17Rac1, N17Cdc42, and N19RhoA as well as untagged p190. All constructs were cotransfected with plasmid pCMV-lacz. Transfected cells were grown overnight in laminin-coated six-well dishes, fixed, and stained for -galactosidase activity. Morphology of transfected cells was scored as described in Materials and
Methods. About 250 cells were scored per well. Numbers represent the mean percentages of two independent experiments. Error bars
indicate S.E.M (n = 2). (D) Expression levels of the transfected constructs in the cell populations as investigated in C. Myc-tagged GTPases (left), Tiam1 (middle), and P190 (right) in N1E-115 cells stably expressing C1199 Tiam1. Total cell lysates were separated on 7.5 or
6% polyacrylamide gels, transferred to nitrocellulose, and immunoblotted. In the anti-myc blot (left), arrows indicate the various transiently expressed GTPases. In the Tiam1 blot (middle), arrows indicate endogenous Tiam1 and C1199 Tiam1. In the p190 blot (right),
the signal present in the control lane represents endogenous p190. Bar, 25 µm.
[View Larger Versions of these Images (74 + 24 + 33K GIF file)]
), markedly increased the
number of flat or neurite-bearing cells. Transfection of
dominant-negative N17 Cdc42 did not affect the Tiam1
phenotype (Fig. 8 C), consistent with Tiam1 activating Rac
rather than Cdc42. Expression of C1199 Tiam1 and each
of these interfering constructs was confirmed by Western blot analysis (Fig. 8 D). Together, these results lead us to
conclude that the induction of these processes by Tiam1
overexpression, is dependent on Rac1- but not Cdc42 activity, and that the relative levels of Rac and Rho activity
determine neuronal morphology of N1E-115 cells. Furthermore, our results suggest the existence of a signaling
pathway in neuronal cells by which activation of Rac antagonizes Rho activity.
Discussion
; Tanaka and Sabry,
1995
). Activation of these small GTPases in response to
extracellular cues is regulated by GEFs. In this paper, we
describe how Tiam1 overexpression affects the morphology of N1E-115 neuroblastoma cells. Transient transfection of a Tiam1 cDNA leads to a sharp change in morphology,
characterized by extensive cell flattening and accompanied by a reorganization of F-actin at the cell periphery.
Expression of constitutively active V12Rac1, but also
V12Cdc42, produces a similar phenotype. The same hierarchical relation between Cdc42 and Rac was shown to
exist in Swiss 3T3 cells (Kozma et al., 1995
; Nobes et al., 1995). Although we find that activation of Cdc42 can produce a Rac phenotype in N1E-115 cells, the effects of Tiam1
can be reversed by coexpressing dominant-negative N17Rac1,
but not N17Cdc42. These results confirm that Tiam1 is involved in Rac signaling, which does not involve Cdc42.
). Moreover, signals downstream of growth factor receptors and integrins converge at several levels to activate
the same downstream effectors (Sastry and Horwitz,
1996
). For instance, the adaptor protein Grb-2 not only associates with activated receptor tyrosine kinases but also
with focal adhesion kinase FAK (Schlaepfer et al., 1994
), a
component of integrin-containing focal adhesion complexes.
Similarly, phosphoinositide-3-kinase is recruited to and activated by growth factor receptors (Cantley et al., 1991
), but also binds FAK (Chen and Guan, 1994
). Furthermore,
lipid second-messengers induced by phosphoinositide-3-kinase have been implicated in the activation of Rac by
PDGF and insulin (Wennstrom et al., 1994
; Hawkins et al.,
1995
), which may involve the specific activation and/or localization of guanine nucleotide exchange factors such as
Tiam1 (Michiels et al., 1997
).
). The phenotypic changes that occur in C1199
Tiam1-expressing N1E-115 cells are remarkably similar to
the early effects of NGF on the morphology of PC12 cells
(Altun-Gultekin and Wagner, 1996
). Since Rac was shown
to be involved in these responses and PC12 cells express
high levels of Tiam1, we are currently using these cells to
study the regulation of endogenous Tiam1 in response to
tyrosine kinase signaling.
6
1 in
Tiam1/Rac1-induced Spreading
6
1. Studies in PC12
cells have demonstrated the importance of the integrin
1
1, a laminin/collagen receptor, in mediating cell adhesion and neurite formation (Tawil et al., 1990
; Tomaselli et
al., 1990
; Zhang et al., 1993
). Rather than affecting integrin expression, C1199 Tiam1 induces the formation of
specific
6
1-containing adhesive complexes at the cell
periphery. These complexes are clearly different from focal contacts, which are also detected in Tiam1-overexpressing cells. In fibroblasts, the presence of stress fibers
and focal contacts is associated with increased Rho activity
and increased contractility (Ridley et al., 1992
; Burridge
and Chrzanowska-Wodnicka, 1996
). In view of our finding
that activation of Tiam1 and consequently Rac1 appears
to induce loss of contractility and inactivation of the Rho
pathway, it may seem contradictory to see prominent focal contacts in the C1199 Tiam1-expressing cells. However, a
direct correlation between Rho activity and the formation
of focal contacts, as it occurs in fibroblasts, does not appear to exist in N1E-115 cells. In these cells, increased contractility, by activation of Rho, results in cell rounding
without the appearance of stress fibers or focal contacts
(Jalink et al., 1994
).
; Kozma et al., 1997
; Gebbink
et al., 1997
). Surprisingly, Tiam1-induced activation of Rac
produces the opposite phenotype: cell spreading and the
formation of neurites. The fact that cells expressing C1199
Tiam1 no longer respond to LPA suggests that activation
of Rac may oppose Rho signaling. A similar conclusion was reached by Kozma et al. (1997)
, who demonstrated
that activation of a muscarinic acetylcholine receptor-
induced pathway in N1E-115 cells is dependent on both
Cdc42 and Rac and prevents LPA-induced neurite retraction. Since we found that this apparent inhibition of the
Rho pathway by Rac is overcome by expression of constitutively active V14RhoA, regulation may occur at the
level of Rho or upstream, rather than downstream. The effector molecules responsible for Rac-induced silencing of
the Rho pathway are not known. Potential candidates include GAPs, which reduce the activity of Rho-like proteins. At least six RhoGAP proteins have been identified,
which show different specificities towards particular Rho
family members (Lamarche and Hall, 1994
; Reinhard et
al., 1995
).
; Ridley et al.,
1993
). By associating with p120RasGAP, p190 may serve
to link the Ras and Rho signaling pathways (McGlade et
al., 1993
; Settleman et al., 1992
). We are currently testing the importance of p190 and other potential GAPs for Rho
in the signaling between Rac and Rho.
). Recently, involvement of Tiam1 in
neuronal development was confirmed by Drosophila genetics. Mutations in the Drosophila gene Still-life (sif),
which appears to be both structurally and functionally homologous to Tiam1, appear to affect synapse formation
during fly development. In addition, transgenic flies which
express truncated SIF proteins show specific defects in axonal extension (Sone et al., 1997
). The results of our study
illustrate how Tiam1, as a specific activator of Rac1, may
determine cytoskeletal architecture and adhesion of neuronal cells. Complex interactions with the extracellular
matrix involving integrins, and probably other cell adhesion molecules, play an important role in this regulation. Furthermore, a regulatory loop between Rac- and Rho-mediated signaling pathways is revealed. The challenge
will be to unravel the molecular mechanisms behind these
phenomena.
Received for publication 2 April 1997 and in revised form 6 August 1997.
Address all correspondence to Dr. J.G. Collard, The Netherlands Cancer Institute, Division of Cell Biology (H1), Plesmanlaan 121, 1066 XC Amsterdam, The Netherlands. Fax: (+31) 20 512 1944. E-mail: JCOLL@ NKI.NLWe thank A. Hall, M. Gebbink, R. Cerione, and M. Symons for providing cDNAs or expression constructs; A. Sonnenberg for providing antibody GoH3; Dr. L. Schrama for B-50/GAP-43 antibodies; Dr. M. Glukhova for antivinculin antibodies; L. Oomen for assistance with the confocal microscope and processing of the images; N. Ong for preparing several of the figures; and E. Sander, P. Hordijk, J. Stam, and A. Sonnenberg for critically reviewing the manuscript. We thank W. Moolenaar for suggesting the use of and providing N1E-115 cells.
This work was supported by grants from the Dutch Cancer Society (NKB 94-878) and The Netherlands Organization for Scientific Research (NWO) to J.G. Collard.
GAP, GTPase activating protein; GEF, guanine nucleotide exchange factor; LPA, lysophosphatidic acid; NGF, nerve growth factor; Tiam1, T-lymphoma invasion and metastasis 1.
1. | Altun-Gultekin, Z.F., and J.A. Wagner. 1996. Src, Ras, and Rac mediate the migratory response elicited by NGF and PMA in PC12 cells. J. Neurosci. Res. 44: 308-327 |
2. | Bogusky, M.S., and F. McCormick. 1993. Proteins regulating Ras and its relatives. Nature (Lond.). 366: 643-654 |
3. | Burridge, K., and M. Chrzanowska-Wodnicka. 1996. Focal adhesions, contractility, and signaling. Annu. Rev. Cell. Dev. Biol. 12: 463-518 . |
4. | Cantley, L.C., K.R. Auger, C. Carpenter, B. Duckworth, A. Graziani, R. Kapeller, and S. Soltoff. 1991. Oncogenes and signal transduction. Cell. 64: 281-302 |
5. | Cerione, R.A., and Y. Zheng. 1996. The Dbl family of oncogenes. Curr. Opin. Cell Biol. 8: 216-222 |
6. | Chao, M.V.. 1992. Neurotrophin receptors: a window into neuronal differentiation. Neuron. 9: 583-593 |
7. |
Chen, H., and
J.L. Guan.
1994.
Stimulation of phosphatidylinositol 3![]() |
8. | Collard, J.G.. 1996. Signaling pathways regulated by Rho-like proteins: a possible role in tumor formation and metastasis. Int. J. Oncol. 8: 131-138 . |
9. | Ehler, E., F. van Leeuwen, J.G. Collard, and P.C. Salinas. 1997. Expression of Tiam1 in the developing brain suggests a role for the Tiam1-Rac signaling pathway in cell migration and neurite outgrowth. Mol. Cell. Neurosci. 9: 1-12 |
10. |
Gebbink, M.F.B.G.,
O. Kranenburg,
M. Poland,
F.P.G. van Horck,
B. Houssa, and
W.H. Moolenaar.
1997.
Identification of a novel putative Rho specific
GDP-GTP exchange factor and a Rho binding protein: control of neuronal
morphology.
J. Cell Biol.
137:
1603-1613
|
11. | Habets, G.G.M., E.H.M. Scholtes, D. Zuydgeest, R.A. van der Kammen, J.C. Stam, A. Berns, and J.G. Collard. 1994. Identification of an invasion-inducing gene, Tiam-1, that encodes a protein with homology to GDP-GTP exchangers for Rho-like proteins. Cell. 77: 537-549 |
12. | Habets, G.G.M., R.A. van der Kammen, J.C. Stam, F. Michiels, and J.G. Collard. 1995. Sequence of the human invasion-inducing Tiam1 gene, its conservation in evolution and its expression in tumor cell lines of different tissue origin. Oncogene. 10: 1371-1376 |
13. | Hawkins, P.T., A. Eguinoa, R.G. Qui, D. Stokoe, F.T. Cooke, R. Walters, S. Wenstrom, L. Claesson-Welsh, T. Evans, M. Symons, and L. Stephens. 1995. PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide-3-kinase. Curr. Biol. 5: 393-403 |
14. |
Hogervorst, F.,
L.G. Admiraal,
C. Niessen,
I. Kuikman,
H. Janssen,
H. Daams, and
A. Sonnenberg.
1993.
Biochemical characterization and tissue distribution of the A and B variants of the integrin ![]() |
15. | Hynes, R.O., and A.D. Lander. 1992. Contact adhesive specificities in the associations, migrations, and targeting of cells and axons. Cell. 68: 303-322 |
16. | Jalink, K., and W.H. Moolenaar. 1992. Thrombin receptor activation causes rapid neuronal cell rounding and neurite retraction independent of classic second messengers. J. Cell Biol 118: 411-419 [Abstract]. |
17. | Jalink, K., T. Eichholtz, F.R. Postma, E.J. van Corven, and W.H. Moolenaar. 1993. Lysophosphatidic acid induces neuronal shape changes via a novel, receptor-mediated signaling pathway: similarity to thrombin action. Cell Growth Diff. 4: 247-255 [Abstract]. |
18. | Jalink, K., E.J. van Corven, T. Hengeveld, N. Morii, S. Narumiya, and W.H. Moolenaar. 1994. Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho. J. Cell Biol 126: 801-810 [Abstract]. |
19. | Jockusch, B.M., P. Bubeck, K. Giehl, M. Kroemker, J. Moschner, M. Rothkegel, M. Rüdiger, K. Schlüter, G. Stanke, and J. Winkler. 1995. The molecular architecture of focal adhesions. Annu. Rev. Cell. Dev. Biol. 11: 379-416 . |
20. | Kozma, R., S. Ahmed, A. Best, and L. Lim. 1995. The Ras-related protein Cdc42HS and Bradykinin promote the formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol. Cell. Biol. 15: 1942-1952 [Abstract]. |
21. | Kozma, R., S. Sarner, S. Ahmed, and L. Lim. 1997. Rho family GTPases and neuronal growth cone remodeling: relationship between increased complexity induced by Cdc42Hs, Rac1 and acetylcholine and collapse induced by RhoA and lysophosphatidic acid. Mol. Cell. Biol. 17: 1201-1211 [Abstract]. |
22. | Kranenburg, O., V. Scharnhorst, A.J. Van der Eb, and A. Zantema. 1995. Inhibition of cyclin-dependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells. J. Cell Biol. 131: 227-234 [Abstract]. |
23. | Lamarche, N., and A. Hall. 1994. GAPs for rho-related GTPases. Trends Genet. 10: 436-440 |
24. | Luo, L., Y.J. Liao, L.Y. Jan, and Y.N. Jan. 1994. Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev. 8: 1787-1802 [Abstract]. |
25. | Luo, L., T.K. Hensh, L. Ackerman, S. Barbel, L.Y. Jan, and Y.N. Jan. 1996. Differential effects of the rac GTPase on Purkinje cell axons and dendritic trunks and spines. Nature (Lond.). 379: 837-840 |
26. | Mackay, D.J.G., C.D. Nobes, and A. Hall. 1995. The Rho's progress: a potential role during neuritogenesis for the Rho family of GTPases. Trends Neurosci. 18: 496-501 |
27. | McGlade, J., D.A. Brunkhorst, D. Anderson, G. Mbamalu, J. Settleman, S. Dedhar, M. Rozakis-Adckock, L.B. Chen, and T. Pawson. 1993. The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion. EMBO (Eur. Mol. Biol. Organ.) J. 12: 3073-3081 [Abstract]. |
28. | Michiels, F., G.G.M. Habets, J.C. Stam, R.A. van der Kammen, and J.G. Collard. 1995. A role for Rac-1 in Tiam1 induced membrane ruffling and T-lymphoma invasion. Nature (Lond.). 375: 338-340 |
29. |
Michiels, F.,
J.C. Stam,
P.L. Hordijk,
R.A. van der Kammen,
L. Ruuls van
Stalle,
C.A. Feltkamp, and
J.G. Collard.
1997.
Regulated membrane localization of Tiam1, mediated by the NH2-terminal pleckstrin homology domain, is required for Rac-dependent membrane ruffling and c-jun NH2-terminal kinase activation.
J. Cell Biol.
137:
387-398
|
30. | Nobes, C.D., and A. Hall. 1995. Rho, Rac and Cdc42 GTP-ases regulate the assembly of multi-molecular focal complexes associated with actin stress fibers, lamellipodia and filopodia. Cell. 81: 53-62 |
31. | Prendergast, G.C., F.R. Khosravi, P.A. Solski, H. Kurzawa, P.F. Lebowitz, and C.J. Der. 1995. Critical role of Rho in cell transformation by oncogenic Ras. Oncogene. 10: 2289-2296 |
32. | Qiu, R.G., J. Chen, D. Kirn, F. McCormick, and M. Symons. 1995a. An essential role for Rac in Ras transformation. Nature (Lond.). 374: 457-459 |
33. | Qiu, R.G., J. Chen, F. McCormick, and M. Symons. 1995b. A role for Rho in Ras transformation. Proc. Natl. Acad. Sci. USA 92: 11781-11785 [Abstract]. |
34. | Reinhard, J., A.A. Scheel, D. Diekmann, A. Hall, C. Ruppert, and M. Bahler. 1995. A novel type of myosin implicated in signalling by rho family GTPases. EMBO (Eur. Mol. Biol. Organ.) J. 14: 697-704 [Abstract]. |
35. | Ridley, A.J., and A. Hall. 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 70: 389-399 |
36. | Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 70: 401-410 |
37. | Ridley, A.J., A.J. Self, F. Kasmi, H. Paterson, A. Hall, C.J. Marshall, and C. Ellis. 1993. Rho family GTPase activating proteins p190, bcr, and rhoGAP show distinct specificities in vitro and in vivo. EMBO (Eur. Mol. Biol. Organ.) J. 12: 5151-5160 [Abstract]. |
38. | Sastry, S.K., and A.F. Horwitz. 1996. Adhesion-growth factor interactions during differentiation: and integrated response. Dev. Biol. 180: 455-467 |
39. | Schlaepfer, D.D., S.K. Hanks, T. Hunter, and P. van der Geer. 1994. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature (Lond.). 372: 786-791 |
40. | Settleman, J., C.F. Albright, L.C. Foster, and R.A. Weinberg. 1992. Association between GTPase activators for Rho and Ras families. Nature (Lond.). 359: 153-154 |
41. | Skene, J.H.P., R.D. Jacobson, G.J. Snipes, C.B. MacGuire, J. Norden, and J.A. Freeman. 1986. A protein induced during nerve growth, GAP-43, is a major component of growth cone membranes. Science (Wash. DC). 233: 783-785 |
42. |
Sone, M.,
M. Hoshiro,
E. Suzuki,
K. Shinya,
K. Kaibuchi,
H. Nakagoshi,
K. Saigo,
Y. Nabeshima, and
C. Hama.
1997.
Still life, a protein in synaptic terminals of Drosophila homologous to GDP-GTP exchangers.
Science (Wash.
DC).
275:
543-547
|
43. | Sonnenberg, A., P.W. Modderman, and F. Hogervorst. 1988. Laminin receptor on platelets is the integrin VLA-6. Nature (Lond.). 336: 487-489 |
44. | Stam, J.C., E.E. Sander, F. Michiels, F.N. van Leeuwen, H.E.T. Kain, R.A. van der Kammen, and J.G. Collard. 1997. Targeting of Tiam1 to the plasma membrane requires the cooperative function of the N-terminal pleckstrin homology domain and an adjacent protein interaction domain. J. Biol. Chem. In press. |
45. | Tanaka, E., and J. Sabry. 1995. Making the connection: cytoskeletal rearrangements during growth cone guidance. Cell. 83: 171-176 |
46. |
Tawil, N.J.,
M. Houde,
R. Blacher,
F. Esch,
L.F. Reichardt,
D.C. Turner, and
S. Carbonetto.
1990.
![]() ![]() |
47. |
Tigyi, G., and
R. Miledi.
1992.
Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in
PC12 pheochromocytoma cells.
J. Biol. Chem.
267:
21360-21367
|
48. |
Tomaselli, K.J.,
D.E. Hall,
L.A. Flier,
K.R. Gehlsen,
D.C. Turner,
S. Carbonetto, and
L.F. Reichardt.
1990.
A neuronal cell line (PC12) expresses two ![]() ![]() ![]() ![]() ![]() |
49. | van Leeuwen, F.N., R.A. van der Kammen, G.G.M. Habets, and J.G. Collard. 1995. Oncogenic activity of Tiam1 and Rac1 in NIH3T3 cells. Oncogene. 11: 2215-2221 |
50. | Van Puijenbroek, A.A.F.L., D.H.J. van Weering, C.E. van den Brink, J.L. Bos, P.T. van der Saag, S.W. de Laat, and J. Hertog. Cell scattering of SK-N-MC neuroepithelioma cells in response to Ret and FGF receptor tyrosine kinase activation is correlated with sustained ERK2 activation. Oncogene. 14:1147- 1157. |
51. | Wennstrom, S., P. Hawkins, F. Cooke, K. Hara, K. Yonezawa, M. Kasuga, T. Jackson, L. Claesson-Welsh, and L. Stephens. 1994. Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling. Curr. Biol. 4: 385-393 |
52. |
Zhang, Z.,
G. Tarone, and
D.C. Turner.
1993.
Expression of integrin ![]() ![]() |