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Biochimica et Biophysica Acta
The Cas family docking protein, HEF1, promotes the formation of
neurite-like membrane extensions
Sharmilla D. Bargon a, Peter W. Gunning a,b, Geraldine M. O’Neill a,b,*
a Oncology Research Unit, The Children’s Hospital at Westmead, 2145, Sydney, Australiab Discipline of Paediatrics and Child Health, University of Sydney, 2006, Australia
Received 17 May 2005; received in revised form 4 October 2005; accepted 21 October 2005
Available online 14 November 2005
Abstract
The Cas family proteins are a family of adhesion docking molecules that mediate protein–protein interactions and contribute to a number of
signal transduction pathways. Recent studies of two family members, p130Cas and Sin, have suggested that they may play a role in neurite
formation. The current study demonstrates that the third family member, HEF1, can also stimulate the formation of neurite-like processes, in the
presence of Rho kinase inhibitors. The HEF1-promoted processes actively extend from the cell body and resemble neurites both in the manner of
process extension and in the distribution of adhesion-associated molecules. The HEF1-promoted processes are dependent on the presence of an
intact microtubule system and can be inhibited by co-expression of either constitutively active Rac or Cdc42 GTPase. Together, our data support a
role for the Cas proteins in regulating cellular morphologies that contribute to tissue specialization.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Focal adhesion; Integrin; Cas protein; Neurite; Migration; GTPase
1. Introduction
The Cas family of docking proteins are grouped together
based on their overall conserved domain structure and high
degree of sequence homology (reviewed in [1]). Included in the
family are p130Cas/BCAR1 [2,3], Sin/Efs [4,5] and HEF1/
Cas-L/Nedd9 [6,7]. Each protein contains multiple interaction
domains that can mediate interaction with partner molecules
containing poly-proline motifs and Src-homology 2 domains
(SH2) and molecules that interact with phosphorylated serine
and threonine consensus motifs (reviewed in [1]). Both
p130Cas and Sin contain additional poly-proline motifs that
can mediate interactions with SH3-domain containing partner
molecules. A number of studies have indicated that p130Cas
and HEF1 can localize to focal adhesions [2,6,8–10] and
together with their overall protein–protein interaction domain
structure this has led to their description as adhesion docking
molecules. Numerous studies have been carried out to identify
0167-4889/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2005.10.008
* Corresponding author. Oncology Research Unit, The Children’s Hospital at
Westmead, 2145, Sydney, Australia. Tel.: +61 2 9845 3116; fax: +61 2 9845
3078.
E-mail address: [email protected] (G.M. O’Neill).
the repertoire of binding proteins (some examples are reviewed
in [1] and [11]) and together these studies have implicated Cas
proteins as playing a role in adhesion signalling and adhesion-
mediated functions.
Currently, it is not clear whether the Cas proteins have
distinct or similar functions. It appears unlikely that they have
redundant function, since knockout of the p130Cas gene in
mice is embryonic lethal [12], suggesting that HEF1 and Sin
cannot compensate for the loss of p130Cas. However, for a
number of functions that have been studied, the molecules
appear to have a similar effect. For example, both p130Cas and
HEF1 have been separately implicated in the promotion of
cellular migration and in both cases this is dependent on an
interaction with Focal Adhesion Kinase (FAK) and requires
intact p130Cas and HEF1 substrate binding domains [13–18].
Both p130Cas and Efs have been shown to stimulate Src kinase
activity and downstream signalling [4,19,20] and all three
molecules have been implicated in the regulation of various
GTPases via recruitment of specific downstream partners
[17,18,21–24]. To date, the majority of the studies have
focused on p130Cas.
Given the large number of molecules known to interact
with Cas proteins (reviewed in [1]), it is probably not
1746 (2005) 143 – 154
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S.D. Bargon et al. / Biochimica et Biophysica Acta 1746 (2005) 143–154144
surprising that the Cas proteins have been implicated in a
wide variety of biological processes. Intriguingly, HEF1 has
been implicated in the apparently unrelated biological
processes of cell migration [13–15] and apoptosis [10,37].
However, a common requirement in both processes is the
integration of information from the external cellular environ-
ment with the regulation of adhesion dynamics and morpho-
logical control. In cell migration, signals received through the
adhesion sites are co-ordinated to regulate adhesion turnover
with lamellipodial protrusion and trailing edge retraction,
while cell rounding during apoptosis is accompanied by
break down of cytoskeletal linkage to cell adhesion sites and
a corresponding loss of focal adhesions. Therefore, we
propose that HEF1 (and likely the other Cas family proteins)
may function by co-ordinating adhesion dynamics that
regulate cellular morphologies required for distinct cellular
functions.
The lack of redundancy implied by the embryonic lethality
of the p130Cas gene knockout [12] suggests that the three
molecules may instead have tissue specific function. Indeed,
while the expression pattern of p130Cas is reported to be
ubiquitous [2], early studies of HEF1 suggested that the
expression of this molecule is more restricted to pre-B and T-
cells and epithelially-derived tissue [6,7,25], while in contrast
Efs is reported to be restricted to brain tissue [5]. However, a
recent study examining the expression pattern of HEF1 mRNA
in rat embryos found that HEF1 is expressed very early in the
developing hindbrain [26]. Together with the fact that one of
the first reports of HEF1 cDNA was in a screen for neural
precursor cell expressed and developmentally down-regulated
molecules (NEDD9 [27]), it therefore appears that HEF1 may
also play a role in neuronal physiology.
Both p130Cas and Sin have been shown to undergo tyrosine
phosphorylation in response to neuritic signals in neuronal cell
types [28–31] and subsequent interaction of Sin with the
adaptor molecule Crk was shown to be important for neurite
formation in cultured PC12 cells [28]. However, Cas protein
involvement in the formation of long neuritic-like processes is
not simply restricted to neuronal-type cells. A number of
studies have now suggested that these molecules may play a
part in the cellular machinery that controls the extension of
long thin membrane processes. For example, a pathway
involving a ternary complex between p130Cas, Crk and the
adaptor molecule CHAT/SHEP1/NSP3 can stimulate the
formation of long branched membrane processes from NIH3T3
fibroblasts [31]. Furthermore, in cultured B-cells and HEK293
cells, over-expression of the GDP-exchange factor (GEF) and
p130Cas/HEF1 interacting partner AND-34/BCAR3/NSP2 can
stimulate the formation of long membrane protrusions [22],
most likely due to the activation of Cdc42 by the GEF activity
of AND-34. Therefore, the Cas proteins may function to
integrate signalling information that then determines cellular
morphologies, including the production of neurites from
neuronal cells. The prevailing view is that activation of Rac
and Cdc42 are required for neurite formation (reviewed in
[32]). Given the array of GTPase regulating molecules that
interact with Cas proteins, including molecules that regulate
Rac and Cdc42 activity [18,21,22,24,31], it is perhaps not
surprising that this family of molecules might contribute to the
formation of neurite-like structures.
In addition to the observation that HEF1 is expressed in the
developing hindbrain, Merrill et al. [26] have reported that
HEF1 mRNA is induced by retinoic acid treatment of
neuroblastoma cells, and this occurs prior to neurite extension.
Therefore, this has led us to ask whether, similar to p130Cas
and Sin, HEF1 can also promote neurite-like membrane
extensions and whether this occurs using similar mechanisms
to that described for p130Cas and Sin. To determine whether
HEF1 can promote membrane process formation we have
examined cells treated with Rho kinase inhibitors, a treatment
previously demonstrated to stimulate neurite-like process
formation in both neuronal cell types [33] and in non-neuronal
cell types [34]. Time-lapse imaging of epithelial cells expres-
sing inducible exogenous HEF1 reveals that HEF1 promotes
active process extension in the presence of Rho-kinase
inhibitors. Comparison of HEF1-promoted processes in epi-
thelial cells with neurites from neuronal cells reveals a similar
sub-cellular distribution of adhesion-associated molecules,
including HEF1, between the two structures, suggesting that
the HEF1 promoted processes are neurite-like. Further,
formation of the HEF1-promoted processes is demonstrated
to require intact microtubules and is inhibited by constitutively
active Rac and Cdc42.
2. Materials and methods
2.1. Cell lines, reagents and antibodies
MCF7 cell lines engineered to express tetracycline-regulatable HEF1 have
been previously described [13]. Cell lines used include HEF1.M1, HEF1.M2
and the vector control cell line CM1. Cells were maintained in Dulbecco’s
Modified Eagles Medium (DMEM) plus 10% Foetal Bovine Serum (FBS)
supplemented with 1 Ag ml�1 tetracycline to repress expression of the
transgene and 2 Ag ml�1 puromycin. For induction of HEF1 expression, cells
were trypsinized and replated into media without antibiotics and grown for 24
to 48 h. B35 rat neuroblastoma cells were maintained in DMEM plus 10%
FBS and 5 mM l-glutamine.
Constitutively active Rac (pEGFP.RacL61) and dominant negative Rac
(pEGP.RacN17) constructs were obtained from Beric Henderson (Westmead
Millenium Institute) with the kind permission of Mark Phillips (New York
University School of Medicine). Constitutively active Cdc42L61 and
dominant negative Cdc42N17 were purchased from Upstate Biotechnology
(Lake Placid, NY, USA). The Cdc42 cDNA inserts were sub-cloned into
EcoR1–XhoI digested pEGFP, to create in-frame fusions with GFP.
Constructs were verified by Western blot analysis of transfected cell lysates.
Monoclonal antibody to paxillin was from BD Transduction Laboratories
(Franklin Lakes, NJ, USA) and to polymerized-tubulin was from Sternberger
monoclonals (Maryland, USA). The monoclonal anti-HEF1 antibody (clone
2G9) is from ImmuQuest (Cleveland, UK) and anti-h1integrin (clone P4C10)
from Invitrogen Life Technologies (Carlsbad, CA, USA). TRITC-phalloidin
was purchased from Sigma Aldrich (St. Lois, MO, USA), vectashield
mounting medium from Vector Laboratories (Berlingame, CA, USA), alexa
488-conjugated donkey anti-mouse and alexa 488-conjugated donkey anti-
rabbit from Jackson Immunological Labs (West Grove, PA, USA). Y-27632
and H-1152 were from Calbiochem (La Jolla, CA, USA). Dibutyryl-cyclic
AMP and Nocodazole were from Sigma Aldrich (St. Lois, MO, USA).
Transfection reagent Lipofectamine 2000 was obtained from Invitrogen Life
Technologies (Carlsbad, CA, USA) and used according to the manufacturer’s
protocol.
S.D. Bargon et al. / Biochimica et Biophysica Acta 1746 (2005) 143–154 145
2.2. Rho kinase inhibition
Cells were either treated for 1, 4 and 24 h as indicated. In initial
experiments, HEF1.M1 cells were grown on coverslips under inducing
conditions for 48 h and then Y-27632 (10 AM) added for 4 h, cells were
then fixed and stained with phalloidin. Rho kinase inhibition with H-1152 was
achieved as described above, with a concentration of 10 AM H-1152.
2.3. Neurite production
B35 cells were plated onto glass coverslips 24 h prior to the addition of
drugs. To stimulate neurite production, either 1 mM dbCAMP or 10 AM Y-
27632 was added to cells in media with 0.5% FBS and cells were incubated for
a further 24 h before fixing and immunofluorescence analysis.
2.4. Immunofluorescence
Cells for immunofluorescence analysis were grown on glass coverslips,
fixed with 4% paraformaldehyde (PFA) in PBS and permeabilized with 0.2%
Triton X-100 in PBS with 0.5% bovine serum albumin (BSA), with the
exception of cells stained with anti-polymerized tubulin. Cell permeabilization
for microtubule staining was performed with �20 -C methanol for 5 min.
Antibody dilutions and immunostaining were performed in 0.5% BSA in
PBS.
2.5. Image capture and analysis
Epifluorescence and phase contrast images were captured using a Spot II-
cooled CCD digital camera (Diagnostics Instruments) mounted on an
Olympus Bx50 microscope with 40� and 60� objectives. DIC time-lapse
imaging was performed using a SPOT RT SE CCD camera mounted on a
Leica DMIRBE inverted microscope with a 40� oil objective. Images were
captured at 15-min intervals. Image analysis was performed using Image-Pro
plus Version 4.0 (Media Cybernetics) and final images prepared in Adobe
Photoshop.
2.6. Monitoring process formation
To analyse the effects of Rac and Cdc42 GTPases on process formation,
HEF1.M1 cells were induced overnight and transfected with GFP fusion
constructs the following day. Cells were incubated for 6 h in the presence of the
transfection complexes, then trypsinized and replated onto coverslips in 6-well
dishes. The following day cells were treated with Y-27632 and incubated for 4
h, then fixed and immunostained as described. Cells were co-stained with DAPI
to monitor nuclear morphology and only cells with healthy (non-apoptotic)
nuclei, were included in the analysis. HEF.M1 cells grown under non-inducing
conditions (that is in the presence of tetracycline) were transfected for 6 h in the
absence of tetracycline and replated in the presence of tetracycline to inhibit
HEF1 production.
2.7. Transwell assay of cellular migration
Prior to assay of cell migration, cells were grown under inducing or non-
inducing conditions for 48 h. 6�104 cells in serum-free media were then
plated into the top chamber of an 8-Am pore transwell filter (Falcon) mounted
in 24-well dishes. The media in the bottom wells contained foetal bovine
serum. Cells under non-induced conditions were supplemented with
tetracycline and other inhibitors were added coincident with plating into the
top well of the trans-well filter. Following 6 h incubation at 37 -C and 5%
CO2 cells from the top of the filter were removed with a cotton bud and cells
on the underside were stained with Diff Quik (Lab Aids, Australia). Filters
were then examined with a 40� objective of a light microscope (Olympus)
and the total number of nuclei positive cells determined for 8 randomly
selected fields. Assays were carried out in triplicate (duplicate samples in
each assay) and the average number of cells migrating under each
experimental condition determined.
3. Results
3.1. HEF1 promotes membrane process formation
In light of recent data suggesting that the Cas proteins
p130Cas and Efs contribute to the formation of membrane
processes [28,29,31,22], we tested whether the third Cas family
protein, HEF1, has similar effects on cell morphology.
HEF1.M1 and HEF1.M2, two cell lines engineered to express
HEF1 under the control of a tetracycline-regulatable promoter
and a vector control cell line CM1 [13], were used as the model
to examine HEF1 effects on cellular morphologies. Cells were
treated with the Rho kinase inhibitor, Y-27632, a treatment
previously shown to promote neurite-like process formation
[33,34]. HEF1.M1 cells were induced for HEF1 expression and
then treated for 4 h with 10 AM Y-27632 [35]. Under these
conditions, and as early as 1 h after drug addition, HEF1.M1
cells exhibited a striking arborized phenotype with numerous
branched processes emanating from the cell body (Fig. 1A).
Confirming that this phenotype is due to increased HEF1
expression, there is little evidence of the branched phenotype in
either CM1 vector control cells or in uninduced HEF1.M1
clone treated with Y-27632 for the same length of time (Fig.
1B). Quantitation of process formation further confirmed that
HEF1 promotes process formation and this occurs for up to 24
h after addition of Y-27632 (Fig. 1C). Interestingly, clone
HEF1.M1, which expresses a higher level of HEF1 protein
than clone HEF1.M2 (Fig. 1D) also shows the greatest number
of processes, therefore suggesting that there may be a dose-
dependent effect of HEF1 on process formation. Finally, HEF1
is normally detected as two bands on western blots and there is
an interesting loss of the upper band in the cells treated with the
Rho kinase inhibitor. This suggests that there may be an
alteration in the phosphorylation of HEF1, similar to results we
have previously described [10].
To test whether the observed phenotype is dependent on
inhibition of Rho kinase activity and not due to non-specific
action of Y-27632, cells were treated with a second Rho kinase
inhibitor, H-1152 [36]. Treatment with H-1152 recapitulated
the results seen with Y-27632 with the cells induced to express
HEF1 again displaying branched processes (Fig. 1E). There-
fore, the inhibition of Rho kinase activity appears to be
sufficient for HEF1-mediated increases in process formation.
Since we have earlier shown that HEF1 expression can
promote apoptosis [10,37], it is possible that the processes
reflect membrane retraction fibres in dying cells. However,
three lines of evidence suggest this is not the case. Firstly, the
length of processes exceeds the size of the original cells.
Secondly, the processes contain bundles of microtubules (see
Fig. 4A) which are absent from retraction fibres [38]. Finally,
time-lapse imaging demonstrates that the majority of the
processes are formed by active membrane protrusion. Analysis
of processes in cells treated with Rho kinase inhibitors
demonstrated that 59% of processes exhibited active membrane
extension (Fig. 2B). There was a mixture of phenotypes, with
some processes representing trailing edges as has been
previously reported by others [39,40], others resulting from
S.D. Bargon et al. / Biochimica et Biophysica Acta 1746 (2005) 143–154146
active protrusion of membrane and the third phenotype was a
combination of the two, that is, active protrusion from the end
of a trailing edge. Examples of active membrane extension are
shown in Fig. 2A (arrows). In the first panel, ruffling
extensions are indicated by the arrows. In progressive frames
the membranes are clearly extending away from the cell body
Fig. 2. Membrane processes are actively extended in HEF1-expressing cells treated with Rho kinase inhibitors. (A) DIC time lapse images of induced HEF1.M1 cells
treated with Y-27632. Frames were taken at 15 min intervals, 40� objective. Indicated are examples of actively extending processes (arrows) and a trailing edge
(arrow head). (B) Membrane process formation was observed in time lapse series and the percentage of distinct process phenotypes calculated. A total of 71
individual processes were measured. (C) Migration of HEF1.M1 cells was assessed using a transwell migration assay. Cells were grown under induced (black bars)
or non-induced (white bars) conditions and the number of cells migrating to the under-side of the filter determined. (D) Transwell migration assays were performed in
the presence of the Rho kinase inhibitors, Y-27632 and H-1152 and the number of cells migrating to the under-side of the filter calculated and expressed as a percent
of the total number of cells migrating in the absence of the inhibitors. HEF1.M1 cells were grown under inducing (black bars) or non-inducing conditions (white
bars). *P <0.05, Student’s t-test.
S.D. Bargon et al. / Biochimica et Biophysica Acta 1746 (2005) 143–154 147
and are reminiscent of neurite-like membrane extensions. An
example of a trailing edge is also indicated in Fig. 2B (arrow
heads). Together, the data suggest that a combination of
behaviours contribute to overall process formation: anchoring
of the trailing edge, active process extension and a combination
of both. Importantly, the majority of the processes (59%)
represent active membrane extension.
Our live cell imaging analysis also suggested that Rho
kinase inhibition blocked HEF1 promoted cell migration. To
quantitate the effects of Rho kinase inhibition on HEF1-
promoted cell migration, we initially confirmed that the
induction of HEF1 expression following tetracycline with-
drawal resulted in approximately 5-fold enhanced cell migra-
tion of the HEF1.M1 cell line under induced conditions when
Fig. 1. HEF1 promotes membrane process formation. (A) Phase contrast images of H
AM Y-27632 for 1 h (right panel). Arrows indicate processes. (B) HEF1.M1 cells a
inducing conditions as indicated. Cells in the bottom panels were all treated with 10 AQuantitation of process formation in two clones, HEF1.M1 and HEF1.M2 and CM
conditions as indicated and then incubated in the presence of Y-27632 for 1, 4 or
counted and expressed per 100 nuclei. (D) Western blot analysis of HEF1 expressio
27632. (E) Phase contrast image of induced HEF1.M1 cells treated with 10 AM H
compared with uninduced control cells (Fig. 2C). Next, we
tested the effect of Rho kinase inhibition on HEF1-promoted
cell migration by using both Y-27632 and H-1152. In transwell
migration assays, the use of both inhibitors significantly
reduced HEF1-promoted cell migration to less than 60% of
that seen in control cells (Fig. 2D). In contrast, Rho kinase
inhibition of cells grown under non-inducing conditions had no
effect on the extent of cell migration (Fig. 2D). These data
correlate with earlier findings that Rho kinase inhibition can
inhibit cell migration by preventing tail retraction [39,40].
However, we note that in our cells we observe loss of the
motile morphology (that is loss of obvious leading and trailing
edges) and a switch to active membrane processes emanating
from around the cell periphery. Therefore, HEF1-promoted
EF1.M1 cells grown under induced conditions (left panel) and treated with 10
nd CM1 vector control cells grown under inducing conditions or control non-
M Y-27632. Cells are stained with TRITC-phalloidin to show cell structure. (C)
1 vector control cell line. Cells were grown under inducing or non-inducing
24 h. Cells were then fixed and immunostained and the number of processes
n in induced HEF1.M1 and HEF1.M2 cells in the presence and absence of Y-
-1152. Arrows indicate processes.
S.D. Bargon et al. / Biochimica et Biophysica Acta 1746 (2005) 143–154148
process formation occurs in the absence of both apoptosis and
cell migration, suggesting that process formation represents a
previously undescribed function of the HEF1 protein.
3.2. HEF1-promoted processes have a similar distribution of
adhesion molecules to that observed in neurites
Since the HEF1-promoted processes are reminiscent of
neurites, the pattern of HEF1 sub-cellular localization in the
HEF1-promoted processes was next compared with the
distribution of HEF1 in neuronal cells induced to produce
neurites. To carry out these experiments, we employed the rat
neuronal cell line B35, which has previously been shown to
produce neurites in response to 1 mM dbCAMP [41]. Western
blot analysis of B35 protein lysates confirms that the HEF1
Fig. 3. HEF1 promoted processes have a similar distribution of adhesion molecules
and induced HEF1.M1 cells probed with anti-HEF1 antibody. (B) Western blot of ex
HEF1.M1 cells grown under induced conditions (left hand panels) and B35 cells (
paxillin and anti-h-1 integrin antibodies as indicated. Arrows indicate focal adhes
membrane accumulation. Scale bar 25 Am. (D) Induced HEF1.M1 cells treated with
and anti-h1 integrin antibodies as indicated. Shown in the middle and bottom panels
fixed and immunostained as above. Images show detail of antibody stained membr
antibody cross-reacts with endogenous rat HEF1 and further
demonstrates that these cells express endogenous HEF1 protein
(Fig. 3A). Interestingly, examination of HEF1 protein expres-
sion in B35 cells treated with dbCAMP reveals a similar loss of
the upper form of HEF1 (Fig. 3B) as was seen in the Y-27632
treated HEF1.M1 and HEF1.M2 lysates (Fig. 1D). However,
no obvious difference is observed in the Y-27632 treated B35
cells (Fig. 3B).
To our knowledge, this is the first examination of HEF1
protein in a neuronal cell type, we therefore initially established
the sub-cellular distribution of HEF1 in control B35 cells.
HEF1 localizes to the perinuclear region of the B35 cells and
can also be detected as discrete punctate staining in the
cytoplasm (Fig. 3C, arrow heads). However, no localization to
classical focal adhesion structures is detected, although the
to that observed in neurites. (A) Western blot of total protein extracts from B35
tracts from control B35 cells (con.) and treated with Y-27632 and dbCAMP. (C)
right hand panels). Cells were fixed and immunostained with anti-HEF1, anti-
ions, arrow heads indicate punctate staining and asterisks indicate regions of
10 AM Y-27632 (top panels) and immunostained with anti-HEF1, anti-paxillin
are B35 neuronal cells treated with 1 mM dbCAMP or 10 AMY-27632 for 24 h,
ane processes enlarged from the inset images. Scale bar, 5 Am.
S.D. Bargon et al. / Biochimica et Biophysica Acta 1746 (2005) 143–154 149
HEF1 antibody can detect adhesion-associated HEF1 as
demonstrated by positive adhesion staining in the HEF1.M1
cells (Fig. 3C arrows). Paxillin staining further confirms that
classical focal adhesion structures do exist in the B35 cells
(Fig. 3C arrows). Hence, in the B35 cells, HEF1 is localized to
punctate regions within the cytoplasm. We next compared the
pattern of HEF1 staining to h1-integrin staining and found that
in contrast, h1-integrin staining appears to accumulate at the
membrane edge in both HEF1.M1 and B35 cells (Fig. 3C,
asterisks).
To determine whether the HEF1-promoted processes have a
similar molecular structure to neurites, the staining patterns of
HEF1 and paxillin were compared between HEF1.M1 induced
cells treated with Y-27632 and B35 cells treated with either Y-
27632 or dbCAMP (Fig. 3D). This analysis revealed firstly that
the HEF1 and paxillin staining patterns appear identical in both
HEF1.M1 and B35 processes (Fig. 3D). Secondly, we can
detect two populations of paxillin staining: punctate staining
throughout the cytoplasm of the process (Fig. 3D arrows) and
more dash-like adhesion staining at regions projecting from the
edges of the processes (Fig. 3D, arrow heads). This is a
prominent feature of the paxillin staining in all three conditions
of process formation. In contrast, HEF1 is restricted to punctate
staining throughout the cytoplasm of the processes in all three
treatments.
The HEF1 staining pattern appears similar to h1-integrinpositive ‘‘point contacts’’ that have been previously reported
[45,46]. Therefore we compared the distribution of HEF1 with
that of h1-integrin. The sub-cellular localization of h1-integrinis indistinguishable between the three conditions of membrane
process formation and further, appears similar to the distribu-
Fig. 4. HEF1-promoted processes require intact microtubules. (A) HEF1.M1 cells gr
an antibody to polymerized tubulin. (B) HEF1.M1 cells grown under inducing co
tubulin antibodies. Note the absence of organized microtubule structure. (C) Phas
presence of nocodazole and following the addition of Y-27632 or H-1152 as indica
tion of HEF1. However, we note that since both antibodies are
mouse monoclonals it is not possible to directly measure
whether HEF1 and h1-integrin are co-localized (Fig. 3C).
Together, these data suggest that the HEF1 promoted processes
have a similar distribution of adhesion molecules to that
observed in neurites. In summary, HEF1 and h1 integrin
display punctate staining along the length of the processes,
while paxillin is additionally localized to peripheral adhesion
structures.
3.3. HEF1-promoted processes require intact microtubules
Characteristic of neurites (reviewed in [38]) and long
membrane processes formed by other, non-neuronal cells
[33,34,42] is the appearance of parallel bundles of micro-
tubules within the processes. Immunostaining of induced
HEF1.M1 cells treated with Y-27632 using an antibody to
polymerized tubulin shows that HEF1-mediated processes also
contain bundles of microtubules (Fig. 4A). Currently, it is not
clear whether microtubules are required for the initiation of
neurite outgrowth, however they do appear to be necessary for
maintaining outgrowth [38] and furthermore were necessary for
process extension in non-neuronal cells [34,42]. Therefore,
induced HEF1.M1 cells treated were treated with nocodazole to
assess the role of polymerized microtubules in HEF1-promoted
process formation. The efficacy of the nocodazole in depoly-
merizing the microtubules is indicated by loss of microtubule
staining (Fig. 4B). Induced HEF1.M1 cells co-treated with both
nocodazole and either Y-27632 or H-1152 display morpholo-
gies that are indistinguishable from cells treated with nocoda-
zole alone and there is an absence of the membrane extensions
own under inducing conditions and treated with Y-27632. Cells are stained with
nditions and treated with 1 AM nocodazole and stained with anti-polymerized
e contrast images of HEF1.M1 cells grown under inducing conditions in the
ted.
S.D. Bargon et al. / Biochimica et Biophysica Acta 1746 (2005) 143–154150
observed following Rho kinase inhibition alone (Fig. 4C). We
note that the cells treated with nocodazole adopt an altered
morphology, with control cells expressing HEF1 losing their
characteristic motile phenotype (Fig. 4C). Similar to other
studies, it appears that HEF1 promoted process formation
requires intact microtubules.
3.4. Active Rac and Cdc42 inhibit HEF1-promoted processes
Previous data have implied a role for Cas proteins in
regulation of Rac and Cdc42 activity ([22] and reviewed in [1])
and given the role of these molecules in control of cell shape
(reviewed in [43]) and neurite outgrowth [32] we questioned
whether HEF1 might mediate different phenotypes via activa-
tion of these molecules. To test this, induced HEF1.M1 cells
were transfected with plasmids expressing either constitutively
active -Rac (Rac1L61) or -Cdc42 (Cdc42L61), and treated with
Y-27632. Surprisingly, expression of both constitutively active
constructs inhibited HEF1-promoted process formation in
the presence of Y-27632 (Fig. 5A and B and 6A and B).
Consequently, HEF1 does not appear to promote process
formation via the activation of either Rac or Cdc42. In contrast,
Fig. 5. Constitutively active Rac inhibits HEF1-mediated process formation. (A) HE
expressing either GFP control, GFP-tagged RacL61 or RacN17, as indicated, and tr
transfected with constructs expressing GFP, GFP-tagged RacL61 or RacN17, we
membrane processes calculated.
induced HEF1.M1 cells transfected with plasmids expressing
dominant negative Rac (RacN17) and Cdc42 (Cdc42N17) and
treated with Y-27632 still display process formation (Figs. 5A
and B and 6A and B). Together, these data suggest that
inhibition of Rac and Cdc42 activities are necessary for process
formation. However, suppression of the activity of Rac and
Cdc42 alone is not sufficient to promote process formation as
HEF1.M1 cells grown under non-inducing conditions and
transfected with either RacN17 or Cdc42N17 then treated with
Y-27632 did not display processes formation (Figs. 5B and
6B). Together, these data suggest that HEF1 does not regulate
process formation via activation of Rac or Cdc42 and indeed
suppression of the activity of these molecules is necessary,
although not sufficient, for HEF1-promoted process formation.
4. Discussion
Following earlier studies suggesting that the Cas proteins
p130Cas and Sin can promote neurite formation, we now show
that the third Cas family member, HEF1, can also promote
neurite-like process formation, in epithelially-derived cancer
cells. These data represent a previously undescribed function
F1.M1 cells grown under inducing conditions were transfected with constructs
eated with Y-27632 (see arrows). (B) Induced and non-induced HEF1.M1 cells
re treated with Y-27632 and the percentage of GFP-positive cells displaying
Fig. 6. Constitutively active Cdc42 inhibits HEF1-mediated process formation. (A) HEF1.M1 cells grown under inducing conditions were transfected with constructs
expressing either GFP control, GFP-tagged Cdc42L61 or Cdc42N17, as indicated, and treated with Y-27632 (see arrows). (B) Induced and non-induced HEF1.M1
cells transfected with constructs expressing GFP, GFP-tagged Cdc42L61 or Cdc42N17, were treated with Y-27632 and the percentage of GFP-positive cells
displaying processes calculated.
S.D. Bargon et al. / Biochimica et Biophysica Acta 1746 (2005) 143–154 151
for HEF1. In earlier data, we and others have shown that HEF1
can variously promote apoptosis [10,37] and migration [13–
15]. In the current study we demonstrate that the formation of
neurite structures occurs in the absence of either apoptosis or
migration. The HEF1-promoted processes are molecularly
similar to neuronal cell-derived neurites, both in their
requirement for microtubules and the distribution of adhe-
sion-related molecules. The formation of such membrane
extensions requires the co-ordinated interaction of multiple
cytoskeletal and cellular signalling molecules. Given the
protein docking function of the Cas proteins, it is possible that
they may play a role in helping to localize molecules required
for this process.
There are many parallels in the processes of cell migration
of non-neuronal cells and neurite extension (reviewed in [44]).
Both processes require the co-ordination of adhesion dynamics
with morphological controls [44,47], and so it is perhaps not
surprising that a molecule that has been shown to stimulate
migration in a number of different cell types [13–15] can also
stimulate the production of neurite-like membrane processes. It
is interesting that HEF1 promoted process formation requires
the inhibition of Rho kinase. In earlier studies induction of
HEF1 expression in the HEF1.M1 cells was shown to stimulate
Rho kinase mRNA [13]. An intriguing question is whether the
increased Rho kinase expression in response to elevated levels
of HEF1 might be required to balance membrane dynamics at
the leading edge. This could serve to promote cell migration
[13] rather than the membrane process extension we have
observed following Rho kinase inhibition. Indeed, the activa-
tion of Rho kinase has been previously suggested to restrict
membrane protrusion to the leading edge of migrating cells
[39].
Importantly, our data extend earlier observations of HEF1
mRNA expression [26] and now demonstrates HEF1 protein
expression in differentiated neuronal cell neurites. Therefore,
HEF1 is expressed at the right time [26] and in the right place
(as demonstrated by our staining of neuritic processes) to play a
role in neuronal cell architecture. Both the HEF1 promoted
processes in the HEF1.M1 cultures and the B35 cell neuritic
processes showed very similar pattern of adhesion molecule
staining: punctate HEF1, paxillin and h1-integrin staining
throughout the extent of the processes and additional discrete
dash-like paxillin staining at small projections from the edges
of the processes. Similar staining patterns to the HEF1 and h1-
S.D. Bargon et al. / Biochimica et Biophysica Acta 1746 (2005) 143–154152
integrin staining have previously been described as point
contacts [45,46], while vinculin has been previously shown to
give a similar distribution in neurites [45] to the results shown
here for paxillin. In the same way that adhesion dynamics are
important for migration of non-neuronal cells [47], they are
also crucial for neuronal cell architecture [44]. A key event
following Rho kinase inhibition in non-neuronal cells, is the
loss of stable focal adhesions and the formation instead of
rapidly turning over, pre-cursor focal complexes [48]. This is
also true in the switch from a non-migratory to a migratory
phenotype in fibroblast cell types where rapidly turning over
focal complexes become predominant [49,50]. Indeed, our data
demonstrate loss of large HEF1 and paxillin positive adhesions
in the HEF1.M1 cells following Rho kinase inhibition,
suggesting altered adhesion dynamics in these cells. Similarly,
dash-like paxillin positive focal adhesions are reduced in size
in B35 cells treated with Y-27632. Crucially, the same pattern
of altered paxillin adhesion staining is also seen in the B35
cells treated with dbCAMP. Therefore it is possible that the
morphological similarities between the HEF1 promoted pro-
cesses and the neuritic processes may be due to altered
adhesion dynamics.
In contrast to current data on the mechanism of neurite
extension suggesting a positive role for Rac and Cdc42
activation (reviewed in [32]), we find that constitutively active
Rac and Cdc42 inhibit HEF1 promoted membrane process
formation, while dominant negative Rac and Cdc42 have no
effect. Currently it is not possible to determine whether this is a
similar mechanism to that for Sin-promoted neurite outgrowth.
Sin-promoted neurite outgrowth requires an interaction with
Crk and while the authors speculated that Rac activation is a
likely target downstream of this interaction [28], it will be
necessary for this to be tested before a direct comparison can be
made with the HEF1 results presented here. However, similar
to our data, neurite-like extensions formed in NIH-3T3 cells in
response to the inhibition of ROCK and the ubiquitin ligase
Cbl were also shown to be independent of Rac and Cdc42
activity [34]. Of note, there is some contradictory data
regarding the absolute requirement for activated Rac and
Cdc42 in neurite extension. Studies have shown that constitu-
tively active Rac can inhibit total neurite length [51] and inhibit
neurite extension in Drosophila [52]. While activated Rac, and
dominant negative and activated Cdc42 have no effect on DRG
neurite outgrowth [53]. These apparently contradictory results
may be due to a requirement for localized activity of GTPases,
at restricted regions within the cell [54]. In our experiments, the
exogenous mutant GTPases are expressed throughout the cell
and presumably normal controls on the cycling of GTP and
GDP throughout the cell are therefore disrupted. However, if
this is the case, it is still difficult to understand why the mutant
GTPase constructs would have a positive effect on neurite
formation in one set of studies and a negative effect in the other
set of studies. An alternative explanation for the apparently
discrepant results is that there may be multiple pathways that
can determine process formation. The activation of each
pathway may be dependent on tissue and developmental
context.
The control of cell morphology is crucial to normal cellular
function and contributes to the specialization of different
tissues. Integral to morphological control is the ability to
regulate the spatial organization of the cytoskeleton and
associated signalling molecules. In the case of neuritic
extension the correct organization of these components is
required for successful neuronal cell function. Together with
this study, it now appears that the Cas family proteins may
contribute to the regulation of neurite extension, although
currently it is not clear to what extent each member of the
family might contribute to neuronal function. In a broader
context, we propose that the Cas proteins may be involved in
the co-ordination of adhesion site dynamics and signalling that
regulates specialized cellular morphologies. Further study of
the interactions between Cas proteins and their cognate
partners should illuminate the role of these molecules in
neurite extension.
Acknowledgments
P. Gunning is a Principal Research Fellow of the NHMRC
(#163626) and G. O’Neill is the NSW Cancer Council
Research Fellow. This study was further supported by a
Howard Florey Centenary Research Fellowship from the
NHMRC of Australia (#147117) to G. O’Neill and an NHMRC
project grant (#117409) to P. Gunning.
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