1 Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg,
Germany
2 Zentrum für Molekulare Biologie Heidelberg (ZMBH), Im Neuenheimer Feld
282, 69120 Heidelberg, Germany
Author for correspondence (e-mail:
walter.nickel{at}urz.uni-heidelberg.de)
Accepted 25 June 2002
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Summary |
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In the second part of this study we made use of the FGF-2 export system described to analyze the fate of biosynthetic FGF-2-GFP following export to the extracellular space. We find that secreted FGF-2 fusion proteins accumulate in large heparan sulfate proteoglycan (HSPG)-containing protein clusters on the extracellular surface of the plasma membrane. These microdomains are shown to be distinct from caveolae-like lipid rafts known to play a role in FGF-2-mediated signal transduction. Since CHO cells lack FGF high-affinity receptors (FGFRs), it can be concluded that FGFRs mediate the targeting of FGF-2 to lipid rafts. Consistently, FGF-2-GFP-secreting CHO cells do not exhibit increased proliferation activity. Externalization and deposition of biosynthetic FGF-2 in HSPG-containing protein clusters are independent processes, as a soluble secreted intermediate was demonstrated. The balance between intracellular FGF-2 and HSPG-bound secreted FGF-2 is shown not to be controlled by the availability of cell surface HSPGs, indicating that the FGF-2 secretion machinery itself is rate-limiting.
Key words: Non-conventional protein secretion, Non-classical protein export, Membrane translocation, Extracellular FGF-2, FGF-2 signalling, FGF-2 microdomains
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Introduction |
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In case of FGF-2, the sodium potassium ATPase (Na-K-ATPase) has been
proposed to play a role in FGF-2 export from cells. This conclusion is based
on the observation that FGF-2 export is partially inhibited in the presence of
ouabain (Florkiewicz et al.,
1998), a drug known to inhibit the Na-K-ATPase
(Jorgensen and Pedersen,
2001
). These results were further substantiated by data
demonstrating that FGF-2 export in the presence of ouabain recovers when an
ouabain-resistant mutant of the
-subunit of the Na-K-ATPase is
expressed (Dahl et al., 2000
).
However, it remains unclear as to whether the Na-K-ATPase plays a direct role
in the translocation mechanism of FGF-2 that results in the release into the
extracellular space.
While export of FGF-1 follows a non-conventional route as well, differences
from FGF-2 export have been reported such as increased secretion of FGF-1
following heat shock treatment (Jackson et
al., 1992; Jackson et al.,
1995
). S100A13, a member of the S100 protein family of
Ca2+-binding proteins (Donato,
2001
) has been shown to affect heat-shock-dependent release of
FGF-1 (Carreira et al., 1998
;
Landriscina et al., 2001
).
Other proteins secreted in an ER/Golgi-independent manner such as IL1ß
and the galectins are increasingly exported upon heat shock as well (for a
review, see Hughes, 1999
);
however, FGF-2 export is unaffected by this treatment
(Mignatti et al., 1992
). Other
differences concern the involvement of intracellular vesicles in the overall
export process of non-conventionally secreted proteins. Studies have shown
that whereas the galectins accumulate underneath the plasma membrane followed
by a release mechanism termed membrane blebbing that appears to involve
exosomes (Mehul and Hughes,
1997
), IL1ß is taken up by intracellular vesicles that have
been defined as an endo-lysosomal compartment
(Andrei et al., 1999
) that
fuses with the plasma membrane in order to release IL1ß. No such data
have been reported for either FGF-1 or FGF-2 [for reviews of the various kinds
of non-conventional protein secretion, see
Cleves, 1997
and
Hughes, 1999
(Cleves, 1997
;
Hughes, 1999
)]. In conclusion,
it may be that distinct mechanisms exist promoting ER/Golgi-independent export
of various secretory proteins from mammalian cells.
In the current study we introduce a novel assay that reconstitutes non-conventional secretion of biosynthetic FGF-2 in living cells. By using stable cell lines and flow cytometry, FGF-2-GFP export can be determined on a quantitative basis. FGF-2-GFP fusion proteins are expressed in a doxicyclin-dependent manner in CHO cells followed by their translocation to the extracellular surface of the plasma membrane. It is shown that both N- and C-terminal GFP-tagging is compatible with this process, which is demonstrated to occur by a controlled mechanism rather than by unspecific release. Based on its design the experimental system described will be useful both for studying the molecular mechanism of FGF-2 secretion and for high throughput screening for inhibitors of this process.
In the second part of this study we made use of the system described in
order to analyze the fate of biosynthetic FGF-2 (rather than exogenously added
FGF-2) following its translocation to the extracellular compartment. Secreted
FGF-2-GFP is shown to accumulate in large HSPG-containing protein clusters
that are distinct from caveolae-like lipid rafts. While CHO wild-type cells
lack high-affinity FGF receptors (Rusnati
et al., 2002), they do express the complex ganglioside
GM1, which has been shown to be required for FGF-2 signalling
(Rusnati et al., 2002
).
Therefore, it can be concluded that FGF receptors are required to target
HSPG-bound FGF-2 to caveolae-like lipid rafts in order to initiate FGF-2
signalling (Davy et al.,
2000
). Consistently, neither endogenous FGF-2-GFP exported to the
cell surface nor exogenously added recombinant FGF-2 stimulate CHO cells with
regard to cell proliferation. FGF-2 export and FGF-2 deposition in
HSPG-containing clusters are not tightly linked processes as we demonstrate a
soluble intermediate that is secreted into the culture medium. Moreover, under
the conditions applied, the availability of HSPGs on the cell surface is not a
rate-limiting step in the overall process of FGF-2 secretion. In conclusion,
the experimental system presented in this study reconstitutes the whole
pathway of FGF-2 biogenesis in living cells, including both non-conventional
secretion and deposition of FGF-2 in HSPG-containing microdomains on the
surface of CHO cells. By the use of CHO cells, FGF-2 export does not cause
autocrine FGF-2 signalling, which makes this system especially suited to the
study of the molecular mechanism of non-conventional FGF-2 secretion.
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Materials and Methods |
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Anti-GFP antibodies were generated by immunization of rabbits with recombinant N-terminally His6-tagged eGFP expressed in E. coli. The resulting anti-serum was incubated with His6-tagged eGFP coupled to epoxy sepharose (Amersham Pharmacia). Bound antibodies were eluted under acidic conditions according to standard procedures. In the same way, affinity-purified anti-FGF-2 antibodies were generated using recombinant N-terminally His6-tagged FGF-2 (18 kDa isoform).
Polyclonal rat anti-CD2 antibodies (LFA-2) as well as polyclonal rabbit
anti-caveolin-1 (N-20) antibodies were obtained from Southern Biotechnology
Associates and Santa Cruz Biotechnology, respectively. Phycoerythrin-coupled
anti-rabbit-IgG were from Molecular Probes. Polyclonal antibodies directed
against the cytoplasmic domain of the Golgi transmembrane protein p23 [#1402,
(Sohn et al., 1996)] were
kindly provided by Felix Wieland (Biochemie-Zentrum Heidelberg).
Generation of model cell lines for the reconstitution of FGF-2
secretion in living cells
CHO wild-type cells (ECACC; Ref. No. 85050302) were genetically modified by
the stable integration of cDNAs encoding the murine cation transporter MCAT-1
(Albritton et al., 1989;
Davey et al., 1997
), the
doxicyclin-sensitive transactivator rtTA2-M2
(Urlinger et al., 2000
), a
truncated version of the cell surface protein CD2
(Liu et al., 2000
), and one of
three reporter molecules (N-FGF-2-GFP-C, N-GFP-FGF-2-C and N-GFP-C) for each
cell line. Throughout this study, the open reading frame of enhanced GFP
(eGFP, Clontech) has been used for the generation of all cDNA constructs. In
the first step the open reading frame of MCAT-1 was subcloned into the vector
pcDNA3zeo+ (Invitrogen), which carries the zeocin-resistance gene.
Following transfection of CHO cells with pcDNA3zeo+-MCAT DNA, cells
were selected with medium containing 400 µg/ml zeozin. After about 14 days
individual clones were pooled to generate a heterogenous population of
zeozin-resistant CHO cells termed CHOMCAT. CHOMCAT cells
were then transduced with retroviral particles produced from HEK-293T cells by
co-transfection of the vectors pVPack-GP and pVPack-eco (Stratagene) as well
as pBI-CD2 (Liu et al., 2000
)
carrying a bicistronic construct encoding the Doxicyclin-sensitive
transactivator rtTA2-M2 and a truncated version of CD2 under the control of a
constitutive promotor based on the vector. After three days, 50,000
CD2-positive cells were isolated by FACS sorting using polyclonal
anti-CD2-antibodies detected by anti-rat secondary antibodies coupled to
Alexa488 (Dianova). This pool of CHO cells was termed CHOTAM2.
Following 7 days of incubation at 37°C, the corresponding population of
cells was transduced with retroviral particles carrying one of the three
reporter molecules mentioned above. In this case, the cDNA constructs
(N-FGF-2-eGFP-C, N-eGFP-FGF-2-C and N-eGFP-C) were subcloned into the vector
pREV-TRE2 (Clontech), which contains a Doxicyclin/transactivator-responsive
element for the initiation of mRNA formation. The open reading frames of FGF-2
and eGFP originated from the vector pT7T3D-Pac (FGF-2, IMAGE Consortium no.
1690025) and the vector pEGFP1 (eGFP, Clontech). Three days after retroviral
transduction, including 12 hours of incubation in the presence of 1 µg/ml
doxicyclin (Sigma), 50,000 cells from each transduction sample were isolated
by FACS sorting based on GFP-derived fluorescence. The three pools of cells
were incubated for 7 days at 37°C in the absence of doxicyclin followed by
the isolation of 50,000 cells from each population that did not display
GFP-derived fluorescence at this point. Each population was now cultured for
another 7 days at 37°C including 12 hours in the presence of 1 µg/ml
doxicyclin at the end of this procedure. Single cells were isolated by FACS
sorting based on GFP-derived fluorescence. These clones were propagated and
used for the preparation of frozen stocks. The newly generated clonal cell
lines were termed CHOFGF-2-GFP, CHOGFP-FGF2 and
CHOGFP, respectively, in order to reflect the reporter molecule
expressed.
Biochemical analysis of FGF-2-GFP secretion
CHOFGF-2-GFP, CHOGFP-FGF-2 and CHOGFP
cells were grown on 6-well plates for 36 hours at 37°C in the presence of
1 µg/ml doxicyclin and 125 µg/ml heparin. Where indicated the cells were
incubated in the presence of 25 µM ouabain (Sigma). The medium was removed
followed by the dissociation of the cells from the culture plates using a
protease-free buffer (Gibco, PBS-based cell dissociation buffer) supplemented
with 125 µg/ml heparin. Following sedimentation of cells, the supernatant
was combined with the original medium, diluted 1:10 in a Tris buffer (10 mM,
pH 7.4) containing 1 mM EDTA and 1% (w/v) Triton X-100. The cells were lysed
in the same buffer. Both the cellular extracts and the corresponding
supernatants were then subjected to FGF-2 affinity purification using heparin
sepharose (Amersham Pharmacia). Bound material was eluted with SDS sample
buffer followed by SDS-PAGE and western blot analysis using affinity-purified
anti-GFP antibodies and ECL detection (Amersham Pharmacia). In the case of
CHOGFP cells, aliquots from both the cellular and the medium
fractions were combined with sample buffer followed by SDS-PAGE and western
blot analysis in order to determine the distribution of the reporter molecule
(GFP) between cells and medium. Since the medium contains about 5-10
µg/µl total protein (derived from the fetal calf serum), it was not
possible to apply more than 1% of the medium (10 µl out of 1000 µl per
sample) to the gel. However, even after prolonged exposure GFP could not be
detected in the medium fraction.
Confocal microscopy
CHOFGF-2-GFP and CHOGFP cells were grown on glass
coverslips for 36 hours at 37°C in the presence of 1 µg/ml doxicyclin.
The cells were then processed, including paraformaldehyde fixation (3% w/v, 20
minutes at 4°C) without permeabilization, followed by antibody processing
as indicated. Alexa546-coupled secondary antibodies (Dianova) were used for
cell surface staining experiments. The specimens were mounted in Fluoromount G
(Southern Biotechnology Associates) and viewed with a Zeiss LSM 510 confocal
microscope.
Fluorescence activated cell sorting
CHOFGF-2-GFP, CHOGFP-FGF-2 and CHOGFP
cells were grown under the conditions indicated in the corresponding figure
legends. To detach the cells from the culture plates without using
protease-based protocols, cell dissociation buffer (Life Technologies) was
used to generate a cell suspension devoid of cell aggregates. Where indicated,
cells were treated with antibodies for 1 hour at 4°C on a rotating wheel.
Wash procedures were carried out by sedimenting the cells at 200
g for 5 minutes at 4°C. Prior to the FACS analysis,
propidium iodide (1 µg/ml) was added in order to detect damaged cells.
GFP- and phycoerythrin-derived fluorescence (Molecular Probes) was analyzed using a Becton Dickinson FACScan flow cytometer. Autofluorescence was determined by measuring non-induced cells that were not treated with phycoerythrin-coupled secondary antibodies. GFP-positive cells (i.e. grown in the presence of 1 µg/ml doxicyclin) that were not treated with antibodies were used to appropriately compensate the FL-2 channel used to detect phycoerythrin-derived fluorescence.
Isolation of detergent-insoluble microdomains
CHOFGF-2-GFP cells were grown on large culture plates (15 cm
diameter) for 36 hours in the presence of doxicyclin (1 µg/ml). The cells
were washed twice with PBS followed by the addition of PBS supplemented with
10% (w/v) sucrose. After dissociation from the culture plates using a rubber
policeman, cell disruption was achieved using a Balch homogenizer
(Balch and Rothman, 1985). The
resulting suspension was subjected to differential centrifugation at 1000
g and 5000 g, respectively. The 5000
g supernatant was loaded onto a 20% sucrose cushion followed
by ultracentrifugation at 100,000 g for 60 minutes at 4°C.
The resulting membrane sediment represents a microsomal membrane fraction
containing intracellular as well as plasma membranes. The preparation of
detergent-soluble and -insoluble fractions as well as the flotation analysis
using sucrose gradients were performed as described
(Gkantiragas et al.,
2001
).
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Results |
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The characterization of CHO clones derived from the procedure described
above is shown in Fig. 1 (see
Materials and Methods). As depicted in Fig.
1A, a PCR analysis of genomic DNA revealed the presence of DNA
fragments of the expected size (lanes 1-3) compared with the vector DNA (lanes
4-6) that was used for retroviral transduction. In
Fig. 1B, total extracts from
each clone were separated on SDS gels followed by western blotting and
immunodetection using affinity-purified anti-GFP antibodies. For each clone,
an immunoreactive band with an apparent migration behaviour corresponding to
about 45 (FGF-2-GFP, GFP-FGF-2) and 26 (GFP) kDa, respectively, was observed
when cells were incubated in the presence of doxicyclin
(Fig. 1B, lanes 2,4,6). By
contrast, no signal was observed when cells were incubated in the absence of
doxicyclin (Fig. 1B, lanes
1,3,5). These results were confirmed by fluorescence microscopy
(Fig. 1C-H) and flow cytometry
(Fig. 1I-K). Upon incubation of
each CHO clone in the presence of doxicyclin, the whole cell population
displayed increased GFP fluorescence (50-100-fold), as determined by flow
cytometry. Based on conventional fluorescence microscopy at low magnification,
FGF-2-GFP and GFP-FGF-2 display both nuclear and cytoplasmic staining. This is
also the case for GFP; however, the ratio of nuclear to cytoplasmic staining
is significantly lower compared with that of FGF-2-GFP and GFP-FGF-2.
|
Biochemical analysis of FGF-2 fusion protein secretion
To functionally characterize the reporter cell lines with regard to
non-classical secretion, we conducted biochemical experiments to assess
extracellular localization of biosynthetic FGF-2-GFP fusion proteins (see
Materials and Methods). For this purpose, cells were exposed to doxicyclin for
48 hours at 37°C in the presence of heparin (to prevent FGF-2 binding to
plasma-membrane-associated HSPGs), followed by their dissociation from the
culture plates by using a protease-free protocol. Residual cell
surface-associated FGF-2 was released by heparin and the corresponding
cell-free supernatant was combined with the original growth medium. In
parallel, detergent extracts from the cellular fractions were prepared.
Following dilution, FGF-2 fusion proteins were affinity-purified from both the
cellular and the medium fractions using heparin sepharose
(Klagsbrun et al., 1987). As
depicted in Fig. 2A, both
FGF-2-GFP (derived from the cell line CHOFGF-2-GFP) and GFP-FGF-2
(derived from the cell line CHOGFP-FGF-2) were readily detectable
in the supernatant of cultured cells (lanes 2 and 4, respectively). Since 1%
of the total material derived from cells (lanes 1 and 3, respectively) and 15%
of the total material derived from the cell supernatant (lanes 2 and 4,
respectively) were applied to the gel, up to 10% of the FGF-2-GFP fusion
proteins expressed were found to be secreted under the experimental conditions
applied. To analyze the specificity of FGF-2 export, the corresponding control
cell line (CHOGFP) was used, which expressed GFP without being
fused to FGF-2. As can be deduced from lanes 5 (cells) and 6 (supernatant) of
Fig. 2A, GFP could not be
detected in the supernatant of CHOGFP cells. As depicted in
Fig. 2B,
CHOFGF-2-GFP cells that were grown in the presence of ouabain, a
known inhibitor of FGF-2 secretion
(Florkiewicz et al., 1998
;
Dahl et al., 2000
), FGF-2-GFP
export to the culture medium was markedly reduced [compare the ratio of the
relative amounts of FGF-2-GFP in lanes 1 and 2 (control) to the corresponding
ratio of lanes 3 and 4 (ouabain)]. These data establish that export of
FGF-2-GFP fusion proteins from CHO cells is a specific transport mechanism
that is compatible with both N-and C-terminal GFP tagging.
|
Determination of FGF-2 translocation to the cell surface based on
flow cytometry
To analyze FGF-2 export to the cell surface in living cells, we have
established a novel assay that is based on flow cytometry (see Materials and
Methods). CHOFGF-2-GFP cells were incubated for 18 hours at
37°C in the presence of doxicyclin. Following dissociation from the
culture plates using a protease-free buffer, cells were incubated at 4°C
under native conditions with affinity purified antibodies directed against
either GFP or FGF-2. Primary antibodies were detected by secondary antibodies
coupled to phycoerythrin (PE) in order to visualize cell surface localization
by PE-derived fluorescence. As depicted in
Fig. 3, GFP-derived
fluorescence rigorously depends on the presence of doxicyclin as shown by dot
blots (compare Fig. 3A and 3B)
as well as the corresponding histogram
(Fig. 3F). Under all
experimental conditions, the degree of doxicyclin-dependent GFP fluorescence
was found to be similar (Fig.
3F). Accordingly, PE-derived fluorescence corresponding to
cell-surface-localized FGF-2-GFP could only be observed when FGF-2-GFP
expression was induced by doxicyclin, which demonstrated the monospecificity
of the affinity-purified anti-GFP antibodies used. Similar results were
obtained with affinity-purified anti-FGF-2 antibodies (data not shown).
|
To further establish that PE-derived fluorescence exclusively represented cell surface localization of FGF-2-GFP, we conducted experiments where native cells were treated with trypsin prior to the FACS analysis (Fig. 3E,G). In addition, experiments were carried out where cells were incubated with heparin in order to elute FGF-2-GFP associated with plasma-membrane-bound heparan sulfate proteoglycans (Fig. 3D,G). In both cases, the majority of PE-derived fluorescence could be removed from the cells demonstrating that the signal was derived from a FGF-2-GFP population associated with the outer surface of the plasma membrane.
FGF-2 translocation to the cell surface depends on the FGF-2 domain
of FGF-2-GFP fusion proteins
To analyze whether the FGF-2 domain is required for the translocation to
the cell surface of FGF-2-GFP fusion proteins, we compared the various CHO
cell lines described in Fig. 1
with regard to their ability to translocate the respective reporter molecule
to the outer surface of the plasma membrane. The three CHO clones were
incubated in the presence of doxicyclin for 18 hours (see Materials and
Methods) followed by antibody processing as described in the legend of
Fig. 3. As shown in
Fig. 4A, total GFP-derived
fluorescence differed in the three cell lines. When the
autofluorescence-corrected GFP signal of CHOFGF-2-GFP cells was set
to 100%, CHOGFP-FGF-2 and CHOGFP cells displayed a
1.4-fold and 2.3-fold higher fluorescence, respectively, compared with that of
CHOFGF-2-GFP cells. By contrast, cell surface localization of the
respective reporter molecules as measured by PE-derived fluorescence
(Fig. 4B) was observed only
with CHOFGF-2-GFP and CHOGFP-FGF-2 cells. Again, the
autofluorescence-corrected signal of CHOFGF-2-GFP cells was set to
100% and shown to be 14-fold higher compared with CHOGFP cells. The
cell surface signal of CHOGFP-FGF-2 was slightly higher than that
of CHOFGF-2-GFP cells; however, the expression level between these
two cell lines also differed to a similar extent. These data establish that
the translocation process depends on the FGF-2 domain of the reporter
molecules, which is consistent with the biochemical data presented in
Fig. 2. Moreover, it is shown
that both N-terminal and C-terminal GFP-tagging of FGF-2 is compatible with
the translocation process in CHO cells.
|
Characterization of FGF-2 translocation to the cell surface
In a series of experiments depicted in
Fig. 5, we characterized the
FACS-based FGF-2 translocation assay with regard to kinetics, unspecific
release as well as sensitivity to ouabain, a known inhibitor of FGF-2 export
(Florkiewicz et al., 1998;
Dahl et al., 2000
). Following
induction of protein expression in the presence of doxicyclin, the amount of
FGF-2 appearing on the cell surface increased in a linear manner for up to 48
hours (Fig. 5A). Afterwards,
the signal turned into saturation, indicating that steady-state conditions
were reached. To rule out that the material found on the cell surface was
derived from damaged cells, experiments were carried out where
CHOFGF-2-GFP cells cultured in the absence of doxicyclin were
incubated with various amounts of a supernatant derived from homogenized
CHOFGF-2-GFP cells cultured in the presence of doxicyclin for 48
hours at 37°C (see Materials and Methods). As shown in
Fig. 5B, the addition of 0,
2.5, 5, 7.5 or 10% (based on cell number; lanes 1-5) of a supernatant derived
from a membrane-free supernatant of homogenized cells to cells not expressing
the reporter molecule accounted for up to 40% of the secretion signal observed
with CHOFGF-2-GFP cells (lane 6). During all FACS experiments the
amount of dead cells was monitored by the addition of propidium iodide (PI), a
low molecular weight dye that enters only damaged cells. Typically, about 2-3%
of the total cell population was found to be positive for PI. Thus, the
population of FGF-2-GFP found on the cell surface (lane 6) cannot be derived
from damaged cells but rather has been secreted by a specific transport
mechanism. This conclusion is further substantiated by the observation that
the appearance of FGF-2-GFP on the cell surface can be partially inhibited by
ouabain (Fig. 5B, lanes 7,8).
The results obtained by this FACS analysis are consistent with the biochemical
secretion experiments shown in Fig.
2.
|
The amount of HSPGs on the cell surface does not limit FGF-2
translocation efficiency
To analyze the influence of HSPG levels on the cell surface with regards to
FGF-2 secretion efficiency we conducted experiments where recombinant
His6-FGF-2 (see Materials and Methods) was titrated into the medium
of both doxicyclin-induced and non-induced CHOFGF-2-GFP cells. As
depicted in Fig. 6, the FGF-2
binding capacity of the cells was not saturated under conditions where
FGF-2-GFP expression and externalization was induced by doxicyclin. Based on
these results, the binding capacity of CHOFGF-2-GFP cells for FGF-2
is at least ten times higher than the amount of FGF-2-GFP externalized under
the conditions described. Therefore, the FGF-2 secretion signal observed is
not limited by the amount of HSPGs available on the cell surface but rather is
a precise measure of the efficiency of the export machinery. Thus, it can be
concluded that the overall process of FGF-2 externalization is not governed by
a balance of intracellular FGF-2 versus extracellular HSPG-bound FGF-2.
Rather, the FGF-2 export machinery appears to be rate-limiting under the
conditions applied.
|
Analysis of FGF-2 translocation to the cell surface by confocal
microscopy
To verify the results obtained by FACS analysis using an independent
method, we conducted experiments based on immunofluorescence confocal
microscopy (see Materials and Methods). CHOFGF-2-GFP and
CHOGFP cells were grown on glass coverslips for 24 hours in the
absence or presence of doxicyclin. Following fixation (without
permeabilization) and antibody processing using affinity-purified anti-GFP
antibodies, the various samples were analyzed by confocal microscopy. As shown
in Fig. 7B,F,
CHOFGF-2-GFP cells incubated in the presence of doxicyclin
displayed both GFP-derived intracellular fluorescence (both the nucleus and
the cytoplasm were found to be positive for FGF-2-GFP) and
plasma-membrane-associated Alexa546-derived fluorescence. By contrast,
CHOFGF-2-GFP cells incubated in the absence of doxicyclin
(Fig. 7A,E) neither showed
GFP-derived fluorescence nor cell surface staining, demonstrating
monospecificity of the antibodies used. Consistent with the FACS experiments
shown in Fig. 3, FGF-2-GFP cell
surface staining can be removed by incubation of the cells with heparin prior
to fixation (Fig. 7C,G), demonstrating that exported biosynthetic FGF-2-GFP associates with HSPGs on
the extracellular surface of CHO cells. Moreover, cell surface staining could
not be observed with CHOGFP cells incubated in the presence of
doxicyclin (Fig. 7D,H). These
results are fully consistent with our FACS analysis establishing specific
translocation from the cytosol to the outer surface of the plasma membrane of
FGF-2-GFP.
|
Biosynthetic FGF-2-GFP exported to the extracellular plasma membrane
surface is targeted to non-lipid raft microdomains
To assess the structural organization of cell-surface-localized FGF-2-GFP
in more detail, we conducted immunofluorescence confocal microscopy at high
magnification. As shown in Fig.
8A (merged image of 16 confocal planes), FGF-2-GFP did not display
a homogenous staining of the plasma membrane but rather appeared in bright
spots representing distinct microdomains. As shown in
Fig. 8B, these microdomains
represent structures exclusively localized to the cell surface since
sequential scanning of focal planes (one of which is shown in
Fig. 8B) revealed the absence
of any intracellular staining. Since cell-surface-associated FGF-2-GFP could
be eluted with heparin (Fig.
7G), these microdomains also contain HSPGs.
|
To analyze the nature of these microdomains with regard to lipid rafts, we
characterized the detergent solubility of plasma membrane-associated
FGF-2-GFP. A plasma membrane-containing microsomal membrane fraction freed of
cell debris, nuclei and soluble cytosolic proteins was isolated by
differential centrifugation (see Materials and Methods). These membranes were
solubilized in a Triton X-100-containing buffer at 4°C. Resuspended
membranes were either subjected to ultracentrifugation in order to sediment
detergent-insoluble complexes or adjusted to 40% (w/v) sucrose followed by
flotation in a sucrose density gradient in order to separate lipid-associated
protein complexes from detergent-soluble material. As shown in
Fig. 9A, FGF-2-GFP almost
exclusively appeared in the soluble fraction (lane 1) of detergent-treated
membranes. As control proteins, the Golgi-localized transmembrane protein p23
(Sohn et al., 1996) was used
as a non-lipid raft marker (Gkantiragas et
al., 2001
) and the plasma-membrane-localized protein caveolin-1
was used as a classical lipid raft marker
(Rothberg et al., 1992
;
Kurzchalia and Parton, 1999
).
Consistently, p23 could be detected only in the soluble fraction (lane 1),
whereas caveolin was almost exclusively found in the insoluble fraction (lane
2). These results were further substantiated by the results from the flotation
experiment performed with detergent-treated membranes. As shown in
Fig. 9B, caveolin-1 could be
detected in the light fractions [corresponding to about 15% (w/v) sucrose] of
the flotation gradient. By contrast, both p23 and FGF-2-GFP were exclusively
localized to the bottom fractions of the gradient demonstrating that the
microdomains observed by confocal microscopy are not related to lipid rafts. A
formal possibility would be that a potential association of FGF-2-GFP with
lipid rafts cannot be detected because the interaction of FGF-2-GFP with cell
surface HSPGs is detergent-sensitive which, in turn, would cause FGF-2-GFP to
appear in the supernatant of detergent-treated membranes. However, as
demonstrated in Fig. 2,
FGF-2-GFP can be affinity-purified from cellular detergent extracts by using
heparin-sepharose, a method that mimics the interaction of FGF-2 with heparan
sulfate proteoglycans (Burgess and Maciag,
1989
). Therefore, FGF-2-GFP-positive microdomains found on the
cell surface of CHOFGF-2-GFP cells are not related to lipid
rafts.
|
Since FGF-2 signalling has been demonstrated to originate from
caveolae-like lipid rafts (Davy et al.,
2000), we analyzed whether exported FGF-2-GFP or exogenously added
recombinant FGF-2 are able to stimulate cell proliferation. As shown in
Fig. 10, neither secreted
FGF-2-GFP nor recombinant FGF-2 added to the culture medium induce cell
proliferation. These data are consistent with the fact that, despite
expressing HSPGs and the FGF-2 co-receptor GM1
(Rusnati et al., 2002
), CHO
wild-type cells do not express high-affinity FGF receptors
(Rusnati et al., 2002
).
Therefore, FGF receptors appear to be required to target FGF-2 to
caveolae-like lipid rafts involved in FGF signalling.
|
Intercellular spreading of exported biosynthetic FGF-2-GFP
To distinguish a translocation mechanism that involves a soluble
intermediate between export and binding to proteoglycans from an integrated
process where the export machinery directly delivers FGF-2 to the proteoglycan
binding site, we conducted experiments where FGF-2-GFP-expressing cells were
cultured together with cells lacking the FGF-2-GFP reporter construct
(CHOMCAT-TAM2 cells; see Materials and Methods). In this way we
were able to analyze intercellular spreading of FGF-2 between different
populations of CHO cells. As depicted in
Fig. 11B,C,
CHOMCAT-TAM2 cells (labeled with an asterisk) not expressing the
FGF-2-GFP fusion protein were found to be positive for cell-surface-localized
FGF-2-GFP. To verify these observations using an independent approach we
prepared a 100,000 gav supernatant from doxicyclin-induced
homogenized CHOFGF-2-GFP cells, which was added to
CHOMCAT-TAM2 cells, and analysed the cell surface staining using
confocal microscopy. FGF-2-GFP appears in bright spots on the cell surface
closely resembling the localization of secreted FGF-2-GFP (data not shown).
These results demonstrate that, following secretion of FGF-2-GFP, a soluble
intermediate exists that subsequently accumulates in cell surface microdomains
based on the interaction with HSPGs.
|
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Discussion |
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---|
In the first part of this study, we implemented a novel experimental system
that will greatly facilitate studies on the molecular machinery of FGF-2
secretion. A key aspect was to reconstitute FGF-2 secretion in living cells
based on a read-out method that provides a precise and quantitative analysis
of this process. Moreover, by using FGF-receptor-deficient CHO cells,
secondary effects based on FGF-2-induced signal transduction can be avoided.
We have established genetically altered cell lines that stably express N- and
C-terminally GFP-tagged FGF-2 in a doxicyclin-dependent manner. Moreover,
these cells express the mouse orthologue of the cationic amino acid
transporter [MCAT-1 (Albritton et al.,
1989; Davey et al.,
1997
)], whose cell surface expression makes non-mouse cells
permissive to ecotropic retroviruses
(Albritton et al., 1989
). This
strategy allows the efficient retroviral transfer of cDNA libraries into
MCAT-expressing mammalian cells of any origin. Following stable integration of
the MCAT-1 cDNA using conventional methods, we took advantage of this approach
by introducing both a doxicyclin-sensitive transactivator
(Urlinger et al., 2000
) and
the various FGF-2-GFP cDNA constructs by using a retrovirus containing an
ecotropic host-range envelope protein. In this way the cDNA constructs were
stably integrated into the genomic DNA of the host cells without the need for
cell selection based on antibiotics.
The resulting clonal cell lines can be transduced with retroviral particles
containing specific cDNAs or cDNA libraries with high efficiency and express
FGF-2-GFP fusion proteins in a strictly doxicyclin-dependent manner. These
cells were functionally characterized with regard to non-conventional
secretion of the FGF-2-GFP reporter molecules. Based on a robust and efficient
FACS-based assay, it is possible to quantitatively assess the amount of FGF-2
released to the extracellular space in living cells. This is because,
following its secretion, FGF-2-GFP binds to the extracellular surface of the
plasma membrane, where it is associated with proteoglycans of the heparan
sulfate type (Burgess and Maciag,
1989; Pellegrini et al.,
2000
; Trudel et al.,
2000
). This allows specific detection of secreted FGF-2-GFP with
affinity purified anti-GFP- or anti-FGF-2 antibodies under native conditions
based on flow cytometry. In this way, GFP-derived fluorescence is used to
normalize the overall expression of the reporter molecule under various
experimental conditions, whereas the secreted population can be exclusively
detected on the cell surface with antibodies coupled to a second fluorophore
such as phycoerythrin.
In the second part of this study, we made use of the experimental system described to study the fate of biosynthetic (i.e. endogenous) FGF-2-GFP following translocation to the extracellular compartment. FGF-2-GFP is shown to accumulate in large macromolecular clusters that appear as bright spots on the cell surface. FGF-2-GFP association with these structures is mediated by HSPGs, as heparin treatment causes a loss of FGF-2-GFP staining on the cell surface. To investigate whether FGF-2 export and deposition in HSPG-containing microdomains are tightly linked processes we analyzed the mode of delivery of FGF-2-GFP to HSPGs following its externalization. A soluble intermediate is demonstrated that allows FGF-2-GFP to spread between different populations of cultured cells. Moreover, FGF-2-GFP prepared as a cell-free supernatant from homogenized CHOFGF-2-GFP cells can associate with non-expressing cells thereby forming morphologically similar microdomains on their surfaces. In conclusion, FGF-2 externalization and deposition in cell surface microdomains do not occur through an integrated process that would restrict cell surface deposition to FGF-2-secreting cells. Rather, a soluble intermediate is released and eventually accumulates in HSPG-containing protein clusters. These data are consistent with our finding that the FGF-2 binding capacity mediated by HSPGs does not influence the balance of intracellular FGF-2 versus extracellular HSPG-bound FGF-2. Rather, the cell surface signal detected provides a precise measure of FGF-2 externalization that is not limited by the availability of HSPGs. Therefore, the FGF-2 export machinery is rate-limiting under the conditions applied.
The presence of FGF-2-GFP in discrete microdomains on the cell surface
implied that these structures might represent protein complexes involved in
FGF-2 signal transduction. In this context, Davy et al. reported that FGF-2
signalling originates from caveolae-like lipid rafts on the cell surface
(Davy et al., 2000). A
functional FGF-2 signal transduction complex consists of FGF-2, HSPGs, the
co-receptor GM1 and high-affinity FGF receptors
(Rusnati et al., 2002
). CHO
wild-type cells do synthesize HSPGs and GM1; however, they do not
express high-affinity FGF receptors
(Rusnati et al., 2002
).
Therefore, it was interesting to study the biophysical properties of the
FGF-2-GFP-positive microdomains observed on the cell surface of CHO cells.
Based on detergent solubility combined with flotation experiments in sucrose
gradients, we can exclude that the FGF-2-positive clusters observed are
related to lipid rafts. Therefore, initial binding of FGF-2 to HSPGs does not
result in the correct targeting to caveolae-like lipid rafts, where FGF-2
signalling is initiated. Rather, FGF receptors are required to direct the core
complex of FGF-2 signaling to lipid rafts. Accordingly, upon
doxicyclin-induced FGF-2-GFP expression and externalization,
CHOFGF-2-GFP cells do not appear to be significantly stimulated
with respect to cell proliferation. Therefore, the large FGF-2-GFP- and
HSPG-containing cell surface clusters appear to represent signalling complex
precursors that, in the presence of high affinity FGF receptors, are converted
into functional signalling complexes. This transition appears to be
accompanied by a targeting of the FGF-2 signalling complex to caveolae-like
lipid rafts.
The FACS-based FGF-2 secretion assay described in this study is a powerful
tool for the analysis of the molecular machinery mediating FGF-2 export. For
example, the systematic testing of candidate proteins (e.g. identified by
interaction studies or genetic screening in mammalian cells) can be carried
out by transiently inhibiting their biosynthesis based on RNA interference
(Elbashir et al., 2001). In
this context, a considerable advantage of the FGF-2-GFP-based system is that
total protein expression (GFP-derived fluorescence) and secreted FGF-2-GFP
(PE-derived cell surface staining) can be measured independently. Therefore, a
phenotype determined by PE-derived cell surface staining can be corrected by
normalization based on the degree of total FGF-2-GFP expression. Due to the
lack of FGF receptors in CHO cells, another unique feature of the experimental
system described is the uncoupling of FGF-2 externalization from FGF-2
signalling. Therefore, FGF-2 export can be studied without the risk of
secondary effects provoked by the action of the secreted product.
Another obvious application is a systematic high throughput screening for
inhibitors (e.g. derived from natural compound libraries) of FGF-2 secretion
and the subsequent functional identification of their cellular targets. Given
the biological function of FGF-2 as a direct stimulator of tumor angiogenesis
(Bikfalvi et al., 1997),
inhibitors of FGF-2 secretion might have strong biomedical implications as
potential lead compounds for the development of anti-angiogenic drugs.
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Acknowledgments |
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Footnotes |
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References |
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