From the
Cholera toxin (CT) consists of a pentameric B subunit which
binds with high affinity to ganglioside GM
Gangliosides are major constituents of the neuronal plasma
membrane (PM)
At the
cell surface, CT is concentrated in non-coated membrane invaginations
(9) recently identified as caveolae
(10) . However, some
controversy exists as to whether CT (and other molecules) are
internalized from caveolae, since caveolae have long half-lives and
have not been directly observed to ``pinch off'' from the
PM
(11) . Moreover, CT rapidly appears in the same endosomal
compartment as that labeled by
In
order to probe further the mechanism of CT internalization, we have
studied the effect of cationic amphiphilic drugs (CADs) such as
chlorpromazine, imipramine, and sphingosine on the internalization of
CT and on the elevation of cAMP in cultured hippocampal neurons. CADs
have previously been shown to disrupt receptor recycling
(16, 17) by stimulating the recruitment of the AP-2 adaptor protein
(18) and of clathrin to an uncoated, late endosomal
compartment
(19) . We now demonstrate that CADs reversibly
inhibit the internalization of CT to the Golgi apparatus and the
CT-stimulated elevation of cAMP.
The
specificity of CT binding to GM
When 3-day-old neurons were labeled
with Rh-CT for 30 min at 16 °C, the cell surface was brightly
labeled (see Fig. 1A). No intracellular fluorescence was
observed if the temperature was maintained at 16 °C. However, upon
subsequent warming to 37 °C for 30 min, Rh-CT accumulated in a
perinuclear area (Fig. 2A) that corresponded to the
Golgi apparatus, as demonstrated by double-labeling studies using
C
We propose the following scheme to describe CT
endocytosis (Fig. 5A) and the effects of CADs
(Fig. 5B). CT binds to GM
In
summary, we demonstrate that the internalization of CT to the Golgi
apparatus proceeds via an endosomal compartment that can acquire
clathrin coats upon treatment with CADs. It will be of interest to
study the effect of CADs on the internalization of other molecules to
the Golgi apparatus, particularly other toxins (i.e. Shiga
toxin
(31) ) that bind to glycosphingolipids on the cell surface.
Five- to six-day-old neurons were
incubated with 5 nM CT for 30 min at 13-16 °C prior
to incubation with either 25 µM chlorpromazine or 25
µM H7 at 37 °C. At the indicated times, cells were
removed from the coverslips by scraping, and levels of cAMP determined
as described under ``Experimental Procedures'' in both cells
and medium. cAMP levels in the medium were less than 6-7% of that
measured in cells and are not included in the table. Results are shown
as fmol/coverslip. The relative stimulation of cAMP by CT is compared
to time = 0 min and shown in parenthesis. Results are mean
± S.D., n = 6 for control cells and n = 3 for chlorpromazine and H7-treated cells.
We thank Rivi Zisling for expert help in preparing
hippocampal cultures and Professor Nava Dekel for help with the cAMP
assay.
, and an A
subunit which stimulates adenylate cyclase, resulting in the elevation
of cAMP. We now examine the effect of cationic amphiphilic drugs (CADs)
on the internalization of rhodamine (Rh)-CT in cultured hippocampal
neurons. CADs have recently been shown to inhibit receptor recycling by
disrupting the assembly-disassembly of clathrin at the plasma membrane
and on endosomes (Wang, L.-H., Rothberg, K. G., and Anderson, R. G.
W.(1993) J. Cell Biol. 123, 1107-1117). Rh-CT was
internalized by an energy- and temperature-dependent (presumably
vesicular) mechanism to the Golgi apparatus. Internalization to the
Golgi apparatus was completely but reversibly blocked by CADs, and the
ability of CT to stimulate the elevation of cAMP was significantly
reduced. In control cells, cAMP levels were elevated 2.3-fold after 20
min of incubation with CT, but in CAD-treated cells cAMP levels were
only elevated 1.3-fold. The effect of CADs on CT internalization was
not due to a direct effect of CADs on the Golgi apparatus. Our data
demonstrate that CADs inhibit vesicular transport of CT to the Golgi
apparatus and imply that the sorting of CT to the Golgi apparatus
occurs in the same endosomal compartment involved in sorting recycling
receptors to the plasma membrane, since both pathways are inhibited by
CADs.
(
)
where they have been postulated
to play a variety of functions
(1) . A large amount of
information is available about ganglioside metabolism
(2) . In
contrast, much less is known about the intracellular transport of
gangliosides (3), due in large part to the lack of suitable probes to
monitor ganglioside traffic. In the case of ganglioside GM
,
some information about the pathways and regulation of transport has
been obtained using cholera toxin (CT). CT consists of a pentameric B
subunit which binds with high affinity to ganglioside GM
and an A-subunit comprising two peptides, A
and
A
, linked by a disulfide bond. The A
-subunit
activates adenylate cyclase via a stimulatory G protein, G
(4). Fluorescent
(5) , horseradish
peroxidase-conjugated
(6) , radioactive
(7, 8) ,
and gold derivatives
(9, 10) of either CT or CT-B (the
B-subunit of cholera toxin) have been used to trace the internalization
of CT from the cell surface to intracellular locations by both
biochemical techniques and by light and electron microscopy.
-macroglobulin
(8) , a ligand that enters cells
via clathrin-coated pits. CT is not excluded from clathrin-coated
pits
(9, 10) , and some evidence exists for the
internalization of a biotinylated derivative of GM
from
clathrin-coated pits (12). Despite the lack of consensus on the
mechanism of internalization of CT, it has recently been unambiguously
shown that a functional Golgi apparatus is necessary for generation of
the A
peptide, which results in stimulation of adenylate
cyclase and associated toxicity ((13, 14), see also Ref. 15)).
Materials
Timed pregnant rats (Wistar) were
obtained from the Weizmann Institute Breeding Center. Fumonisin B was purchased from the Division of Food Science and Technology,
CSIR, Pretoria, South Africa. CT was from Calbiochem, La Jolla, CA.
N-{6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl}-D-erythro-sphingosine
(C
-NBD-ceramide) and
N-[5-(5,7-dimethylBodipy)-1-pentanoyl]-D-erythro-sphingosine
(C
-Bodipy-ceramide) were from Molecular Probes Inc.,
Eugene, OR. Chlorpromazine, imipramine, sphingosine, H7, and
3-isobutyl-1-methylxanthine were from Sigma. Ganglioside GM
was supplied by Fidia Research Laboratories, Abano T., Italy.
Protein A and an anti-cAMP antibody were from BioMakor, Rehovot,
Israel. Acetic anhydride and triethylamine were obtained from BDH,
Dorset, United Kingdom. Adenosine 3`,5`-cyclic
3-[
I]iodotyrosine methyl (2000 Ci/mmol) was
obtained from Amersham International plc, Amersham, U.K.
Incubation of Hippocampal Neurons with a Fluorescent
Analog of CT
Hippocampal neurons cultured at low density
(12,500-25,000 cells/13-mm glass coverslip)
(20, 21) were incubated with Rhodamine (Rh)-conjugated cholera toxin
(Rh-CT) (containing the A- and B-subunits) or the B-subunit of CT
(Rh-CT-B) (prepared as in Ref. 5), for 20-30 min at 13-16
°C in HEPES-buffered medium (minimal essential medium containing 50
mM HEPES (pH 7.3), 4 mM NaHCO, 11 mg/ml
pyruvic acid, 1 mM glutamine, 0.6% (w/v) glucose) and 0.1%
(w/v) bovine serum albumin, and then warmed to 37 °C for various
times. In one experiment (see Fig. 1C), neurons were
incubated with exogenous ganglioside GM
(5 nM).
GM
was dissolved in ethanol, dried under N
, and
dissolved in phosphate-buffered saline followed by sonication.
Figure 1:
Rh-CT labels cell
surface GM in cultured hippocampal neurons. Neurons were
incubated with 10 µM fumonisin B
immediately
after transferring the coverslips to multiwell dishes containing
cocultures of glial cells. After 3 days in culture, coverslips were
removed and incubated with 5 nM Rh-CT in HEPES-buffered medium
for 30 min at 16 °C. Rh-CT is barely detectable at the cell surface
after fumonisin B
-treatment (B), but is detected
on the cell body, dendrites and axons of control cells (A).
Addition of exogenous GM
(5 nM) to the medium 18 h
before incubation with Rh-CT restores the binding of Rh-CT at the cell
surface (C). The bar corresponds to 20
µm.
Incubation of Hippocampal Neurons with
C
Neurons were washed in
HEPES-buffered medium, cooled to 13-16 °C and incubated with
either a bovine serum albumin/C-NBD-ceramide and
C
-Bodipy-ceramide
-NBD-ceramide or a bovine
serum albumin/C
-Bodipy-ceramide complex (molar ratio 1:1,
in 10 mM HEPES (pH 7.3)) for 30-40 min, washed, and
further incubated for various times at 37 °C; both ceramide analogs
specifically label the Golgi apparatus
(22, 23) . For
long incubations, neurons were placed in Multiwell dishes containing
glial cocultures in the CO
incubator. Prior to observation,
neurons were washed and ``back-exchanged'' with 0.34% or 2%
(w/v) defatted-bovine serum albumin (3
10 min, 37 °C) for
C
-NBD-ceramide and C
-Bodipy-ceramide,
respectively.
Determination of cAMP
Neurons cultured at high
density (230,000 cells/24-mm glass coverslip)
(24) were used for
cAMP determination. 5-6-day-old neurons were washed in
HEPES-buffered medium for 15 min at 37 °C. Neurons were
subsequently incubated for 5 min at 13-16 °C in
HEPES-buffered medium containing 0.2 mM
3-isobutyl-1-methylxanthine and 0.01% (w/v) bovine serum albumin prior
to addition of 5 nM CT for 30 min at 13-16 °C. Drugs
were added to the medium for 5 min prior to warming to 37 °C. After
various times, cells were removed from the coverslips by scraping with
a rubber policeman into 7 mM sodium acetate buffer (pH 6.5)
containing 0.2 mM 3-isobutyl-1-methylxanthine, lyophilized,
and resuspended in distilled water to give a final concentration of 70
mM sodium acetate (pH 6.5). Cells were frozen in liquid
nitrogen and thawed four times to completely release cAMP into the
buffer; samples were stored at -80 °C. cAMP was determined by
radioimmunoassay as described
(25) .
Microscopy
Fluorescence microscopy was performed
using Plan Apochromat 63X/1.4 and Plan Neofluar 40X/1.3 oil objectives
of a Zeiss Axiovert 35 microscope equipped with filters for rhodamine
or Bodipy fluorescence. Cells were photographed using a Contax 167MT
camera and Kodak Tmax p3200 film.
RESULTS
The Internalization of Rh-CT
Initial experiments
were performed to characterize the mechanism of internalization of CT
in cultured hippocampal neurons. These neurons are a particularly
suitable model to analyze CT internalization since they contain high
levels of ganglioside GM. Moreover, the rate of
internalization of CT to the Golgi apparatus is rapid on early days in
culture and decreases as neurons mature.
(
)
was determined by
incubating neurons with fumonisin B
, a specific inhibitor
of sphingolipid synthesis
(20, 26) . After 3 days of
incubation with fumonisin B
, levels of Rh-CT binding to the
PM were significantly reduced (Fig. 1B), whereas in
control cells, Rh-CT labeled the somatodendritic and axonal PMs
(Fig. 1A). Addition of exogenous GM
(5
nM) to fumonisin B
-treated cells
(Fig. 1C) restored the binding of CT. Identical results
were obtained using either intact Rh-CT or Rh-CT-B. Preincubation with
a 10-fold excess of unlabeled CT also significantly reduced Rh-CT
labeling to the cell surface.
-NBD-ceramide (not shown). Preincubation with sodium azide
(5 mM) and 2-deoxyglucose (50 mM) abolished
internalization to the Golgi apparatus (Fig. 2B) at 37
°C. Together, these results demonstrate that Rh-CT is internalized
when bound to GM
via an energy- and temperature-dependent,
presumably vesicular mechanism to the Golgi apparatus in cultured
hippocampal neurons.
Figure 2:
Rh-CT
is internalized by an energy-dependent mechanism to the Golgi
apparatus. Six-day-old neurons were preincubated for 15 min at 37
°C with sodium azide (5 mM) and 2-deoxyglucose (50
mM) in HEPES-buffered medium minus glucose. Neurons were
subsequently cooled to 16 °C for 5 min, incubated with 5
nM Rh-CT for 30 min, and then warmed to 37 °C for 30 min.
The metabolic inhibitors were present throughout the incubation with
Rh-CT. In control cells (A), Rh-CT is internalized to the
Golgi apparatus, but is not internalized from the PM in cells treated
with metabolic inhibitors (B). The bar corresponds to
10 µm.
The Effect of CADs on Rh-CT
Internalization
Neurons were incubated with Rh-CT for 30 min at
13-16 °C prior to addition of chlorpromazine for 5 min at
13-16 °C, followed by warming to 37 °C for 20 min. In
chlorpromazine-treated cells, Rh-CT was detected in punctate, vesicular
structures (Fig. 3, C and D), whereas in
untreated cells, intense Golgi apparatus-labeling was detected
(Fig. 3, A and B). Golgi apparatus labeling
could be restored in chlorpromazine-treated cells by washing and
addition of fresh medium that did not contain chlorpromazine
(Fig. 3, E and F). Two other CADs, sphingosine
(Fig. 3, G and H) and imipramine (not shown)
had identical effects to chlorpromazine inasmuch as Rh-CT
internalization to the Golgi apparatus was inhibited, and Rh-CT
accumulated in punctate structures. In contrast, the protein kinase C
inhibitors, H7 (Fig. 3, I and J) and
staurosporine (not shown) had no effect on Rh-CT internalization.
Similarly, these compounds had no effect on the distribution of
clathrin and the AP-2 adaptor in cultured fibroblasts (19).
Figure 3:
CADs inhibit the internalization of Rh-CT
to the Golgi apparatus. Six-day-old neurons were incubated with 5
nM Rh-CT in HEPES-buffered medium for 30 min at 13-16
°C. Neurons were then incubated with 25 µM
chlorpromazine (C and D), 5 µM
sphingosine (G and H), or 25 µM H7
(I and J) for 5 min at 13-16 °C, prior to
incubation at 37 °C for 20 min. Untreated cells are shown in
panels A and B. In panels E and F,
cells were incubated with 25 µM chlorpromazine as above,
washed to remove chlorpromazine, and placed in multiwell dishes
containing a glial monolayer in the CO incubator for an
additional 90 min. Neurons were fixed with 4% paraformaldehyde for 20
min at 37 °C prior to observation. The left-hand panels show phase contrast micrographs, and the right-hand panels show immunofluorescence. The bar corresponds to 20
µm.
To
exclude the possibility that CADs abolished Golgi apparatus labeling by
affecting the integrity of the Golgi apparatus, 3-day-old neurons were
first incubated with either Rh-CT (Fig. 4, A and
B) or C-Bodipy-ceramide (Fig. 4, C and D), and then treated with chlorpromazine for 20 min
(Fig. 4, B and D). No changes in Golgi
apparatus morphology were observed upon treatment with chlorpromazine;
similar results were seen in 6-day-old neurons. These data confirm that
chlorpromazine inhibits Rh-CT accumulation in the Golgi apparatus by
disrupting a step of Rh-CT endocytosis between the PM and the Golgi
apparatus.
Figure 4:
CADs
have no effect on Golgi apparatus morphology. Three-day old neurons
were incubated with 5 nM Rh-CT (A and B) or
5 µM C-Bodipy-ceramide (C and
D) for 30 min at 13-16 °C, followed by washing and
warming to 37 °C for 2.5 or 15 h, respectively. Cells were
subsequently incubated in the absence (A and C) or
presence (B and D) of 25 µM
chlorpromazine for 20 min at 37 °C. Neurons labeled with Rh-CT were
fixed with 4% paraformaldehyde for 20 min at 37 °C prior to
observation. The bar corresponds to 10
µm.
The Effect of CADs on CT-stimulated Elevation of
cAMP
CT mediates its cytotoxic effects by stimulating adenylate
cyclase, resulting in intracellular accumulation of cAMP
(4) .
Initial experiments determined that Rh-CT and CT have identical effects
on the elevation of cAMP, whereas CT-B was totally ineffective. To
determine the effect of CADs on this process, neurons were incubated
with CT at 13-16 °C prior to incubation with CADs or other
drugs at 37 °C. No elevation in cAMP levels were observed in either
control or CAD-treated cells for 10 min after warming, similar to the
lag period observed in the elevation of cAMP in other
cells
(5, 27) . However, after 20 min, cAMP levels were
elevated 2.3-fold in control cells (). In contrast, cAMP
levels were only elevated 1.3-fold in chlorpromazine-treated cells.
After 30 min, cAMP levels were elevated 3.1- and 1.7-fold,
respectively, in control and chlorpromazine-treated cells
(). Likewise, 20 and 30 min after imipramine-treatment (100
µM), cAMP levels were only elevated 1.4- and 2.2-fold,
respectively, and after sphingosine treatment (5 µM), cAMP
levels were elevated 1.7- and 2.1-fold. The effect of chlorpromazine on
cAMP was reversible (). H7, an inhibitor of protein kinase
C () and staurosporine (not shown) had no effect on CT
stimulation of cAMP. Thus, in addition to inhibiting the vesicular
transport of Rh-CT to the Golgi apparatus, CADs also inhibit the
ability of CT to elevate cAMP.
DISCUSSION
The major finding of the current study is that
internalization of CT to the Golgi apparatus, and the subsequent
elevation of cAMP, is inhibited by three CADs, namely chlorpromazine,
imipramine, and sphingosine. CADs have recently been shown to effect
endocytosis by disrupting the assembly-disassembly of clathrin from
coated pits and endosomes
(19) . Clathrin binding to coated pits
is mediated by the AP-2 adaptor protein. AP-2 binding to the membrane
is itself mediated via an integral membrane protein, the AP-2
receptor
(18) , that exists in two states, one that binds AP-2
with high affinity and one that does not bind AP-2
(28) .
Switching between these two states is believed to regulate the
assembly-disassembly of clathrin coats. Thus, soon after formation of
clathrin-coated vesicles, the AP-2 receptor switches to its non-binding
state, resulting in the release of AP-2 and clathrin into the cytosol
and the generation of non-coated vesicles. In cells treated with CADs,
the AP-2 receptor becomes activated on a non-coated endosomal
compartment
(19, 28) , leading to the recruitment of AP-2
and clathrin. The recycling of receptors for low density
lipoprotein
(19) , transferrin
(16) , and
-macroglobulin and epidermal growth
factor
(17) , is inhibited, although the receptors do not
accumulate in the same endosomal compartment that acquires clathrin
(19). Our current data suggest that the sorting of CT to the Golgi
apparatus occurs in the same endosomal compartment involved in sorting
recycling receptors to the plasma membrane, since both processes are
inhibited by CADs.
on the external
leaflet of the PM via a high affinity binding site on the B-subunit of
CT. At low temperature, CT accumulates in caveolae on the cell
surface
(9, 10) , although it is not excluded from
clathrin-coated pits
(10) . No internalization of CT occurs at
low temperature (i.e. 13-16 °C, see
Fig. 1A). Upon warming to 37 °C, CT is internalized
from either clathrin-coated pits (Fig. 5, Ai) or from
caveolae (Fig. 5, Aii). Since these two pathways
converge
(8, 29) (Fig. 5, Aiv) after
removal of clathrin from clathrin-coated vesicles (Fig. 5,
Aiii), the exact site of internalization has no bearing on the
subsequent fate of CT in the endocytic pathway. Non-coated vesicles
move along the endocytic pathway to a sorting compartment
(Fig. 5, Av). CT is sorted away (Fig. 5,
Avii) from recycling membrane components (Fig. 5,
Avi) and components targeted to lysosomes (not shown), and
targeted to the Golgi apparatus (Fig. 5, Aviii) where it
is processed to produce the
A
-subunit
(13, 15) . The accumulation of
coated endosomes upon CAD treatment disrupts the further processing of
CT to the Golgi apparatus (Fig. 5, Bv). Since CADs have
no effect on the initial uncoating of coated vesicles
(19) (Fig. 5, Biii), cells are able to
internalize CT (Fig. 5, Bi) at least as far as early
endosomes (Fig. 5, Biii and iv).
Figure 5:
Schematic representation of the pathways
of internalization of CT in the absence (A) and presence (B)
of CADs. For details, see ``Discussion.'' CT is
represented by the symbol T, and clathrin coats by thick
lines.
Although no
labeling of the Golgi apparatus by CT was observed after CAD treatment
(see Fig. 3, D and H), there was a small
increase in CT-stimulated cAMP levels (). It is possible
that small amounts of CT, below the limit of resolution of the light
microscope, are nevertheless delivered to the Golgi apparatus in the
presence of CADs. Alternatively, CT could elevate cAMP levels in a
compartment prior to the Golgi apparatus. Biochemical studies have
indicated that activation of adenylate cyclase requires CT
internalization and processing in an acidic-endosomal
compartment
(7) . However, the fraction used in this study was a
``Golgi-endosome'' fraction that is often used as a source of
enriched Golgi apparatus membranes
(30) . Moreover, in Caco-2 and
SK-N-MC cells, chloroquine had no effect on CT stimulation of cAMP
accumulation
(14) although it did inhibit cAMP generation in
hepatocytes
(7) . In PtK1 cells, in which the Golgi apparatus but
not endosomes are resistant to Brefeldin A, no changes were observed in
the ability of CT to stimulate adenylate cyclase
(13) . Together,
these data suggest that transport to the Golgi apparatus is normally
necessary for CT to mediate its effects on adenylate cyclase.
Table:
CT-stimulated elevation of cAMP after
chlorpromazine or H7 treatment
-Bodipy-ceramide,
N-[5-(5,7-dimethylBodipy)-1-pentanoyl]-D-erythro-sphingosine;
CAD, cationic amphiphilic drug; C
-NBD-ceramide,
N-{6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl}-D-erythro-sphingosine;
CT, cholera toxin; CT-B, B-subunit of cholera toxin; Rh, rhodamine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.