Department of Neurobiology, University of Heidelberg, D-69120 Heidelberg, Germany
We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18°C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18°C with the membraneimpermeant, cleavable sulfo-NHS-SS-biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37°C originated exclusively from the membranes containing the MesNaaccessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37°C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18° or 37°C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18°C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.
The synaptic vesicle cycle has served as one of the
paradigms for understanding the molecular basis of
vesicular traffic in the eukaryotic cell (for reviews
see Kelly, 1993b It is for these reasons that studies addressing the biogenesis of synaptic vesicles have mostly been carried out with
neuroendocrine cell lines, in particular PC12 cells. These
cells contain synaptic-like microvesicles (SLMVs),1 the neuroendocrine counterpart of synaptic vesicles (Navone et al., 1986 To dissect the molecular machinery involved in SLMV
biogenesis, we have reconstituted this process in a cell-free
system derived from PC12 cells that will be described in a
separate report (Schmidt, A., and W.B. Huttner, manuscript in preparation). In the course of establishing this
cell-free system, we performed a detailed characterization
of the compartment into which cell surface-labeled synaptophysin is internalized before its appearance in SLMVs. Surprisingly, we find that SLMVs originate from a novel
compartment that is distinct from the transferrin receptor-
containing endosome and connected to the plasma membrane via a narrow membrane continuity.
Biotinylation and Chase of PC12 Cells
PC12 cells were grown in DME supplemented with 10% horse serum and
5% fetal calf serum as described (Tooze and Huttner, 1990 For biotinylation at 37°C, the rinse was removed, and 3.5 ml PBS2+ at
37°C was added per dish. The biotinylation was started by the addition to
each dish of 500 µl PBS2+ containing 4 mg of either sulfo-NHS-LC-biotin
(Pierce, Rockford, IL) or sulfo-NHS-SS-biotin (Pierce; final concentration 1 mg/ml), followed by incubation at 37°C for 2-30 min as indicated.
Biotinylation was terminated by removal of the medium, and dishes were
either placed on ice for further treatment at 4°C and analysis, as described in the following sections, or subjected to chase. Dishes were rinsed with 10 ml 37°C PBS2+ followed by 10 ml of 37°C growth medium and chased at
37°C in 10 ml of PC12 cell-conditioned and centrifuged growth medium
for various times as indicated in the figure legends. At the end of chase,
the medium was removed, and dishes were placed on ice.
For biotinylation at 18°C, the 37°C rinse was removed, the dishes were
rinsed with 5 ml of PBS2+ preequilibrated at 18°C, and 3.5 ml of 18°C
PBS2+ added per dish. The dishes were placed in a waterbath set at 18°C
in a cold room, and the biotinylation was started as above by the addition
to each dish of 500 µl PBS2+ containing 4 mg of either sulfo-NHS-LC-biotin or sulfo-NHS-SS-biotin. Biotinylation was carried out at 18°C for the
times indicated in the figure legends (usually 30 min) and terminated by
removal of the medium. When indicated (see figure legends), dishes were rinsed after biotinylation with 10 ml of PBS2+ at 18°C and chased for 5 min
at 18°C with 5 ml PBS2+ containing 20 mM glycine. Dishes were then subjected to chase at 37°C as described above, or placed on ice. In some experiments, dishes were incubated for various times at 18°C as above, except that the biotinylation reagent was omitted, followed by biotinylation with sulfo-NHS-LC-biotin for 30 min at 4°C.
Dishes were then subjected to further treatment at 4°C (avidin quench,
MesNa [2-mercaptoethanesulfonic acid sodium salt] treatment) and analysis. In some experiments, the MesNa treatment at 4°C was followed by a
chase at 37°C, and dishes were then placed on ice.
Avidin Quench
All steps were performed at 4°C, and 10 ml per dish of each solution was
used. After biotinylation with sulfo-NHS-LC-biotin, dishes were rinsed with
and incubated for 15 min in PBS2+ containing 20 mM glycine. Dishes were
then incubated for 60 min with PBS2+ containing 0.2% BSA and 50 µg/ml
avidin (Boehringer-Mannheim GmbH, Mannheim, Germany), rinsed with
PBS2+/0.2% BSA, incubated for another 30 min in PBS2+/0.2% BSA containing 100 µg/ml biocytin (Sigma Chemical Co., St. Louis, MO) to quench
the free biotin binding sites of avidin, and rinsed extensively with PBS2+/
0.2% BSA. Control dishes were treated identically except that avidin was
omitted. In some experiments, incubation with avidin was performed for
15 min at either 18° or 4°C. Dishes were then either subjected to chase at
37°C as described in the above section, or the cells were harvested and homogenized as described below.
MesNa Treatment
Treatment with MesNa (Sigma Chemical Co.) was performed by modifying the procedure of Carter et al. (1993) For the kinetics of the acquisition of MesNa inaccessibility, 6-cm dishes
of PC12 cells were used. Biotinylation, chase, and MesNa treatment were
performed as described above, except that the volumes per dish reduced
to 0.75 ml for the biotinylation, 2 ml for the chase, and 1 ml for the MesNa
treatment. After MesNa treatment and washing in NT buffer, the steps (at
4°C) were as follows. Cells were rinsed twice in homogenization buffer
(150 mM NaCl, 0.2 mM MgCl2, 1 mM EDTA, 10 mM Hepes, pH 7.2) and
scraped from the dish in 1 ml of this buffer using a rubber policeman. The
cell suspension was centrifuged for 5 min at 850 g, and each pellet was resuspended in 500 µl of solubilization buffer (1% Triton X-100, 100 mM
NaCl, 2 mM EDTA, 50 mM Tris-HCl, pH 7.4). The cell lysates were kept
on ice for 30 min and centrifuged at maximal speed in an Eppendorf
bench-top centrifuge for 15 min. Aliquots (100 µl) of the cleared cell lysates were diluted fivefold with solubilization buffer and subjected to
streptavidin-agarose adsorption.
Subcellular Fractionation
All steps were performed at 4°C. Dishes were rinsed with homogenization
buffer containing 0.5 mM PMSF and scraped from the dish in 5 ml of this
buffer using a rubber policeman. The cell suspension was centrifuged for 5 min at 850 g, and the pellet from one dish of cells was resuspended in 1 ml
of homogenization buffer with PMSF. This suspension was passed six
times back and forth through a 0.70 × 30 mm needle attached to 1-ml syringe followed by eight passages through a ball-bearing homogenizer
(cell-cracker, 12 µm clearance, European Molecular Biology Laboratory).
The homogenate was centrifuged for 10 min at 1,350 g, and the resulting
postnuclear supernatant was centrifuged for 15 min at either 12,000 g
(15,000 rpm) or 66,000 g (35,000 rpm) in an Optima TL centrifuge (Beckman Instrs., Inc., Fullerton, CA) using a TLA-45 rotor, as indicated in the figure legends. The 12,000 and 66,000 g pellets were resuspended in 200 µl of
homogenization buffer containing 1 µg/ml leupeptin and 2 µg/ml aprotinin. The 12,000 and 66,000 g supernatants were loaded onto a 5-ml, 5-25%
glycerol gradient (Clift-O'Grady et al., 1990 Streptavidin-Agarose Adsorption
Unless indicated otherwise, all steps were performed at 4°C. Aliquots of
the postnuclear supernatants (30-60 µl), the resuspended 12,000 and
66,000 g pellets (5 µl), and the 12,000 and 66,000 g supernatants (50 µl)
were mixed with half a volume of 3X concentrated solubilization buffer
(see above) and brought to 200 µl with solubilization buffer. Glycerol gradient fractions (500 µl) were mixed with 250 µl of 3X solubilization buffer.
The samples were incubated for 30 min on ice followed by addition of 20 µl
of a 1:1 slurry of streptavidin-agarose (Sigma Chemical Co.) in solubilization buffer. In initial experiments, this amount of streptavidin-agarose was shown to be sufficient for maximal recovery of biotinylated proteins from
the above aliquots of the various subcellular fractions. Samples were incubated for 60 min on a rocking platform and briefly centrifuged in benchtop centrifuge to pellet the agarose beads. The supernatants were collected
for the determination of streptavidin-unbound proteins. The agarose pellets were washed three times with 500 µl of solubilization buffer. After
addition of 15 µl of 3X Laemmli sample buffer to the streptavidin-agarose
pellets (10 µl) and the residual wash (~20 µl), the beads were gently resuspended, the samples were incubated for 4 min at 95°C, and the fluid
phase above and around the beads was collected with a 100-µl Hamilton syringe while the samples were kept at 95°C on a heating block. The eluates from the streptavidin-agarose beads were analyzed by SDS-PAGE
followed by immunoblotting.
Immunoblotting
SDS-PAGE (9% acrylamide) was performed using the BioRad Laboratories (Richmond, CA) mini-gel system. Proteins were transferred in 20%
methanol, 0.1% SDS, 20 mM Tris base, 150 mM glycine, pH 8.3, onto nitrocellulose (BA85; Schleicher & Schuell, Inc., Keene, NH, 0.45 µm) using a Genie electrophoretic blotter (Idea Sci., Minneapolis, MN). The nitrocellulose was incubated for 30 min in blocking buffer (PBS containing
5% low fat milk powder and 0.2% Tween-20). The nitrocellulose was cut
between the 69- and 45-kD Rainbow markers (Amersham Intl., Buckinghamshire, UK) to separate the synaptophysin-containing portion of the
membrane from that containing transferrin receptor and SV2; the membranes were then incubated for 60 min at room temperature in blocking buffer containing 25 ng/ml of the anti-synaptophysin monoclonal antibody
Sy38 (Boehringer Mannheim GmbH), 1 µg/ml of the anti-transferrin receptor monoclonal antibody HTR 68.4 (White et al., 1992 Quantification of Data
Multiple exposures of the immunoblots were made on Hyperfilm-MP
(Amersham Intl.). Exposures whose signals were in the linear range of the
procedure as determined from standard curves were quantitated by densitometric scanning using an LKB Ultroscan XL. The values obtained were
expressed as arbitrary units. "Biotinylated synaptophysin" and "biotinylated transferrin receptor" are defined as synaptophysin and transferrin
receptor, respectively, recovered bound to streptavidin-agarose (except
for experiments with avidin quenching, in which the term "streptavidinbound" is used instead of "biotinylated"). "Total synaptophysin" and "total transferrin receptor" are defined as the sum of streptavidin-agarosebound plus streptavidin-agarose-unbound synaptophysin and transferrin receptor, respectively, present in either the postnuclear supernatant, the
sum of the 12,000 g pellet plus supernatant, or the sum of the 66,000 g pellet plus supernatant; only a minor proportion of synaptophysin and transferrin receptor was recovered in the nuclear pellet (data not shown). "Total
biotinylated synaptophysin" is defined as the streptavidin-agarose-bound
synaptophysin present in either the postnuclear supernatant, the sum of
the 12,000 g pellet plus supernatant, or the sum of the 66,000 g pellet plus supernatant.
Transferrin Uptake and Fluorescence Microscopy
Transferrin Uptake.
PC12 cells were grown on polylysine-coated, 9-mm glass
coverslips. The growth medium was replaced with DME containing 0.2% BSA, and cells were incubated for 30 min at 37°C. The medium was replaced by DME/0.2% BSA containing 50 µg/ml of Texas red-labeled transferrin (Molecular Probes, Inc., Eugene, OR), and cells were incubated for 30 min at 37°C followed by fixation and fluorescence analysis.
Synaptophysin Immunofluorescence.
Unless specified otherwise, all procedures were performed at room temperature. After removal of the transferrin-containing medium, cells were rinsed once with PBS and fixed for
15 min with 3% (wt/vol) paraformaldehyde in PBS. Coverslips were
rinsed with and incubated for 10 min in PBS containing 50 mM NH4Cl.
Coverslips were then incubated for 10 min with 100 µl of PBS containing
2 mM EDTA, 0.5 mM PMSF, 10 µg/ml aprotinin, 40 µg/ml leupeptin, and
either 0.3% (vol/vol) Triton X-100 (Sigma Chemical Co.) or 50 µg/ml digitonin (Fisher Scientific Co., Pittsburgh, PA; added from a 50 mg/ml stock
in DMSO) as indicated. Coverslips were then incubated for 10-20 min in
PGA buffer (0.2% [wt/vol] gelatin, 0.02% [wt/vol] azide in PBS) followed
by 30 min in PGA buffer containing 0.4 µg/ml of the monoclonal anti-synaptophysin antibody Sy38 (Boehringer-Mannheim GmbH). The coverslips were washed three times in PGA buffer and incubated for 30 min
with 3.3 µg/ml Cy2-conjugated goat anti-mouse IgG secondary antibody
(Biotrend, Köln, Germany) in PGA buffer. The coverslips were then sequentially washed three times in PGA buffer, three times in PBS, dipped
twice in distilled water, and finally mounted in Mowiol.
Confocal Microscopy.
Fluorescent images were obtained using a Leica
TCS4D confocal laser scanning microscope with a 63X 1.4 numerical aperture objective. The confocal microscope settings were such that the photomultipliers were within their linear range. The images shown were prepared from the confocal data files using Adobe Photoshop, without any alteration of the original fluorescence data.
Synaptophysin Extraction after Fixation
PC12 cells grown on coverslips in 10-cm dishes were biotinylated for 30 min at 18°C using 1 mg/ml sulfo-NHS-LC-biotin. Controls were treated
identically except that sulfo-NHS-LC-biotin was omitted. After the chase
in PBS2+/glycine, dishes were rinsed once with 18°C PBS2+, and cells were
subjected to one of the following three protocols. (a) Cells were fixed by
the addition of 3% (wt/vol) paraformaldehyde in PBS at 18°C and transferred to room temperature. After 15 min fixation, coverslips were incubated with NH4Cl and treated with Triton X-100, as described under Synaptophysin Immunofluorescence, except that 10 mM N-ethyl maleimide
was included in the Triton X-100 buffer. All subsequent steps were performed at 4°C unless indicated otherwise. The Triton X-100 extract (100 µl) was cleared by centrifugation in a bench-top centrifuge, incubated for
2 h with 50 µl of a 1:1 slurry of streptavidin-agarose in PBS containing 0.3% Triton X-100 and 2 mM EDTA. The beads were washed in PBS/Triton/EDTA, incubated for 2 h with 100 µl of PBS/Triton/EDTA containing
0.2 µg/ml of the monoclonal anti-synaptophysin antibody Sy38, washed in
PBS/Triton/EDTA, incubated for 2 h with 100 µl of PBS/Triton/EDTA containing 1.6 µg/ml of goat anti-mouse IgG antibody conjugated to
HRP, and washed again. Peroxidase activity associated with the beads was
revealed by incubation for 5 min at room temperature in 750 µl of 50 mM
sodium phosphate, pH 5, 0.1% Triton X-100, 0.003% H2O2, 100 µg/ml
O-dianisidine. Reactions were stopped with 0.7% azide. Absorbances
were determined at 455 nm and converted to values of ng HRP-IgG by
comparison to a standard curve. (b) Unfixed cells were treated with Triton
X-100 as described above except that 10 mM N-ethyl maleimide was included, and the extraction was performed for 1 h on ice. The Triton X-100
extract was then processed as in a. (c) Cells were fixed by the addition of
3% (wt/vol) paraformaldehyde in PBS at 18°C and transferred to room temperature. After 15 min fixation, coverslips were sequentially incubated
with NH4Cl, digitonin, and the monoclonal anti-synaptophysin antibody
Sy38 and washed with PGA buffer, as described under Synaptophysin Immunofluorescence. Controls were treated identically except that anti-synaptophysin was omitted. Coverslips were then incubated in the Triton X-100
buffer and the resulting extract processed as in a, except that the 2 h incubation with the anti-synaptophysin monoclonal antibody was omitted.
Immunoisolation
For the immunoisolation of SLMVs, the procedure of Burger et al. (1989) Electron Microscopy
Immunoisolated Membranes.
Immunobeads and control beads were
washed with 100 mM cacodylate buffer, pH 7.4, fixed with 1% glutaraldehyde in cacodylate buffer, postfixed in 1% (wt/vol) OsO4 plus 1.5% (wt/
vol) magnesium ferricyanide, and contrasted with 1.5% (wt/vol) magnesium uranyl acetate. Samples were then dehydrated in ethanol and embedded in Epon. Ultrathin sections were contrasted with uranyl acetate and lead citrate and observed in a Zeiss EM 10 CR electron microscope.
Immunogold Labeling of Ultrathin Cryosections.
PC12 cells were incubated for 30 min at 18° or 37°C with sulfo-NHS-LC-biotin as described
above, followed by a 5 min chase in PBS2+/20 mM glycine at the respective temperature. The dishes (10 ml per dish, all steps at 4°C) were rinsed
twice with PBS2+/20 mM glycine, once with PBS2+, and once with 0.25 M
Hepes-NaOH, pH 7.4, and cells were fixed on the dish by addition of 8%
paraformaldehyde in the latter buffer at 4°C. After 1 h at 4°C, the fixed
cells were scraped off the dish and centrifuged for 5 min at 10,000 g. The
cell pellets were incubated in fixative for another 3 h at 4°C, infiltrated
with 2.1 M sucrose in PBS, and frozen in liquid nitrogen. Fixation at 4°C
was chosen to allow a comparison of synaptophysin immunoreactivity between the cells incubated at 18° and 37°C; the epitope recognized by the
monoclonal antibody Sy38 is known to be sensitive to the extent of aldehyde fixation (Hoog et al., 1988 Synaptophysin Biotinylated at the Surface of PC12
Cells Is Rapidly Transported to SLMVs
To study the donor membrane from which SLMVs originate, we chose to label synaptophysin of PC12 cells by surface biotinylation, exploiting the fact that the newly synthesized form of this protein travels via the plasma membrane
to SLMVs (Régnier-Vigouroux et al., 1991
We next determined the kinetics of appearance of cell
surface-biotinylated synaptophysin in SLMVs. PC12 cells
were biotinylated for 5 min, chased for various times, and
analyzed by glycerol gradient centrifugation to separate
SLMVs from other membranes. This showed that cell surface-biotinylated synaptophysin appeared very rapidly in
SLMVs. Already without chase, i.e., at the end of the 5 min biotinylation, a small proportion (~2%) of the biotinylated synaptophysin was detectable in the SLMV-containing fractions of the glycerol gradient (Fig. 3 A). A similar observation was made after 3 min of chase (Fig. 3 B;
2.5% of total biotinylated synaptophysin recovered in SLMV
fractions). Within 10 min of chase, the appearance of biotinylated synaptophysin in SLMVs approached a value corresponding to ~13% of the total biotinylated synaptophysin, which remained constant for at least 3 h (Fig. 3, A and C).
Surface-biotinylated Synaptophysin Does Not Appear in
SLMVs at 18°C
The kinetics of appearance in SLMVs of cell surface-biotinylated synaptophysin was much more rapid than that of
newly synthesized synaptophysin sulfate labeled in the TGN
(Régnier-Vigouroux et al., 1991 Glycerol gradient analysis of PC12 cells incubated for 30 min at 18°C with sulfo-NHS-LC-biotin showed that the reduced temperature indeed blocked the appearance of biotinylated synaptophysin in SLMVs. In contrast to an incubation at 37°C after which biotinylated synaptophysin was
found in both bottom and middle fractions of the gradient
(Fig. 4 A), synaptophysin biotinylated at 18°C was found
in the bottom fractions of the gradient (Fig. 4 B) containing early endosomes, as indicated by the presence of biotinylated transferrin receptor (Fig. 4 C), but not in the middle
fractions (Fig. 4 B) containing SLMVs formed before biotinylation, as indicated by the presence of nonbiotinylated synaptophysin (Fig. 4, D and E; two distinct procedures of differential centrifugation before the glycerol
gradient; see figure legend). A chase for 30 min at 37°C,
performed after the 30 min biotinylation at 18°C, resulted in the appearance of biotinylated synaptophysin in SLMVs
(~15% of the total biotinylated synaptophysin; Fig. 4 F),
showing that the temperature block was reversible. Our
observations are consistent with the results of Desnos et
al. (1995)
Characterization of the 18°C Compartment
Given that synaptophysin biotinylated at the cell surface
did not appear in SLMVs at 18°C, we next investigated
whether it was internalized at this temperature. For this
purpose, we studied the accessibility of biotinylated synaptophysin to extracellularly added avidin. As a positive control, PC12 cells were rapidly cooled from 37° to 4°C (a
temperature preventing further membrane traffic), biotinylated for 60 min at 4°C, and then exposed to avidin at 4°C.
In this condition one would expect to biotinylate only
those synaptophysin molecules that are exposed at the cell
surface; these should be accessible to avidin and, hence,
not be able to bind to streptavidin-agarose subsequently.
Only ~2% of the total synaptophysin was biotinylated in
this condition, in line with previous observations showing
that only very little of the total synaptophysin is present at
the surface of PC12 cells (Johnston et al., 1989
Studies on the endocytosis of clathrin-coated vesicles in
nonneuroendocrine cells (Carter et al., 1993
The proportion of the total synaptophysin biotinylated
after 30 min at 18°C (~10% in Fig. 5 B) was at least five
times that biotinylated for 60 min at 4°C (Fig. 5 A). Biotinylation at the latter temperature for 30 min yielded the
same value (~2% of the total synaptophysin) as that carried out for 60 min (data not shown), showing that within
30 min at 4°C, every synaptophysin molecule accessible to
sulfo-NHS-LC-biotin was biotinylated to an extent sufficient to allow binding to streptavidin. This in turn suggested that the greater proportion of biotinylated synaptophysin obtained at 18° as opposed to 4°C reflected the
delivery, at 18°C, either of unbiotinylated synaptophysin
molecules from an intracellular pool to the site where biotinylation occurred, or of sulfo-NHS-LC-biotin to an intracellular site where synaptophysin resided. This conclusion was supported by studying the time course of biotinylation
of synaptophysin at 18°C, which revealed a continuous increase in the amount of biotinylated synaptophysin over
time (Fig. 6, closed circles). Together with the accessibility
of the majority of this synaptophysin to MesNa (Fig. 5 C),
these results indicate that upon incubation at 18°C, biotinylated synaptophysin accumulated in a membrane compartment connected to the plasma membrane. Interestingly, when PC12 cells incubated for various times at 18°C
were subsequently biotinylated for 30 min at 4°C, only
~2% of the total synaptophysin was biotinylated, irrespective of the incubation time at 18°C (Fig. 6, closed triangles).
This suggested that upon cooling the cells to 4°C, the lumen of this membrane compartment became inaccessible to sulfo-NHS-LC-biotin, as was the case for avidin (Fig.
5 B) but not MesNa (Fig. 5 C). Biotinylation of the transferrin receptor at 18°C also increased over time, reaching a
value of ~35% of total by the end of the 30-min incubation (Fig. 6). Given the inaccessibility of the transferrin receptor to MesNa (Fig. 5 D), this implied its continuous delivery to the plasma membrane and endocytosis at 18°C.
We next investigated whether SLMV biogenesis involved the MesNa-sensitive or -protected population of biotinylated synaptophysin. PC12 cells biotinylated at 18°C
were treated with MesNa and then chased for 10 min at
37°C. In contrast to the control, virtually no biotinylated
synaptophysin appeared in SLMVs after MesNa treatment (Fig. 7), although ~30% of the biotinylated synaptophysin
was protected from MesNa (Fig. 5 C). Hence, the MesNasensitive population of biotinylated synaptophysin molecules rather than the protected one participated in SLMV
biogenesis.
SV2, Another Membrane Protein of SLMVs, Is Present
in the MesNa-accessible Compartment
If the MesNa-accessible compartment gives rise to SLMVs,
one would expect it to contain SLMV-specific membrane
proteins other than synaptophysin. We investigated this issue by analyzing whether one such protein, SV2, showed a
similar accessibility to MesNa after cell surface biotinylation for 30 min at 18°C. As shown in Fig. 8, MesNa accessibility of biotinylated SV2 was indeed comparable to that
of synaptophysin (the latter confirming the data shown in
Fig. 5 C) and in marked contrast to the almost complete MesNa inaccessibility of the transferrin receptor (confirming the data shown in Fig. 5 D).
Synaptophysin Passes through a
Compartment Continuous with the Plasma
Membrane at Physiological Temperature
Together, the data presented so far suggested that the
SLMVs formed upon reversal of the 18°C block originated
from a compartment that (a) was continuous with the
plasma membrane and (b) lacked transferrin receptor. It
was important to determine whether such a compartment
also exists in PC12 cells maintained at 37°C. To investigate
this issue, PC12 cells were biotinylated at 37°C for a very
brief period of time (2 min) and chased for 10 min. Analysis of the biotinylated synaptophysin chased to SLMVs for
sensitivity to MesNa treatment showed that it was not accessible to MesNa, as expected (Fig. 9 A). However,
~90% of the biotinylated synaptophysin recovered in the
66,000 g pellet, which contained the bulk of the nonSLMV synaptophysin, was still MesNa sensitive at the end of the 10 min chase (Fig. 9 B), although inaccessible to avidin (Fig. 9 C). In contrast, the biotinylated transferrin receptor in the 66,000 g pellet was inaccessible to MesNa
(Fig. 9 D), consistent with its rapid endocytosis. These observations show that not only at 18° but also at 37°C, synaptophysin is internalized into a compartment that is characterized by a narrow continuity with the plasma membrane
and is devoid of transferrin receptor.
To obtain information about the kinetics with which
synaptophysin passes through this compartment, PC12
cells were biotinylated for 2 min at 37°C, chased for increasing periods of time, and subjected to MesNa treatment (Fig. 10). Biotinylated synaptophysin became inaccessible to MesNa with a t1/2 of 15-20 min. Acquisition of
MesNa inaccessibility by the transferrin receptor occurred much faster, with half of the transferrin receptor being
protected from MesNa already at the end of the 2-min biotinylation and maximal protection being reached after 7.5 min of chase (Fig. 10).
A Sub-plasmalemmal, Tubulo-cisternal Membrane
System Containing Synaptophysin but Not Transferrin
Receptor: Relationship to the 18°C Compartment
By immunocytochemistry, synaptophysin has been previously shown to be colocalized with transferrin receptor in
PC12 cells (Cameron et al., 1991
We exploited (a) the selective access of the anti-synaptophysin antibody to the peripheral synaptophysin upon
digitonin permeabilization of paraformaldehyde-fixed PC12
cells and (b) the ability to extract synaptophysin from
fixed cells, to demonstrate that the peripheral synaptophysin immunoreactivity included the biotinylated synaptophysin accumulated in the pre-SLMV compartment at 18°C.
When Triton X-100 extracts of fixed and nonfixed PC12
cells that had been biotinylated at 18°C were adsorbed to
streptavidin-agarose, virtually the same amount of synaptophysin immunoreactivity was detected on the beads (Fig.
12 A). This showed that Triton X-100 quantitatively extracted from paraformaldehyde-fixed PC12 cells, and the
synaptophysin biotinylated at 18°C. Next, PC12 cells biotinylated for 30 min at 18°C were fixed, permeabilized with
digitonin, and exposed to anti-synaptophysin antibody.
The biotinylated synaptophysin was then extracted by Triton X-100, adsorbed to streptavidin-agarose, and analyzed
for bound anti-synaptophysin antibody. If the peripheral synaptophysin that is selectively tagged by anti-synaptophysin addition to digitonin-permeabilized fixed cells (see
Fig. 11, A and a) included the synaptophysin biotinylated
at 18°C, one would expect to detect anti-synaptophysin
immunoreactivity associated with the streptavidin-agarose beads in this condition. This was indeed the case (Fig.
12 B).
Electron microscopy of immunogold-labeled ultrathin
cryosections revealed synaptophysin immunoreactivity associated with a pleiomorphic subplasmalemmal membrane system in both PC12 cells biotinylated for 30 min at
18°C (Fig. 13, A-D) and cells kept at 37°C (Fig. 13, E-H).
Most membranes immunoreactive for synaptophysin had
a tubular or cisternal morphology and often were localized in very close proximity to the plasma membrane. Connections between these membranes and the plasmalemma
could not be convincingly demonstrated, presumably because the fixation had to be carried out at 4°C to allow a
comparison of synaptophysin immunoreactivity between
the cells incubated at 18° and 37°C (see Materials and Methods); at 4°C, these connections become very narrow
(Fig. 6 and Discussion). Little synaptophysin immunoreactivity was detected on the plasma membrane itself, consistent with the results of biotinylation at 4°C (Fig. 5 A).
We have characterized the compartment from which
SLMVs originate, using cell surface-biotinylated synaptophysin and SV2 as markers. Our results lead to a modification of the concept about the biogenesis of SLMVs in neuroendocrine cells, which is illustrated by the model shown
in Fig. 14. According to our results, SLMVs originate from
a subplasmalemmal tubulo-cisternal membrane system, referred to as SLMV donor compartment, that is (a) distinct from the transferrin receptor-containing endosome
and (b) connected to the plasmalemma via a narrow membrane continuity.
The SLMV Donor Compartment Is Connected to the
Plasma Membrane
The key observation, which provided the basis for the
identification and characterization of the SLMV donor
compartment, was that upon cell surface biotinylation at
18°C, a temperature which blocked the appearance of synaptophysin in SLMVs, all of the biotinylated synaptophysin was present in avidin-protected membranes, the
majority of which were accessible to MesNa and therefore
in continuity with the plasmalemma. This allowed us to
show, by chasing such biotinylated synaptophysin following MesNa treatment at 37°C, that SLMVs originate from
the latter membranes. Further evidence is provided by our
experiments in which PC12 cells were biotinylated at 18°C,
treated with avidin or MesNa, and then perforated to perform cell-free SLMV biogenesis; MesNa but not avidin pretreatment abolished the appearance of biotinylated synaptophysin in SLMVs (Schmidt, A., and W.B. Huttner,
manuscript in preparation).
SLMVs originated from membranes in continuity with
the plasmalemma not only upon reversal of the 18°C
block, but also when cells were maintained at physiological temperature. After cell surface biotinylation at 37°C
for 2 min followed by a 10 min chase, some (~5% in Fig.
9 A) of the biotinylated synaptophysin had already appeared in SLMVs, but the vast majority (>90% in Fig. 9, B
and C) was still present in the faster sedimenting membranes from which these SLMVs must have been originating. The latter synaptophysin was protected from avidin
but largely sensitive to MesNa.
The membrane connection between the SLMV donor
compartment and the plasma membrane must be fairly
narrow at 18°C because biotinylated synaptophysin was
protected from avidin added at this temperature. This connection may, however, allow passage of other proteins (with
less crosslinking potential than avidin) into the SLMV donor compartment given that Desnos et al. (1995) In two out of seven experiments, ~60% of the synaptophysin biotinylated at 18°C was found to be accessible to
avidin (data not shown). While we do not know the reason
for this variability of results (which may be due to differences in the batch of cells, passage number, state of confluency, batch of growth medium, etc.), it raises the possibility that the membrane connection between the SLMV
donor compartment and the plasma membrane is subject to dynamic changes and may open and close, similar to the
membrane dynamics of caveolae during potocytosis (Anderson, 1993 The SLMV Donor Compartment: A Tubulo-cisternal
Membrane System beneath the Plasma Membrane
A characteristic feature of the SLMV donor compartment
at the light microscopic level was the synaptophysin immunofluorescence at the cell cortex. Visualization of this synaptophysin required the use of the mild detergent digitonin as permeabilizing agent on fixed cells, which allowed
the selective detection of the peripheral but not the perinuclear synaptophysin. Use of the stronger detergent Triton X-100 resulted in the quantitative extraction of the peripheral synaptophysin from fixed cells. Consistent with
these immunofluorescence data, synaptophysin labeled
in the SLMV donor compartment by biotinylation at 18°C
followed by fixation could be tagged with an antibody upon digitonin permeabilization and was quantitatively
extracted by Triton X-100.
At the electron microscopic level, the correlate of the
peripheral synaptophysin immunofluorescence, and hence
of the SLMV donor compartment, was a tubulo-cisternal
membrane system beneath the plasmalemma, observed in
both cells maintained at physiological temperature and
cells cooled to 18°C. Our observations confirm and extend
those of other investigators who described synaptophysinimmunoreactive subplasmalemmal membrane structures
in PC12 cells (Johnston et al., 1989 The narrow connection of the SLMV donor compartment to the plasma membrane and its tubular structure
would slow down the entry into and spreading within this
compartment of extracellularly added fluid phase markers.
This provides an explanation why HRP internalized for 5 min had been detected in SLMVs after long (3 h) but not
short (7 min) chase, an observation previously taken to indicate the origin of SLMVs from an endosomal compartment distinct from the plasma membrane (Bauerfeind et al.,
1993 By both immunoblotting and immunofluorescence,
PC12 cells were found to lack caveolin (data not shown),
and hence their subplasmalemmal tubulo-cisternal membrane system is distinct from the caveolin-positive caveolae.
The morphological appearance of the synaptophysinimmunoreactive membrane structures seen in cells incubated at 18°C clearly speaks against the possibility that the
SLMV donor compartment corresponded to nascent SLMVs
that had not yet pinched off from the plasma membrane.
This possibility was also contradicted by the observation that the vast majority (>80%) of the synaptophysin biotinylated at 18°C was recovered, upon chase at 37°C, in membranes larger than SLMVs, as revealed by their sedimentation properties.
Segregation of Synaptophysin and the
Transferrin Receptor
SLMVs are devoid of transferrin receptor (Clift-O'Grady
et al., 1990 Consistent with our biochemical results, double immunofluorescence of digitonin-permeabilized PC12 cells revealed a distinct subcellular localization of the transferrin
receptor and synaptophysin. However, previous double
immunofluorescence studies have shown colocalization of
synaptophysin and transferrin receptor in PC12 cells, observed mostly in the perinuclear area (Cameron et al., 1991 Synaptophysin Traffic beyond the SLMV
Donor Compartment
How then, does the synaptophysin in the transferrin receptor-containing endosome fit into the present concept about
SLMV biogenesis (Fig. 14)? In addressing this question,
the following observations are relevant. First, after biotinylation for 30 min at 18°C, some of the labeled synaptophysin was found in subcellular fractions also containing
transferrin receptor (Fig. 4, B and C), and ~30% of it was
resistant to MesNa. Second, synaptophysin biotinylated for 2 min at 37°C became progressively inaccessible to MesNa. Third, upon chase at 37°C, only a minor fraction (up to
15%) of the synaptophysin biotinylated at 18° or 37°C appeared in SLMVs; the majority was associated with larger
membranes (as judged from their sedimentation properties), some of which were MesNa inaccessible and recovered in subcellular fractions also containing transferrin receptor (Fig. 9 A). These observations are consistent with
synaptophysin moving from the SLMV donor compartment to an endosome not in continuity with the plasma
membrane, such as that containing transferrin receptor
(Fig. 14). However, other explanations are also conceivable, for example, that synaptophysin moves into tubules
in continuity with the SLMV donor compartment but inaccessible to MesNa under the present conditions.
Sources of Synaptophysin Undergoing Biotinylation
Approximately 28% of the total synaptophysin became biotinylated within 30 min at 37°C (data not shown), and 16%
of this synaptophysin (i.e., ~4.5% of total) was recovered
in SLMVs (Fig. 4 A, legend). At most, 1% of synaptophysin is newly synthesized in this time period given a
half-life of the protein in PC12 cells of ~24-48 h (Rehm et
al., 1986 Since only ~2% of synaptophysin was exposed on the
plasma membrane, most of the synaptophysin biotinylated
for 30 min at 37° or 18°C (up to ~15% of total) was derived from intracellular pools. The proportion of nonbiotinylated synaptophysin in SLMVs at the end of a 30-min incubation at 18°C (Fig. 4 D, 14% of total synaptophysin) was within the range of that of cells kept at 37°C (18.6 ± 4.3 SD, n = 3). Since there appeared to be no SLMV formation at 18°C (as judged from the absence of biotinylated
synaptophysin in these organelles), the maintenance of
SLMV levels (as judged from synaptophysin levels) implies a low rate of exocytosis of SLMVs at this temperature. This in turn suggests that at 18°C, synaptophysin undergoing biotinylation was derived from an intracellular
pool other than SLMVs. This must have been the case also
for part of the synaptophysin biotinylated at 37°C, because
recycling SLMVs alone (containing 19% of the total synaptophysin) cannot account for the ~28% of total synaptophysin that was biotinylated within 30 min at this temperature.
The intracellular non-SLMV pool of synaptophysin
could reside (a) in the SLMV donor compartment itself,
implying that the biotinylation reagent spread within this
compartment with time; (b) in the transferrin receptor-
containing endosome, implying cycling of synaptophysin
between this endosome, the plasma membrane, and the
SLMV donor compartment (Régnier-Vigouroux et al., 1991 Implications for Synaptic Vesicle Biogenesis in Neurons
Given that SLMVs of neuroendocrine cells are highly related to synaptic vesicles of neurons (Navone et al., 1986 Recently, Takei et al. (1996); Bennett and Scheller, 1994
; Jahn and Südhof, 1994
; Südhof, 1995
; De Camilli and Takei, 1996).
Three principal aspects of the membrane traffic of synaptic vesicle proteins can be distinguished: (a) the de novo
biogenesis of synaptic vesicles, (b) their fusion with the presynaptic plasma membrane, and (c) the recycling of the
synaptic vesicle membrane after exocytosis for the re-formation of synaptic vesicles. The latter two processes, in
particular, have been intensively studied and are relatively
well understood (Heuser and Reese, 1973
; Bennett and
Scheller, 1994
; Fesce et al., 1994
; Jahn and Südhof, 1994
;
O'Connor et al., 1994
; Rothman, 1994
; Südhof, 1995
; De
Camilli and Takei, 1996). By contrast, comparatively little
is known about the molecular events underlying the de
novo biogenesis of synaptic vesicles. Reasons for this scarcity of information include the need for studies involving
biosynthetic labeling of synaptic vesicle proteins. Such experiments, however, are not possible with the isolated
nerve terminal preparations (synaptosomes) that have
been so useful for exocytosis and recycling studies and are
technically demanding in the case of primary neuronal cultures because of the numbers of cells required.
; Wiedenmann et al., 1988
; Clift-O'Grady et al.,
1990
; De Camilli and Jahn, 1990). The current view of SLMV
biogenesis, based on work from several laboratories, is that
SLMVs of neuroendocrine cells originate from the transferrin receptor-containing early endosome. The evidence
that has lead to this view can be summarized as follows.
First, synaptophysin, a major membrane protein of synaptic vesicles (Jahn et al., 1985
; Wiedenmann and Franke,
1985
) and SLMVs (Navone et al., 1986
), has the intrinsic
tendency to travel to transferrin receptor-containing endosomes, as revealed by its expression in fibroblasts (Johnston et al., 1989
; Cameron et al., 1991
; Linstedt and Kelly,
1991
). Second, synaptophysin endogenous to neuroendocrine cells is present not only in SLMVs but is also found
in membranes containing transferrin receptor (Cameron
et al., 1991
) and HRP after a 7-min internalization (Bauerfeind et al., 1993
). Third, newly synthesized synaptophysin
is delivered from the TGN to the cell surface via the constitutive secretory pathway, cycles several times between the
plasma membrane and an internal compartment, and then
appears in SLMVs (Régnier-Vigouroux et al., 1991
). Fourth,
HRP is detected in early endosomes but not SLMVs after
a pulse internalization (5 min) and short chase (7 min) but
appears in the latter organelles after a longer chase (3 h;
Bauerfeind et al., 1993
). Fifth, VAMP/synaptobrevin, another membrane protein found in synaptic vesicles and
SLMVs (Trimble et al., 1988
; Baumert et al., 1989
; Trimble, 1993
), is internalized at 15°C into a compartment that
is distinct from the plasma membrane and that generates
SLMVs in vivo and in vitro (Desnos et al., 1995
).
Materials and Methods
) and used until
passage 16. Subconfluent 15-cm dishes were rinsed three times at 37°C
with 10 ml PBS2+ (136 mM NaCl, 2.5 mM KCl, 1.5 mM KH2PO4, 6.5 mM
Na2HPO4, 0.5 mM CaCl2, 2 mM MgCl2), incubated for 20 min in 10 ml
PBS2+ at 37°C, and rinsed again with 10 ml PBS2+ at 37°C.
as follows. All steps were performed at 4°C, and 10 ml per dish of each solution was used. After biotinylation with sulfo-NHS-SS-biotin, dishes were rinsed with PBS2+ followed
by NT buffer (150 mM NaCl, 1 mM EDTA, 0.2% BSA, 20 mM n-tris(hydroxymethyl)methyl-3-aminopropane-sulfonic acid, pH 8.6). Dishes were
incubated for 10 min in NT buffer, for 30 min in NT buffer containing
freshly dissolved 10 mM MesNa, and two times for 60 min each in fresh
NT/MesNa buffer. Dishes were rinsed extensively with NT buffer to remove MesNa. In some experiments, residual MesNa possibly left on the
dish was inactivated by the addition of 20 mM iodoacetic acid in NT buffer
for 15 min; this treatment gave the same results as extensive rinsing in NT
buffer alone. Control dishes were treated identically except that MesNa
was omitted. Dishes were then subjected to chase at 37°C, or the cells were harvested and homogenized.
) in homogenization buffer
poured on top of a 5-ml, 1.8 M sucrose cushion in homogenization buffer.
The gradients were then centrifuged for 55 min at 40,000 rpm (285,000 gmax) in a SW40Ti rotor (Beckman Instrs., Inc.). Fractions (500 µl) were collected from the bottom of the gradient (sucrose/glycerol interface, fraction
n 1) to its top (fraction n 10, without load) by introducing a needle
through the wall of the centrifuge tube just below the sucrose/glycerol interface. Qualitatively similar results were obtained with the 12,000 and
66,000 g supernatants analyzed by glycerol gradient centrifugation; however, after the 66,000 g centrifugation, the immunoblot signals in the endosome-containing fractions of the glycerol gradient relative to those in
the SLMV-containing fractions were decreased as compared to the 12,000 g
centrifugation.
; a kind gift from
Trowbridge, I. [Salk Institute, San Diego, CA] and P. DeCamilli [Yale
University, New Haven, CT]), or a 1:3,000 dilution of an ascites containing
the anti-SV2 monoclonal antibody (Buckley and Kelly, 1985
; a kind gift
from De Camilli, P.). Bound monoclonal antibodies were revealed with
HRP-conjugated goat anti-mouse IgG antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA; 0.25 µg/ml in blocking buffer)
followed by the ECL detection system (Amersham Intl.).
was adopted. Affinity purified goat-anti mouse IgG (5 mg; Cappel Laboratories, Cochranville, PA) was coupled to Eupergit C1Z beads (1 g; RöhmPharma GmbH, Rodleben, Germany) by incubation for 8 h at room temperature followed by quenching with 1 M glycine overnight. The beads
were washed sequentially with three cycles of 0.5 M NaCl, 0.1 M acetate,
pH 4.5, followed by 0.5 M NaCl, 0.1 M Tris, pH 8.0, and then rinsed with
150 mM NaCl, 10 mM Tris/HCl, pH 7.3, and stored at 4°C until use. On
the day of SLMV isolation, 400 µl of a 1:1 suspension of beads was washed
in homogenization buffer/0.2% BSA and resuspended in 1 ml of this buffer; 500 µl of the bead suspension was incubated for 2 h at room temperature with 500 ng of the monoclonal anti-synaptophysin antibody Sy38 followed by washing in homogenization buffer/0.2% BSA (immunobeads), while the other 500 µl of the bead suspension was kept as control beads.
Two pools of fractions from the glycerol gradient were prepared, one containing the five fractions corresponding to the SLMV peak (2.5 ml) and
the other the two bottom fractions (1 ml), and half of each pool was incubated for 3 h at 4°C on a rocking platform with 250 µl of either immunobeads or control beads. After immunoadsorption, the beads were washed
with ice-cold homogenization buffer/0.2% BSA and examined by electron
microscopy.
). Ultrathin cryosections (Griffiths, 1993
) were
prepared using a Reichert FCS cryoultramicrotome and collected on Formvar carbon-coated grids. Sections were blocked for 10 min with 20 mM
glycine in PBS supplemented with 10% fetal calf serum and subsequently
incubated with the primary anti-synaptophysin monoclonal antibody (1 µg/ml; Boehringer-Mannheim GmbH), the secondary rabbit anti-mouse
IgG antibody (65 µg/ml; Cappel Laboratories), and protein A coupled to
9-nm gold, each for 30 min in blocking solution containing only 5% fetal
calf serum. Sections were treated with 0.3% uranyl acetate and 1.8% methyl
cellulose, dried, and observed in the electron microscope.
Results
). We first ascertained that biotinylation of synaptophysin, which modifies lysine residues, does not interfere with its ability to be
sorted to SLMVs. The appearance in SLMVs of newly synthesized synaptophysin, sulfate labeled in the trans-Golgi
network, approaches a plateau after 3 h of chase (RégnierVigouroux et al., 1991). We therefore labeled PC12 cells
for 5 min at 37°C by addition of the membrane-impermeant sulfo-NHS-LC-biotin to the medium, followed by a
3-h chase. After glycerol gradient centrifugation which
separates SLMVs from larger membranes (Clift-O'Grady
et al., 1990
), two differentially sedimenting populations of
biotinylated synaptophysin (determined from its binding
to streptavidin-agarose) were recovered in the bottom
and middle fractions of the gradient, respectively (Fig. 1, A
and B), both of which also contained nonbiotinylated synaptophysin (Fig. 1 B). The bottom, but not the middle,
fractions also contained biotinylated transferrin receptor
(Fig. 1 A), a membrane protein constitutively cycling between the plasma membrane and early endosomes (Trowbridge et al., 1993
). Electron microscopy of the membranes
immunoadsorbed to beads coated with an antibody against
the cytoplasmic tail of synaptophysin showed that the bottom fractions contained synaptophysin in membrane structures of various sizes (Fig. 2 A). Given the presence of
transferrin receptor in these fractions, and in line with previous reports (Clift-O'Grady et al., 1990
; Cameron et al.,
1991
), the biotinylated synaptophysin in the bottom fractions was presumably present in membranes of early endosomes. Electron microscopy of the synaptophysin-containing membranes in the middle fractions of the glycerol
gradient showed that these had the expected morphology
of SLMVs (Fig. 2 B), corroborating previous conclusions
based on biochemical data (Clift-O'Grady et al., 1990
; Cameron et al., 1991
; Linstedt and Kelly, 1991
). We conclude
that synaptophysin biotinylated at the cell surface, like
nonbiotinylated synaptophysin, is sorted to SLMVs.
Fig. 1.
Biotinylated synaptophysin is sorted to SLMVs. PC12
cells were incubated with sulfo-NHS-LC-biotin for 5 min at 37°C
and chased for 180 min at 37°C. The 12,000-g supernatant prepared from the cells was subjected to glycerol gradient centrifugation, and the fractions (n 1, bottom) were analyzed for biotinylated (A and B, closed circles, same data points for both panels)
and nonbiotinylated (B, open triangles) synaptophysin and biotinylated transferrin receptor (A, open circles) by streptavidin-agarose adsorption followed by immunoblotting of bound and unbound material with the respective antibodies. Bar, SLMVs.
[View Larger Version of this Image (26K GIF file)]
Fig. 2.
Electron microscopy of glycerol gradient
fractions. The 12,000 g supernatant prepared from PC12
cells was subjected to glycerol gradient centrifugation,
and the pools of fractions 1 and 2 (A) and fractions 5-9
(B) were subjected to immunoisolation using anti-synaptophysin beads followed by
fixation and processing for electron microscopy. Note
the heterogeneous membrane structures in A and the
homogenous population of
50-nm vesicles in B. No membranes were adsorbed
to the beads if the anti-synaptophysin antibody was
omitted (not shown). Bars,
300 nm.
[View Larger Version of this Image (88K GIF file)]
Fig. 3.
Time course of appearance of biotinylated synaptophysin in SLMVs. PC12
cells were incubated with
sulfo-NHS-LC-biotin for 5 min at 37°C and chased at
37°C for various times as indicated (A) or for 3 min (B)
and 180 min (C). The 66,000 g
(A) or 12,000 g (B and C) supernatants prepared from
the cells were subjected to
glycerol gradient centrifugation, and the fractions were
analyzed for biotinylated
synaptophysin by streptavidin-agarose adsorption followed by immunoblotting of
bound material with antisynaptophysin. (A) Synaptophysin immunoreactivity in the SLMV-containing fractions is expressed as percent of total biotinylated synaptophysin. Data are
the mean of two (0, 10, 180 min) or three (60 min) independent experiments; bars indicate the variation of individual values from
the mean or the standard error, respectively. (B and C) Immunoblots; 2.5% (B) and 11% (C) of the total biotinylated synaptophysin was recovered in the SLMV-containing fractions (B, n 5-8;
C, n 4-8).
[View Larger Version of this Image (29K GIF file)]
). This raised the question
whether in PC12 cells, SLMVs originate indeed from early
endosomes as previously assumed (Johnston et al., 1989
;
Clift-O'Grady et al., 1990
; Cameron et al., 1991
; Linstedt
and Kelly, 1991
; Régnier-Vigouroux et al., 1991
; Bauerfeind et al., 1993
) or perhaps directly from the plasma membrane. In nonneuroendocrine cells, reduced temperature
(16-20°C) has been shown to differentially affect certain
membrane traffic steps in the endocytic system (Dunn et al.,
1980
; Wolkoff et al., 1984
; Mueller and Hubbard, 1986
). We
therefore explored whether a reduction in temperature to
18°C could be used to accumulate cell surface-biotinylated synaptophysin in, and hence to facilitate characterization of, the membrane compartment from which SLMVs originate.
reported while the present work was in progress,
which show that an epitope-tagged VAMP/synaptobrevin mutant labeled by an antibody bound to its extracellular
domain does not appear in SLMVs at 15°C but does so at
37°C.
Fig. 4.
Biotinylated synaptophysin does not appear in
SLMVs at 18°C. PC12 cells
were incubated with sulfoNHS-LC-biotin either for 30 min at 37°C (A), for 30 min at
18°C (B-E), or for 30 min at 18°C followed by a 30-min
chase at 37°C (F). The 12,000 g
(A-D) or 66,000 g (E and F)
supernatants prepared from
the cells were subjected to
glycerol gradient centrifugation, and the fractions were
analyzed for biotinylated (BSy; A, B, and F) and nonbiotinylated (NB-Sy; D and E)
synaptophysin and biotinylated transferrin receptor (BTfR; C) by streptavidin-agarose adsorption followed by
immunoblotting of bound
(A-C and F) and unbound
(D and E) material with the
respective antibodies. The
immunoblots shown in (B) and (C) were obtained from the same
filter. 16% (A) and 15% (F) of the total biotinylated synaptophysin was recovered in the SLMV-containing fractions (A, n
5-8; F, n 4-8); the immunoblot shown in F is a longer exposure relative to that shown in A. Note that the ratio of SLMVs to the larger membranes recovered in the bottom fractions of the gradient is greater in E than D, because a 66,000 g and a 12,000 g supernatant, respectively, was subjected to glycerol gradient centrifugation.
[View Larger Version of this Image (28K GIF file)]
), and most
of the biotinylated synaptophysin was accessible to avidin
(Fig. 5 A). In contrast, when PC12 cells biotinylated for 30 min at 18°C and chased for 5 min at 18°C were exposed to
avidin at 4°C, the amount of biotinylated synaptophysin that subsequently could be adsorbed to streptavidin-agarose was not reduced compared to control (Fig. 5 B). Furthermore, the same amount of synaptophysin bound to
streptavidin-agarose when PC12 cells biotinylated for 15 min at 18°C were exposed to avidin at either 18° or 4°C
(data not shown). These results indicated that the synaptophysin biotinylated at 18°C was no longer exposed at the
cell surface.
Fig. 5.
Differential sensitivity to extracellular probes
of synaptophysin and transferrin receptor biotinylated
at 18°C. PC12 cells were incubated with sulfo-NHS-LC-
biotin (A and B) or sulfoNHS-SS-biotin (C and D)
for 60 min at 4°C (A) or for
30 min at 18°C (B-D), chased for 5 min at 18°C in the presence of glycine (B-D) or not
chased (A), and incubated at
4°C in the absence () or
presence (+) of extracellularly
added avidin (A and B) or MesNa (C and D). Synaptophysin and transferrin receptor in the postnuclear supernatants were analyzed for
binding to streptavidin-agarose by immunoblotting of bound and unbound material with the respective antibodies. Streptavidin-bound
biotinylated synaptophysin and transferrin receptor present in the postnuclear supernatant is expressed as percentage of total (sum of
streptavidin-bound and streptavidin-unbound synaptophysin and transferrin receptor, respectively). (A) Data are the mean of two independent experiments; bars indicate the variation of the individual values from the mean. (B-D) Data are the mean of three independent
experiments; bars indicate SD.
[View Larger Version of this Image (24K GIF file)]
) have revealed the existence of an intermediate, referred to as a
deeply invaginated coated pit, which is thought to still be
connected to the plasma membrane via a narrow neck, because the lumen of the pit is not accessible to the 68,000-D
protein avidin but is sensitive to the small (150 D) membrane-impermeant, thiol-reducing agent MesNa. Given this
finding and the inaccessibility of the synaptophysin biotinylated at 18°C to avidin, we investigated whether or not this synaptophysin was sensitive to MesNa added to the
medium. For this purpose, we used sulfo-NHS-SS-biotin
in which the biotin moiety after crosslinking to protein can
be cleaved off by thiol reduction. As was the case for sulfoNHS-LC-biotin, labeling of synaptophysin with sulfo-NHSSS-biotin did not interfere with its ability to be sorted to
SLMVs (data not shown; see Fig. 7). When PC12 cells biotinylated for 30 min at 18°C with sulfo-NHS-SS-biotin and
chased for 5 min at 18°C were treated with MesNa at 4°C,
~70% of the biotinylated synaptophysin was found to be
MesNa sensitive, as indicated by the decrease in binding
to streptavidin-agarose compared to the control (Fig. 5
C). In contrast, transferrin receptor biotinylated in the
same condition was completely protected from MesNa
(Fig. 5 D). These results show that the majority of synaptophysin biotinylated at 18°C is internalized into a compartment that has a narrow connection to the plasma membrane allowing the passage of MesNa but not avidin and in
which there is no detectable transferrin receptor.
Fig. 7.
SLMV biogenesis
involves the MesNa-sensitive population of synaptophysin molecules biotinylated
at 18°C. PC12 cells were incubated with sulfo-NHS-SS-biotin for 30 min at 18°C, chased
for 5 min at 18°C in the presence of glycine, incubated at
4°C in the absence (Control)
or presence of extracellularly added MesNa, and chased for 10 min at 37°C. The 66,000-g supernatants prepared from the cells
were subjected to glycerol gradient centrifugation, and fractions
were analyzed for biotinylated synaptophysin by streptavidin- agarose adsorption followed by immunoblotting of bound material with anti-synaptophysin antibodies. 11% and 4% of the total
synaptophysin present in the sum of the 66,000 g pellet plus supernatant bound to streptavidin-agarose without and with MesNa
addition before the 37°C chase, respectively.
[View Larger Version of this Image (18K GIF file)]
Fig. 6.
Time course of accumulation of biotinylated
synaptophysin and transferrin receptor at 18°C. PC12
cells were incubated with (open and closed circles) or
without (closed triangles)
sulfo-NHS-LC-biotin at 18°C
for the indicated times. The
cells incubated without sulfoNHS-LC-biotin at 18°C were
subsequently biotinylated for
30 min at 4°C (closed triangles). Postnuclear supernatants prepared
from the cells were analyzed for biotinylated and nonbiotinylated synaptophysin and transferrin receptor by streptavidin-
agarose adsorption, followed by immunoblotting of bound and
unbound material with the respective antibodies. Biotinylated
synaptophysin and transferrin receptor is expressed as percent of
total (sum of biotinylated plus nonbiotinylated synaptophysin
and transferrin receptor, respectively). Data points without error
bars represent single determinations. Data points with error bars
represent the mean of three (transferrin receptor), four (synaptophysin at 10 and 30 min), or two (synaptophysin at 20 min) independent determinations; bars indicate SD or the variation of the
individual values from the mean.
[View Larger Version of this Image (26K GIF file)]
Fig. 8.
SV2 is accessible to
MesNa after biotinylation at
18°C. PC12 cells were incubated with sulfo-NHS-SS-
biotin for 30 min at 18°C,
chased for 5 min at 18°C in
the presence of glycine, and incubated at 4°C in the absence (control) or presence
of extracellularly added MesNa. Transferrin receptor, synaptophysin, and SV2 in the postnuclear supernatants were analyzed
for binding to streptavidin-agarose by immunoblotting of bound
and unbound material with the respective antibodies. Streptavidin-bound biotinylated transferrin receptor, synaptophysin, and
SV2 present in the postnuclear supernatant were calculated as
percentage of total (sum of streptavidin-bound plus streptavidinunbound transferrin receptor, synaptophysin, and SV2, respectively), and the individual values obtained after MesNa treatment were expressed as percentage of control. Data are the
mean of three independent experiments; bars indicate SD. The
mean values of biotinylated protein for the control condition were 25.3% (transferrin receptor), 7.5% (synaptophysin), and
3.1% (SV2).
[View Larger Version of this Image (38K GIF file)]
Fig. 9.
SLMVs originate
from an avidin-protected,
MesNa-accessible compartment at 37°C. PC12 cells
were incubated with sulfoNHS-SS-biotin for 2 min at
37°C, chased for 10 min at
37°C, and incubated at 4°C in
the absence (Control) or
presence of extracellularly
added MesNa (A, B, and D)
or avidin (C). The 66,000 g
pellets and supernatants were
prepared from the cells and
the supernatants subjected to glycerol gradient centrifugation. Synaptophysin in the
glycerol gradient fractions (A)
and transferrin receptor and
synaptophysin in the 66,000 g
pellets (B-D) were analyzed
for binding to streptavidin- agarose by immunoblotting of
bound and unbound material
with the respective antibodies.
(A) A representative experiment showing biotinylated
synaptophysin in the glycerol
gradient fractions. (B-D) Biotinylated synaptophysin (B
and C) and transferrin receptor (D) in the 66,000 g pellet
is expressed as percent of total
(sum of streptavidin-bound
plus streptavidin-unbound synaptophysin and transferrin receptor, respectively, present
in the sum of 66,000 g pellet
plus supernatant). Data are
the mean of four (MesNa) or
two (Avidin) independent experiments; bars indicate SD
or the variation of the individual values from the mean.
[View Larger Versions of these Images (20 + 36K GIF file)]
Fig. 10.
Kinetics of the acquisition of MesNa inaccessibility. PC12 cells were incubated with sulfo-NHS-SS-
biotin for 2 min at 37°C, chased for the indicated
times at 37°C, and incubated
at 4°C in the absence (controls, 0 and 30 min chase
only) or presence of MesNa.
Synaptophysin and transferrin receptor in the cell lysates were analyzed for binding to
streptavidin-agarose by immunoblotting of bound and unbound
material with the respective antibodies. Synaptophysin and transferrin receptor bound to streptavidin-agarose were calculated as
percentage of total (sum of streptavidin-bound plus streptavidinunbound synaptophysin and transferrin receptor, respectively)
and are expressed as percentage of control (mean of the control
values at 0 and 30 min of chase, which were very similar to each
other). Data are the mean of two independent experiments; bars
indicate the variation of the individual values from the mean and,
for some time points, are within the size of the symbol. In the
control, 4.2 ± 0.3% and 11.0 ± 1.3% of the total synaptophysin and transferrin receptor were biotinylated, respectively.
[View Larger Version of this Image (21K GIF file)]
). Given the above biochemical data, we reinvestigated the subcellular localization of synaptophysin. Confirming previous observations
(Cameron et al., 1991
), synaptophysin was indeed found to
be colocalized with internalized transferrin when fixed PC12 cells were permeabilized with Triton X-100 and analyzed by double fluorescence using confocal microscopy
(Fig. 11, C, D, c, and d). Synaptophysin immunoreactivity
and fluorescent transferrin were predominantly observed
in a perinuclear location. Surprisingly, a strikingly different subcellular localization of synaptophysin was observed
when fixed PC12 cells were permeabilized with digitonin (Fig. 11, A, B, a, and b). In this condition, synaptophysin
immunoreactivity was confined to the cell periphery (Fig.
11, A and a) and was remarkably distinct from the largely
perinuclear localization of transferrin fluorescence (Fig.
11, B and b) or immunofluorescence for the transferrin receptor using an antibody against its cytoplasmic domain
(data not shown). This difference in the intracellular pattern of synaptophysin immunoreactivity depending on the
detergent used reflects a phenomenon that will be described in detail elsewhere (Hannah, M.J., and W.B. Huttner, manuscript in preparation) and is just briefly summarized here. Upon digitonin permeabilization of fixed PC12
cells, the anti-synaptophysin antibody has access only to
synaptophysin localized at the cell periphery but not to
that localized in the perinuclear area. By contrast, Triton
X-100 permeabilization results in the virtually complete
extraction, despite paraformaldehyde fixation, of synaptophysin from the cell periphery and allows access of the
anti-synaptophysin antibody to synaptophysin in the perinuclear area. (This extration of synaptophysin from fixed
cells can be demonstrated biochemically [Fig. 12 A]; Hannah, M.J., and W.B. Huttner, manuscript in preparation.)
Fig. 11.
Double fluorescence analysis of PC12 cells
for synaptophysin and internalized transferrin. PC12
cells were incubated with Texas red-labeled transferrin for 30 min at 37°C, fixed
with paraformaldehyde, permeabilized with either digitonin (A, B, a, and b) or Triton X-100 (C, D, c, and d), and
labeled with anti-synaptophysin followed by Cy2-
labeled secondary antibody.
Cells were analyzed by confocal laser scanning microscopy for synaptophysin immunoreactivity (A, a, C, and
c) or internalized transferrin
(B, b, D, and d). Single optical sections in the xy (A-D)
or xz (a-d) planes of the
same cells are shown. The white triangular indentations at the margins of the panels indicate the approximate positions of the respective corresponding plane. The same confocal microscope parameters were used for the image pairs shown in panels A and C, B and D,
a and c, and b and d. Note that upon permeabilization with digitonin, synaptophysin immunoreactivity is detected at the cell periphery but not in the perinuclear area which, however, shows transferrin fluorescence. After permeabilization with Triton X-100, the synaptophysin immunoreactivity at the cell periphery is decreased concomitant with its appearance in the perinuclear area, where it is largely colocalized with transferrin.
[View Larger Version of this Image (68K GIF file)]
Fig. 12.
Synaptophysin biotinylated at 18°C is quantitatively
extracted from paraformaldehyde-fixed PC12 cells by Triton
X-100 (A) and is accessible to anti-synaptophysin after digitonin
permeabilization of fixed cells (B). (A) PC12 cells were incubated
without () or with (+) sulfo-NHS-LC-biotin for 30 min at 18°C,
chased for 5 min at 18°C in the presence of glycine, and fixed (+)
or not fixed (
), and Triton X-100 extracts were subjected to
streptavidin-agarose adsorption. Specific immunoreactivity due
to the binding of biotinylated synaptophysin to streptavidin-agarose was determined by incubating the beads without (
) or with
(+) anti-synaptophysin antibody (
-Sy) followed by HRP-conjugated goat anti-mouse IgG antibody. Data are the mean of values obtained from three coverslips; bars indicate SD. (B) PC12
cells were incubated without (
) or with (+) sulfo-NHS-LC-
biotin for 30 min at 18°C, chased for 5 min at 18°C in the presence
of glycine, fixed, permeabilized with digitonin, incubated without
(
) or with (+) anti-synaptophysin (
-Sy), and extracted with
Triton X-100. The Triton extracts were subjected to streptavidin-
agarose adsorption, and anti-synaptophysin bound to the beads
via biotinylated synaptophysin was detected by HRP-conjugated
goat anti-mouse IgG antibody. Data are the mean of values obtained from three coverslips; bars indicate SD. The lower synaptophysin immunoreactivity in B than A (compare ordinate scales)
presumably reflects incomplete accessibility of the anti-synaptophysin to its epitope when added to digitonin-permeabilized fixed
cells as compared with anti-synaptophysin addition after Triton
X-100 extraction of synaptophysin and its adsorption to streptavidin-agarose beads.
[View Larger Version of this Image (29K GIF file)]
Fig. 13.
Morphology of the SLMV donor compartment. PC12 cells incubated at 18° (A-D) or 37°C (E-H) with sulfo-NHS-LC-biotin
for 30 min followed by a 5-min chase were fixed at 4°C. Ultrathin cryosections were immunogold-labeled for synaptophysin and analyzed by electron microscopy. Note the synaptophysin immunoreactivity associated with tubulo-cisternal membrane structures beneath
the plasma membrane. Little synaptophysin immunoreactivity is found at the plasma membrane itself. Bars, 100 nm.
[View Larger Version of this Image (110K GIF file)]
Discussion
Fig. 14.
Model for the biogenesis of SLMVs from a subplasmalemmal compartment connected to the plasma membrane. T,
transferrin receptor; S, synaptophysin. Segregation of synaptophysin from the transferrin receptor occurs at the plasma membrane. The transferrin receptor is internalized via endocytic vesicles
to endosomes located predominantly in the perinuclear region
(MesNa protected), from which it recycles to the plasma membrane. Both endocytosis and recycling occur at 18°C. Synaptophysin, but not the transferrin receptor, moves into the SLMV
donor compartment via lateral mobility and/or membrane invagination. The SLMV donor compartment, located at the periphery of the cell, is connected with the plasma membrane via a narrow membrane continuity allowing entry of MesNa, but not avidin, at 4°C. From the SLMV donor compartment, a minor proportion of
synaptophysin (10-15%) is incorporated into SLMVs. This process is blocked at 18°C. The majority of synaptophysin is either
delivered to perinuclear, MesNa-protected endosomes from which
it recycles, like the transferrin receptor, to the plasma membrane
(broken arrows) or moves to MesNa-inaccessible membranes
connected to the SLMV donor compartment (not illustrated). For further details, see Discussion.
[View Larger Version of this Image (26K GIF file)]
observed
SLMV biogenesis upon reversal of a 15°C temperature
block during which VAMP/synaptobrevin, tagged with a
monoclonal antibody, had been internalized. Further constriction of this membrane connection appears to occur
upon cooling cells to 4°C; during biotinylation at 18°C, biotinylated synaptophysin accumulated in the SLMV donor
compartment, whereas sulfo-NHS-LC-biotin added at 4°C
to cells preincubated at 18°C did not reach the synaptophysin in this compartment.
). Perhaps, as a means of regulation, the SLMV
donor compartment even pinches off from the plasma membrane and reconnects with it. This would be reminiscent of
the acid-secreting gastric parietal cell, for which the reversible connection of an internal membrane compartment (rather than an exocytic-endocytic vesicle) with the plasma
membrane has been hypothesized (Forte and Yao, 1996
;
Pettitt et al., 1996
).
); occasionally, these
were seen in continuity with the plasma membrane, consistent with our biochemical results.
). Given its structure, the SLMV donor compartment
is also likely to become physically separated from the plasma
membrane upon homogenization of cells. This would explain why the SLMV donor membranes from PC12 cells
incubated at 15°C sedimented differently from the plasma membrane (Desnos et al., 1995
).
; Cameron et al., 1991
). Given the SLMV donor
compartment described here, where does the segregation
of synaptophysin and transferrin receptor occur? A key
observation of the present study was that the SLMV donor
compartment did not contain detectable levels of biotinylated transferrin receptor, as judged from its inaccessibility
to MesNa at both 37° and 18°C. This suggests that the transferrin receptor and synaptophysin segregate at the plasma membrane, with transferrin receptor being internalized directly from the plasma membrane via endocytic vesicles
and synaptophysin moving into the SLMV donor compartment by lateral mobility and/or membrane invagination
(Fig. 14).
). This apparent discrepancy to our data is resolved by
the finding that despite formaldehyde fixation, Triton X-100,
which is commonly used to permeabilize cells for immunofluorescence analysis (Cameron et al., 1991
), quantitatively extracts synaptophysin from the SLMV donor compartment. Hence, in the previous study (Cameron et al.,
1991
), only the subpopulation of synaptophysin molecules
present in transferrin receptor-containing membranes, i.e., endosomes, was seen.
; Johnston et al., 1989
). It follows that de novo biogenesis of SLMVs accounted for only a fraction of the biotinylated synaptophysin that appeared in SLMVs. If we assume that in our experiments at 37°C the cells were in
steady state, this in turn implies that the biotinylated synaptophysin in SLMVs that could not be accounted for by de
novo biogenesis reflected the recycling of SLMV membrane.
); or (c) in both.
;
Wiedenmann et al., 1988
; Clift-O'Grady et al., 1990
; De
Camilli and Jahn, 1990), it is likely that the SLMV donor
compartment described here for PC12 is related to the
compartment implicated in synaptic vesicle biogenesis in
nerve cells. Neurons are thought to contain at least two
types of endosomes, the somatodendritic, transferrin receptor-containing, "housekeeping" endosome and the axonal, transferrin receptor-lacking, "specialized" endosome
thought to be involved in synaptic vesicle biogenesis and
recycling (Cameron et al., 1991
; Kelly, 1993a
; Mundigl
et al., 1993
). Our data showing the absence of detectable
levels of transferrin receptor in the SLMV donor compartment of PC12 cells further support its close relationship to
the axonal endosome. Moreover, our data support the notion (Kelly, 1993a
) that the somatodendritic and axonal
types of endosomes can coexist in the absence of obvious
cell polarity.
proposed, on the basis of electron microscopic observations of K+-depolarized neurons
in culture and isolated nerve terminals, that the axonal endosome may be in continuity with the plasma membrane,
and that the reformation of synaptic vesicles after exocytosis may occur in a single vesicle budding step from these deep membrane invaginations of the presynaptic plasma
membrane. Our results concerning the donor compartment of SLMVs are fully consistent with the observations
of Takei et al. (1996)
. Together, the latter report and the
present study suggest that a plasma membrane-connected
SLMV/synaptic vesicle donor compartment may have a
central role in both SLMV/synaptic vesicle de novo biogenesis and reformation.
Received for publication 15 October 1996 and in revised form 10 February 1997.
1. Abbreviation used in this paper: SLMV, synaptic-like microvesicle.We thank Drs. P. DeCamilli and I. Trowbridge for their generous gifts of monoclonal antibodies, Andrea Hellwig for performing the electron microscopy, Ruth Jelinek for excellent technical assistance, and Alan Summerfield for artwork.