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The Journal of Neuroscience, November 1, 2000, 20(21):7986-7993
Clathrin-Mediated Endocytosis near Active Zones in Snake
Motor Boutons
Haibing
Teng and
Robert S.
Wilkinson
Department of Cell Biology and Physiology, Washington University
School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
We have used the activity-dependent probe FM1-43 with electron
microscopy (EM) to examine endocytosis at the vertebrate nerve-muscle synapse. Preparations were fixed after very brief neural stimulation at
reduced temperature, and internalized FM1-43 was photoconverted into an
electron-dense reaction product. To locate the reaction product, we
reconstructed computer renderings of individual terminal boutons
from serial EM sections. Most of the reaction product was seen in
40-60 nm vesicles. All of the labeled vesicles were clathrin-coated,
and 92% of them were located within 300 nm of the plasma membrane,
suggesting that they had undergone little processing after retrieval
from their endocytic sites. The vesicles (and by inference the sites)
were not dispersed randomly near the plane of the membrane but
instead were clustered significantly near active zones.
Additional reaction product was found within putative macropinosomes;
these appeared to form from deep membrane invaginations near active
zones. Thus two mechanisms of endocytosis were evident after brief
stimulation. Endocytosis near active zones is consistent with the
existence of local exo/endocytic cycling pools. This mechanism also
might serve to maintain alignment of active zones with postsynaptic
folds during periods of activity when vesicular and plasma membranes
are interchanged.
Key words:
clathrin; endocytosis; nerve terminal; neuromuscular
junction; neurosecretion; optical probes; vesicle processing
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INTRODUCTION |
The cycle of transmitter exocytosis
and subsequent endocytosis of spent vesicular membranes first was
described nearly three decades ago (Couteaux and Pecot-Dechavassine,
1970 ; Heuser and Miledi, 1971; Ceccarelli et al., 1973, 1979; Heuser
and Reese, 1973). Although exocytosis was associated with active zones
(AZs; Couteaux and Pecot-Dechavassine, 1970, 1974), the endocytic
process was unclear in two respects: the type of endocytosis that was used under physiological conditions and the location at which, relative
to the AZ, endocytosis took place (see Heuser, 1989). Additional
information about exocytosis, endocytosis, and their coupling has
become available from a variety of recent techniques (for review, see
Angleson and Betz, 1997). However, the questions first debated by
Heuser, Ceccarelli, and their colleagues have not been answered completely.
Three endocytic mechanisms have been proposed. Clathrin-mediated
endocytosis, evidenced by coated pits and coated vesicles (Heuser and
Reese, 1973), is the standard model. Macropinocytosis (Kadota et al.,
1994; Takei et al., 1996), or bulk endocytosis of plasma membrane, is
probably responsible for the formation of large "cisternae" within
terminals; clathrin-mediated budding from the cisternae (and perhaps
also from noninternalized membrane invaginations; Takei et al., 1996)
then produces vesicles similar or identical to those endocytosed
directly from the plasma membrane. "Kiss and run" transmitter
release (Fesce et al., 1994; Ales et al., 1999; Daly et al., 2000)
refers to the putative process whereby exocytosis is followed by
endocytosis of the same membrane via a single fusion pore.
Activity-dependent probes taken up by kiss and run necessarily would
appear at or near AZs, the precise location reported by Ceccarelli et
al. (1973, 1979) for the uptake of dextran and for the formation of
pits in freeze-fracture replicas. In contrast, clathrin-mediated
endocytosis and macropinocytosis could occur anywhere in the plasma
membrane of the terminal, but observations of Heuser and colleagues
indicated that retrieval of vesicle membrane was spatially separate
from exocytosis (Heuser, 1989). More recent indications are that two or
more endocytic mechanisms might coexist (and perhaps be spatially
distinct), with their recruitment depending on factors such as the rate
at which the nerve terminal must recycle membrane (von Gersdorff and
Matthews, 1994; Koenig and Ikeda, 1996; Matthews, 1996; Takei et al.,
1996; Kuromi and Kidokoro, 1998, 1999; Roos and Kelly, 1999; Teng et
al., 1999).
Ascertaining the relative importance of these disparate mechanisms, for
example by viewing endocytosis directly, has proven difficult. Even
small stimuli can elicit responses in which processing proceeds well
beyond the stage of initial endocytosis, obscuring the association of
activity-labeled structures with the endocytic site. We describe here a
recent synaptic preparation that overcomes this limitation. Motor
terminals of the garter snake comprise ~60 discrete boutons, which
avidly take up activity-dependent probes and become labeled with <100
low-frequency stimuli (Teng et al., 1999). Reduced temperature
(~7°C) slows vesicle processing so that vesicles remain near their
sites of endocytosis. We found that under these conditions internalized
FM1-43 was in vesicles distributed just inside the presynaptic membrane
and significantly clustered around AZs. All FM1-43-positive vesicles
were clathrin-coated, as were numerous membrane pits from which
internalized vesicles presumably arose. A few additional labeled
structures appeared to be macropinosomes that had formed from deep
membrane invaginations near AZs. Endocytosis near AZs, if appropriately
regulated, could help to stabilize the AZs and provide other advantages
to the recycling pathways.
Parts of this paper have been published in abstract form (Teng and
Wilkinson, 1999).
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MATERIALS AND METHODS |
Garter snakes (Thamnophis sirtalis) were killed by
rapid decapitation. Several contiguous segments of the
single-fiber-thick transversus abdominis muscle were dissected from the
animals, placed in reptilian saline solution, and divided as needed to provide individual three segment nerve-muscle preparations. Details of
the anatomy of the muscle, dissection procedure, and saline composition
are described elsewhere (Wilkinson and Lichtman, 1985).
Electrical stimulation and activity-dependent staining.
Preparations were placed in a dish on the stage of an inverted
microscope equipped with differential interference contrast optics. In
most experiments the dish was surrounded by ice so that the bath
temperature was ~7°C to slow clathrin-related activity (Teng et
al., 1999). For electrical stimulation the cut end of the muscle nerve
was drawn into a hook-in-oil electrode. Negative rectangular pulses (200 µsec) were delivered from an isolated stimulator. The amplitude of the pulses was set supramaximal as judged by visible contraction of
the muscle (2-5 V). A minimal number of stimuli (two to three) were
delivered to confirm function of the nerve; then the preparation was
allowed to rest for ~5 min so that endocytosis could return to
baseline (unstimulated) levels.
The styryl dye FM1-43 (Molecular Probes, Eugene, OR) was prepared as a
stock solution (4 mg/ml in 100% dimethylsulfoxide) and applied to the
bath (13.0 µM, ~7°C) before a timed period of
electrical stimulation (usually 5 Hz for 30 sec, or 150 stimuli in
total). After stimulation the preparation was allowed to remain in the
dye-containing bath for either 0.5 or 1.0 min and then washed in 4°C
reptilian saline for 10 sec (frequent solution changes) before fixation
in 2% gluteraldehyde/100 mM sodium phosphate buffer (PB)
solution for 20 min. To reduce the interference of glutaraldehyde autofluorescence with the photoconversion process, we washed the fixed
preparation further in 100 mM glycine/PB for 1.0-1.5 hr, in 100 mM ammonium chloride/distilled water for 5 min, and
in PB for 10 min (Harata et al., 1998).
Horseradish peroxidase (HRP; Type VI; 2 mg/ml; Sigma, St. Louis, MO),
was bath-applied as an alternative endocytic probe. After electrical
stimulation the dye remained in the bath for 10 sec or 1 min. Then the
preparation was washed in 4°C reptilian saline for 10 sec (frequent
solution changes) and fixed in 2% glutaraldehyde and 1%
paraformaldehyde/PB for 20 min, followed by three rinses in PB (10 min
each). Diaminobenzidine (DAB; powder, 0.5 mg/ml in PB; Sigma) and 0.2 µl/ml of H2O2 solution
(30%) were applied to the preparation at room temperature (RT) for 20 min, followed by a washing in PB.
Photoconversion of FM1-43. FM1-43 staining was
photoconverted to an electron-dense reaction product, using the
protocol of Harata et al. (1998). Incubation with filtered ice-cold DAB
(tablets, 1.3 mg/ml; Sigma) was for 15 min in the dark at RT. Then the
preparation was transferred to an upright microscope equipped with
fluorescein epifluorescence optics (100 W mercury lamp) and a water
immersion objective (40×; 0.55 numerical aperture). A small region of
the endplate band of the stimulated muscle was "mapped" so that it could be identified later for embedding and electron microscopy (EM).
Illumination of several labeled terminals in this region continued
until fluorescence staining was bleached completely and the DAB
reaction product was visible (9-15 min). During this time the
preparation was kept cool by changes of DAB solution every 5 min. The
time of illumination was critical to produce a suitably dense reaction
product without concomitant ultrastructural damage. After
photoconversion a final rinse in PB (three times for 10 min each) was
done before preparation for EM.
To exclude the possibility that the photoconverted product that was
seen was artifact, we either did not illuminate additional FM1-43-stained nerve terminals after DAB incubation (n = 2) or illuminated them without incubation in DAB reagent
(n = 1). EM confirmed that no structures were labeled.
Electron microscopy. Preparations were post-fixed in 1 or
3% osmium dissolved in PB for 1 hr and rinsed with PB buffer (6×). Previously "mapped" regions containing illuminated terminals
(FM1-43) or regions of the endplate band that were visualized with a
dissecting microscope (HRP) were cut from the muscle, dehydrated in
acetone and propylene oxide, embedded in Epon (Araldite 502), and
processed by standard methods (see Wilkinson and Nemeth, 1989). Thin
sections (~65 nm) were cut and examined with a JEM-1200EX electron
microscope. Portions of 24 boutons were sectioned in four HRP
preparations, and portions of 34 boutons were sectioned in six FM1-43
preparations. Among the latter were serial sections obtained from 16 boutons in two preparations. Serial sections from one of these
preparations were post-stained with 1% uranyl acetate in 1% sodium
acetate and Reynold's lead citrate to enhance the visibility of the
AZs (see Results).
Electron micrographs were taken at 8000-50,000× magnification;
negatives were scanned (Agfa DuoScan, Belgium) into files on the
magnetic disk of a personal computer. Measurements such as perimeter of
synaptic vesicles and surface area of bouton sections were acquired
with Scion Image software (Scion, Frederick, MD; http://scioncorp.com).
Computerized rendering. Six sets of serial EM sections, each
representing a portion of one bouton, were reconstructed into three-dimensional renderings by the following method. Features common
to two or more sections, such as the bouton plasma membrane and
postjunctional secondary folds, were aligned first with IGL Align
(http://synapses.tch.harvard.edu). This software permits features
in each of two images (adjacent EM sections) to be seen while moving
one of them. In this way all of 22-31 sections representing one bouton
were aligned in sequence. Then objects in those aligned images
(postjunctional folds, AZs, labeled vesicles, endosomes, etc.) were
traced by IGL Trace. The locations of specific points, such as the
centers of labeled vesicles (LVs), centers of AZs, and the center of
the folds at their intersection with the synaptic cleft (FCs), were
recorded; these same coordinates comprised the three-dimensional
(x, y, z) data sets for use in statistical analyses (see
below). LVs and AZs were rendered as uniform spheres (50 nm diameter)
and thin slabs (actual dimensions of AZ density), respectively. Other
objects, such as the plasma membrane of the bouton, postjunctional
folds, deep membrane invaginations, and labeled or unlabeled endosomes,
were rendered as surfaces. A final three-dimensional image of a bouton
or an object (i.e., endosome) was integrated, colored, and modified
with three-dimensional Studio Max R3 software (Discreet, Montreal,
Quebec; http://www2.discreet.com).
Statistics. Statistical tests were performed to assess the
spatial relationship among LVs, AZs, and FCs within the presynaptic membrane. The tests compared the actual distribution of LVs with a
uniform random distribution of points of the same number and occupying
the same area (two-dimensional analyses) or volume (three-dimensional analyses). For the coordinates of each actual LV, or for those of each
randomly generated LV point, the distance to the nearest AZ was
calculated. From this a mean nearest neighbor (NN) distance was
calculated for all of the LVs in one bouton. The same AZ coordinates were used to calculate a similar mean NN distance for each of 250 random LV coordinate sets. Then the mean NN distance for LVs was ranked
among the mean NN distances of the 251 data sets. Thus the relative
rank order (rank in order of ascending mean distances/251) was taken as
an empirical p value regarding the likelihood that LVs were
located nearer AZs than would be expected were the LVs distributed
randomly. The likelihood that LVs were clustered around FCs was
computed in an identical manner. Finally, the disposition of AZs (as
candidates) with respect to FCs (as targets) was assessed. This
calculation served as a control for our measurements and statistical
analyses because the well known coincidence of AZs with folds was
evident in all individual serial sections.
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RESULTS |
We previously reported experiments in which snake motor terminals
were stimulated in the presence of an activity-dependent probe at a
reduced temperature, fixed, and viewed at the light level (Teng et al.,
1999). Two types of structures containing internalized dye were seen.
The experiments described below were intended to find and characterize
the ultrastructural correlates of those structures. Although several
boutons were studied with either HRP or FM1-43 as the endocytic marker
(see Materials and Methods), only six (all labeled with FM1-43) were
reconstructed from serial sections. In three of these boutons the EM
serial sections were left unstained to maximize the contrast of FM1-43 photoconversion product. Because AZs were not always visible, we were
unable to quantitate the spatial relation between internalized dye and
AZs. Subsequent serial EM sections of three other boutons were stained
lightly so that AZs became clearly visible, yet photoconverted FM1-43
LVs remained distinguishable from unlabeled ones. In those boutons the
location of endocytosed dye relative to AZs was studied directly. HRP
was used primarily to compare results from this well established
endocytic marker with those from the photoconversion of FM1-43.
Electron microscopy with FM1-43 and HRP
Figure 1 compares typical thin EM
sections from boutons labeled with HRP and with FM1-43. Both
preparations received 150 stimuli (5 Hz for 30 sec) in their respective
baths. Morphology was compromised by photoconversion, presumably caused
by free radical formation via the interaction of intense light with
FM1-43. Interestingly, vesicles labeled with HRP, a fluid phase marker,
often appeared hollow, whereas those labeled with FM1-43, which stains
primarily the vesicular membrane, often appeared filled (compare Fig.
1A,B, black arrowheads). We presume that
the hollow appearance of some HRP-containing vesicles was attributable
to the affinity of the enzyme for membranes, whereas the solid
appearance of some FM1-43 vesicles was attributable to spreading of the
photoreaction into the lumen of the vesicle or to photoreaction of
FM1-43 that had equilibrated into the aqueous contents of the lumen.
Fuzzy clathrin coats were seen more easily in HRP preparations but were
visible in photoconverted ones as well. Virtually all small vesicles
labeled with either marker were clathrin-coated. The main difference
between HRP and FM1-43 was the number of vesicles that were stained.
Fewer vesicles seemed to be labeled with HRP, although we cannot
quantitate this observation because only FM1-43 preparations were
sectioned serially. Consistent with this, numerous clathrin-coated but
unlabeled vesicles were seen in HRP preparations (23% of 116 coated
vesicles in 31 EM sections; white arrowhead in Fig.
1A), but few coated vesicles that did not also
contain visible reaction product (2.4% of 289 coated vesicles in 22 EM
sections) were seen in FM1-43 preparations.
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Figure 1.
Electron micrographs (65 nm tissue sections)
comparing two activity-dependent endocytic probes. A,
Horseradish peroxidase (HRP) provided satisfactory contrast to
visualize active zones (AZs, white arrows) and vesicles
labeled with reaction product (LVs). All LVs were clathrin-coated
(black arrowheads), but some coated vesicles were not
labeled (white arrowhead). Coated pits (black
arrows) and a deep membrane invagination
(asterisk) are also present in this section. Note the
affinity of HRP for the plasma membrane, particularly coated pits and
AZs. B, Photoconversion reaction product of FM1-43
(without post-staining). LVs were clathrin-coated (black
arrowheads). Coated pits (black arrow) were
visible, but putative AZs (white arrow) exhibited
low contrast without post-staining (compare Fig.
2A), possibly because FM1-43 rinsed more easily
from the AZ densities than did HRP. Note LVs in B and
coated pit in A that were close to the Schwann cell
(SC; see Results). m, Mitochondrion.
Scale bar, 500 nm.
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As shown in Figure 2, most, but not all,
LVs were the same size as uncoated vesicles. LV effective diameters
(measured perimeters/ ) were distributed over a broader range than
those of unlabeled vesicles from the same preparation (Fig.
2E). Although some LVs seemed to be "doublets"
(Fig. 2C), most of the ~8% of LVs that were larger than
unlabeled ones exhibited no special features. As discussed further
below, internalized coated vesicles were found predominantly near AZs.
This was often true of the coated pits (omega profiles) seen in the
membrane as well (Table 1; examples in
Fig. 2A,D).
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Figure 2.
Size distribution of LVs. A,
Virtually all LVs were clathrin-coated (black
arrowheads). Note LV and coated pit (black
arrow) near AZ (white arrow;
asterisk marks center of postjunctional fold). B,
C, Examples of LVs that were larger than the unlabeled vesicles
surrounding them. D, Large coated pit near AZ
(white arrow) is possible precursor to "doublet"
LVs, as shown in B and C.
E, Distribution of vesicle sizes in 57 sections from one
animal, showing substantial contribution of larger LVs to population.
Effective diameters were calculated as measured perimeters/. Shown
are FM1-43 preparations with post-staining. Scale bar, 100 nm.
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EM sections revealed numerous flattened endosomal structures
(cisternae), which generally were not labeled (Table 1; Fig. 3D,E). Endosomes are a common
feature of resting motor terminals. However, even with the brief
stimulation that was used, there was evidence for the formation of new
endosomes. Examples are in Figure 3A-D. Occasionally, fully
internalized endosomes containing FM1-43 reaction product were seen
(four structures in three serially sectioned boutons; Fig.
3D; see also Fig. 4). More often, deep unstained
invaginations of the plasma membrane were evident in one or more serial
sections (seven in three boutons; Fig. 3A-C). Like LVs,
often they were found close to AZs (see below). However, although LVs
were found over the entire presynaptic membrane, invaginations were
found more often near the outer edges of the membrane than near its
center. This was confirmed by examining all EM sections containing
invaginations (including those from preparations that were not
sectioned serially) that had been cut perpendicular to the synaptic
cleft. In this configuration the entire circumference of the bouton was
visible, appearing as an oval-shaped line. Approximately one-half of
the line was opposite the cleft (the presynaptic membrane), with the
rest opposite the Schwann cell cap. With the presynaptic portion of the
oval divided into four equal quarters, invaginations were found most
often in the two quarters near the margins (71%) rather than in the two quarters nearest the center of the presynaptic membrane (29%; n = 21). Figure 3A shows two views of a
typical invagination rendered from eight contiguous serial sections.
Four of these sections (e.g., Fig. 3B) contained the
clathrin-coated bud at the tip (arrows in Fig.
3A,B; clathrin not shown in rendered image). Such buds were
sometimes present at the tip of these deep invaginations (4 of 7), but
no coated vesicles were found nearby. Thus the structures were probably
incipient macropinosomes from which clathrin-mediated budding was about
to begin. Alternatively, we cannot rule out that the clathrin could
have been involved directly in the invagination process itself.
Clathrin-mediated budding was, however, seen unambiguously in fully
internalized endosomes, whether labeled (Fig. 3D) or not
(Fig. 3E). Figure 4 is a
stereo image of two such endosomes from which numerous vesicles were
budding at the time of fixation.
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Figure 3.
Endosomes and deep membrane invaginations in
FM1-43 preparations. A, Two views of a deep membrane
invagination, rendered from eight EM sections. Black
arrow points to budding vesicle. B, One of five
contiguous EM sections from rendering in A, showing
continuity with synaptic cleft (arrowhead); note
clathrin coat on bud (arrow). C, Double
membrane invaginations (white arrowheads) near AZ. The
invaginations and their openings to the cleft were seen in six
contiguous EM sections. D, Labeled fully internalized
endosome (left) and unlabeled fully internalized
endosome (arrowhead). Note coated LVs that may have
budded from the endosomes. E, Unlabeled endosome
containing clathrin-coated buds (arrows). Scale bars:
A, 250 nm; B-E, 500 nm.
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Figure 4.
Two budding endosomes in HRP preparation. Stereo
view is from stack of four EM sections. SC, Schwann
cell. Scale bars, 500 nm.
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Rendering of boutons
Three-dimensional reconstructions of computer tracings were made
to analyze the spatial relationship between AZs and internalized structures. Specifically, each image data set comprised the
x, y, and z coordinates of three
structures: LVs, AZs, and the centers of postjunctional folds (FCs)
where they intersect the synaptic cleft. As detailed in Table 1, the
majority (83%) of LVs was found within 300 nm of the
presynaptic membrane. Most of the remainder was found near the plasma
membrane at the back of the bouton (i.e., underlying Schwann cells;
9%) or near endosomes (5%). An example (Bouton 3 in Table 1) is shown
in Figure 5A. Each section was cut in a plane approximately parallel to that of the page; the image is
oriented vertically and rotated slightly to the left for ease of
viewing the stereo pair. A part of the bouton (which would appear
closest to the viewer; we estimate approximately one-third) was not
sectioned and rendered. Figure 5A contains most of the
postjunctional fold structure (blue; at left),
the bouton plasma membrane (gray-green), and two deep
membrane invaginations and four endosomes (all shown gray;
see Table 1). The muscle fiber (data not shown) coursed vertically left
of the bouton, which partially indented its surface. AZs (red
slabs) can be seen to lie in the presynaptic membrane, which
opposed the folds. A tiny subset of the vesicles of the bouton (LVs;
those labeled with FM1-43 and therefore recently internalized; see Fig.
1) is depicted as uniform 50 nm spheres. Figure 5B-D shows
FCs, AZs, and 118 of 141 LVs (i.e., excluding those >300 nm from the
presynaptic membrane) as they appear in two-dimensional projections
onto a plane that corresponds approximately to the presynaptic membrane in Figure 5A. Thus the horizontal dimension in Figure
5B-D corresponds to depth in Figure 5A, whereas
the vertical dimensions in Figure 5B-D and Figure
5A are equivalent. The positions of AZs (red
squares) and FCs (blue squares) are shown in Figure
5B. A few AZs appeared without folds, possibly because the
latter were not visible in certain EM sections. Because each red square
depicts the location of an AZ in a single section, AZs that were seen
opposite the same fold in two or more contiguous sections appear as
contiguous red squares. Figure 5C shows the relation of LVs
(white spheres) with AZs (red squares). In this
and in similar projections from two other boutons (data not shown), LVs
appeared to be clustered near, but usually not directly over, AZs.
Consistent with this, larger areas of the presynaptic membrane that
contained no AZs tended to contain no LVs either. The relation between
LVs and folds (data not shown) was similar: LVs seemed to be associated with FCs and were absent from areas not containing FCs. Another consistent feature seen among both LV-AZ comparisons (three boutons) and LV-FC comparisons (six boutons) was that some AZ/fold regions were
"occupied" by endocytosed vesicles whereas others were not (see
Discussion). Examples of the locations of coated pits
(yellow spheres) and deep membrane invaginations
(arrows) relative to AZs are shown in Figure 5D
(same bouton as in Fig. 5A-C) and Figure 5E
(Bouton 1 in Table 1). Relatively few of these structures were seen,
but their positions also appeared to be near AZs.
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Figure 5.
Endocytosis occurs near AZs in snake motor
boutons. A, Rendering of bouton portion from 31 serial
EM sections. Stereo pair shows postjunctional folds of muscle fiber at
left (blue). AZs (red) lie
on presynaptic membrane. LVs (white), shown as 50 nm
spheres, were found predominantly near the presynaptic membrane. Also
shown are four endosomes and two deep membrane invaginations
(gray). B, C, Projections of fold
centers (FCs; blue squares) AZs (red
squares), and LVs (white spheres) onto a
plane corresponding to that of the presynaptic membrane (see Results).
B, AZs appeared near (or in direct apposition to) FCs.
Some fold regions were not occupied by AZs. C, LVs were
found clustered near AZs (and therefore near folds as well). D,
E, Locations of coated pits (yellow
spheres) and deep membrane invaginations (gray;
arrows) relative to AZs in two rendered boutons. Bouton in
D is the same as that in A-C. Scale
bars: A, 1 µm; B-E, 1 µm.
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Statistical tests
To assess whether LVs were clustered significantly near AZs, we
asked whether the average distance separating an LV from the nearest AZ
was smaller than would be expected from random chance. For each
rendered partial bouton the positions of LVs, AZs, and FCs were
compared with 250 random data sets as described below and in Materials
and Methods. LVs located >300 nm from the presynaptic membrane,
including those that budded from endosomes, were excluded (see Table
1). In some analyses only the two dimensions corresponding to the
"plane" of the presynaptic membrane were considered (i.e., the
projections shown in Fig. 5B-E; the coordinate representing distance perpendicular to the membrane was set to zero). This simplification distorted (shortened) distances near the bouton margins
(where the presynaptic membrane was curved) but did so equally for all
three types of structures. In other analyses we represented actual and
random positions in three dimensions. This eliminated the distortion
described above but was also less than ideal because, among LVs that
budded directly from the membrane, distances traveled perpendicular to
the membrane had no meaning.
As shown in Table 2, the two-dimensional
tests confirmed the association of LVs with AZs at high levels of
significance (p < 0.004; n = 3 boutons). Similar analyses were performed to test the spatial relation
between LVs and FCs (p < 0.008 in 5 of 6 boutons). Finally, we took advantage of known morphological features of
AZs and FCs for use in verifying the statistical tests. Because AZs are
known to oppose folds precisely (as was evident in individual EM
sections), AZ and FC data were known to be associated. Consistent with
these observations, tests confirmed the correspondence of AZs and FCs
at high significance (p < 0.004;
n = 3 boutons; Table 2). All three-dimensional tests
corresponding to those described above indicated nonrandom dispositions
as well (p < 0.004; data not shown).
Correspondence with light level observations
We previously observed two types of endocytic structures in
confocal images of briefly stimulated boutons (Teng et al., 1999). Small punctate hot spots of endocytosed optical probe (sulforhodamine 101 or FM1-43) increased in size and brightness, but not in number, with increasing stimulation. The hot spots tended to oppose
postjunctional folds as do AZs, but their location relative to AZs
(which are known to be scattered along folds; Walrond and Reese, 1985)
(see also Fig. 5B) was not determined. A second class of
structures (which were larger and brighter than the first) appeared
after additional stimulation (>20 sec at 5 Hz). On the basis of the results presented above, the larger structures were fully internalized endosomes, whereas the smaller structures were vesicle clusters that
had appeared as single structures at light level. To test the latter
hypothesis, we modified EM-based renderings of internalized vesicles to
account for the diffraction limit (~200 nm) of confocal light
microscopy. An example is shown in Figure
6. A confocal image of part of an
FM1-43-labeled terminal is shown in Figure 6A, with a
magnified region shown in Figure 6B. Stimulation in the presence of FM1-43 was for the same duration, and under the same
conditions, as in the current study (see Teng et al., 1999). It can be
appreciated that the light-level hot spots were complex structures and
that some of the background staining also appeared punctate. Shown in
Figure 6C is a projection, as in Figure 5C, of
the rendering of Bouton 3 showing FM1-43 LVs. To emulate the diffraction-limited image of vesicles, we rendered each LV as a 200 nm
disk for which the brightness decreased radially from the center. The
disks were made semi-transparent so that two or more overlapping disks
(corresponding to vesicles spaced with centers <200 nm apart) became
proportionally brighter in the regions of overlap. In Figure
6D, the same image is shown slightly blurred and with
contrast and brightness adjusted to approximate optical conditions in
which only overlapping vesicle images are clearly visible. The
resemblance of Figure 6D to 6B
argues that vesicle clusters can appear as endocytic hot spots when
viewed with the light microscope.
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Figure 6.
Clusters of LVs appear as punctate hot spots of
FM1-43 staining at light level. A, Confocal
photomicrograph of FM1-43 uptake by snake motor terminal in response to
150 stimuli delivered at 5 Hz. B, Magnified view of
bracketed region in A showing large,
bright, irregular spots plus small circular spots in background.
C, Region of rendered projection from EM as in Figure
5C (same stimulation as the terminal in
A). LVs with actual diameter of ~50 nm are depicted as
200 nm partially transparent shaded disks to emulate
diffraction-limited detail in light image (see Results).
D, Contrast in C was adjusted manually to
match the image in B best. Note similarity of large
bright spots (overlapping vesicles) and small background spots
(individual vesicles) to structures in B. Scale bars:
A, 2 µm; B-D, 500 nm.
|
|
| |
DISCUSSION |
We have shown that endocytosed vesicles in snake motor terminals
cluster near active zones. The locations of virtually all LVs and AZs
in each of three serially sectioned boutons were mapped. The fraction
of each bouton that was sectioned and rendered was 50-75%, giving a
large sample size and consequent high levels of statistical
significance. Moreover, because AZs are numerous in snake boutons and
therefore separated by only ~400 nm, the association of LVs with
individual AZs revealed by statistical tests was demonstrably quite
strong. The close spacing of AZs made it difficult to appreciate the
clustering around individual AZs visually (see Fig. 5C).
However, it was evident that clathrin-mediated endocytosis occurred
quite near AZs collectively, as opposed to the classical model that has
it well away, near the Schwann cell margins (Heuser, 1989).
The question arises as to whether labeled vesicles are representative
of all of the vesicles that were endocytosed. For example, if FM1-43
were sequestered within folds, its activity (effective concentration)
might be high near AZs (which oppose folds) yet insufficient elsewhere
to produce reaction product. However, unlabeled internalized vesicles
would still be identifiable by their clathrin coat, yet virtually no
coated but unlabeled vesicles were found by using FM1-43 as a marker.
Our ability to argue in this way that all internalized vesicles were
labeled was attributable to an advantage of FM1-43 over the classical
marker HRP, which failed to label 23% of coated vesicles. This failure
may have been attributable to the high molecular weight of HRP and
limited solubility, creating the possibility that some vesicles were
internalized without even a single HRP molecule. It is also possible to
estimate the total number of endocytosed vesicles that was expected and
compare this with the actual number that was found. Under low-frequency
stimulus conditions similar to those that were used here, one snake
motor bouton releases, on average, 1.4 quanta per stimulus (Wilkinson et al., 1996). Thus ~210 quanta (vesicles) per bouton would have been
released during our stimulus protocol, which should equal the number
that was retrieved (Smith and Betz, 1996). In our renderings of partial
boutons we saw, on average, 144 LVs, 27 coated pits, plus one to two
labeled endosomes (or membrane invaginations) from which budding was in
progress at the time of fixation. This represents, at the least, a
substantial fraction of the membrane likely to have been exocytosed and
therefore argues that the majority of endocytosed vesicles was near AZs.
LVs remained close to the bouton membrane and were still coated with
clathrin. Interestingly, their size range was somewhat greater than
that of the general vesicle population. This suggests that later steps
in processing contribute to uniformity, although it remains possible
that the larger coated vesicles comprised a separate population (see De
Camilli and Takei, 1996). We infer from these observations that
processing had progressed little after internalization and that
clustering near AZs suggests internalization near AZs as well. The
location of coated pits predominantly near AZs supports the same
argument, but because too few coated pits were seen to permit
statistical analysis, it remains possible that the vesicles were
internalized elsewhere (e.g., randomly) and then traveled toward AZs
before they were fixed. Endocytosis near AZs was reported first by
Ceccarelli et al. (1973, 1979), but the activity-labeled vesicles and
profiles that were seen could not be demonstrated to contain clathrin.
Thus it was argued that either kiss-and-run exo/endocytosis was
evidenced or that the profiles and vesicles that were seen were
actually exocytotic, with endocytosis taking place elsewhere in the
terminal (see Heuser, 1989). More recently, the association of
endocytosis with active zones has been described in
Drosophila synapses either directly (the shibire
mutant; Koenig et al., 1998; Koenig and Ikeda, 1999) or indirectly via
immunofluorescence labeling of putative endocytic machinery hot spots
(Roos and Kelly, 1999). However, ours is the first demonstration (1)
that the majority of endocytosis occurs near AZs, particularly at a
conventional fast chemical synapse, and (2) that endocytosis near AZs
is clathrin-dependent (and not, for example, kiss and run). Thus our
results support the argument that regeneration of transmitter vesicles
relies exclusively on clathrin (Heuser, 1989; Takei et al., 1996).
Although endocytosis was associated with active zones at high levels of
significance, the converse did not appear to be true. As can be seen in
the example of Figure 5C, some regions of the rendered
presynaptic membrane contained AZs, but no LVs. Presumably these AZs
were silent, or nearly so, during the brief stimulation. If so, release
probability varied among AZs as it apparently does at the frog
neuromuscular junction (NMJ; see Zefirov et al., 1995). Alternatively,
some AZs might have appeared silent because relatively few stimuli were
delivered. We also emphasize that a significant fraction of LVs (9%)
and of coated pits (14%) was found within 300 nm of the Schwann cell
side of the bouton membrane. These vesicles represent a population
endocytosed far from AZs that might increase and perhaps even dominate
with more prolonged stimulation, consistent with previous observations
at the frog NMJ (Heuser, 1989). Alternatively, exocytosis might have
occurred at these sites, perhaps in some regulatory role (Robitaille,
1998). This scenario would support the principle of endocytosis near
release sites generally.
In addition to coated vesicles, three types of endosomal structures
were seen. The first type was free of reaction product, indicating that
a reserve of membrane already was internalized in the terminal before
stimulation. Second, additional endosomes contained reaction product
and therefore were formed during or just after stimulation. Finally,
deep invaginations of the plasma membrane were seen, usually near AZs.
When rendered in three dimensions, these putative incipient
macropinosomes resembled in their complex shape the other classes of
endosomes, excepting that their lumens were continuous with the
extracellular space. Another similarity among all three structures was
that each exhibited clathrin-mediated budding. These common features
suggest that the fully internalized endosomes were formed from membrane
invaginations by macropinocytosis (see also Heuser, 1989; Takei et al.,
1996) (for review, see De Camilli and Takei, 1996), and not by fusion
of vesicles (Heuser and Reese, 1973).
We found previously (Teng et al., 1999) that putative endosomes seen at
light level formed only after vesicular endocytosis had already begun,
and then only when a sufficient number of stimuli were delivered. We
now can identify this secondary endocytic pathway with
macropinocytosis. Thus the vertebrate NMJ exhibits two endocytic modes,
as do goldfish bipolar terminals (von Gersdorff and Matthews, 1994) and
motor terminals of Drosophila shibire (Koenig and Ikeda, 1996). We therefore propose a general model in which
clathrin-mediated budding from the plasma membrane is used at low
levels of activity, with macropinocytosis reserved for bursting or
prolonged activity when larger areas of membrane must be recycled
quickly. Deep membrane invaginations evidently participate in both
schemes by clathrin-mediated budding from their tips and by fission
(macropinocytosis) from the plasma membrane. The fact that
endosomes and membrane invaginations were seen to be budding after only
150 stimuli argues that macropinocytosis at the NMJ is recruited
rapidly, perhaps in anticipation of sustained activity.
Clathrin-mediated budding of individual vesicles also might expand away
from AZs and into the margins with increased activity (Heuser, 1989),
which was not studied.
Although we have visualized light-level hot spots (putative vesicle
clusters) with stimulation at room temperature (Teng et al., 1999), it
should be noted that the relative contributions of clathrin-mediated
endocytosis and macropinocytosis probably were influenced by our
deliberate use of low temperature. Clathrin-mediated endocytosis in
mouse fibroblasts is slowed only slightly below 15°C, but the ability
of vesicles to shed their clathrin coat is arrested almost totally
(Illinger et al., 1991). Snake nerve terminals may behave similarly,
because all of the labeled vesicles that were seen were still coated.
Blockade of decoating by temperature decrease might have shifted the
equilibrium between endocytic pathways toward macropinocytosis. It
would be interesting to learn whether this is an adaptation of
reptilian synapses, which necessarily function at lower temperatures
than those of mammals.
Why endocytosis occurs near AZs is unknown, but the scheme provides
several potential advantages. First, matching of endo- and exocytosis
might be achieved more rapidly (or more precisely) by several
short-range interactions limited to AZ microdomains (e.g., via
Ca2+ signaling) than by global signaling
throughout the bouton. Second, removal of membrane exactly where it is
added, if properly regulated, could ensure that the AZ remains fixed
relative to the postjunctional fold without stress on putative
cell-to-cell structural anchors (Marques et al., 2000). Third,
vesicular membrane proteins could remain separate from those in the
plasma membrane if they were recycled quite near (and soon after)
exocytosis. Thus the need for protein sorting might be reduced (see
Roos and Kelly, 1999). Last, proximity of exo- and endocytic "active
zones" permits local cycling pools (Kuromi and Kidokoro, 1998). With
this arrangement a fraction of vesicles released at a particular AZ
could be refilled for immediate use at the same AZ, without the need to
enter the reserve pool. Alternatively, the clusters of internalized
vesicles might mark entry points for transport to the reserve pool by
as yet unidentified motors. Further experimentsaimed at determining how the endocytic machinery is localized as well as the fate of endocytosed structures at later time pointsare in progress to investigate these various possibilities.
| |
FOOTNOTES |
Received July 7, 2000; revised Aug. 17, 2000; accepted Aug. 18, 2000.
This work was supported by United States Public Health Service Grant
NS-24752. We thank J. Cole and J. Heuser for helpful discussions,
P. Bridgman and G. Phillips for help with electron microscopy, D. Bishop for help with three-dimensional rendering, and K. Schechtman for
help with statistics.
Correspondence should be addressed to Dr. Robert S. Wilkinson,
Department of Cell Biology and Physiology, Washington University School
of Medicine, Box 8228, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: wilk{at}cellbio.wustl.edu.
| |
REFERENCES |
-
Ales E,
Tabares L,
Poyato JM,
Valero V,
Lindau M,
de Toledo GA
(1999)
High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism.
Nat Cell Biol
1:40-44[Medline].
-
Angleson JK,
Betz WJ
(1997)
Monitoring secretion in real time: capacitance, amperometry, and fluorescence compared.
Trends Neurosci
20:281-287[Web of Science][Medline].
-
Ceccarelli B,
Hurlbut WP,
Mauro A
(1973)
Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction.
J Cell Biol
57:499-524[Abstract/Free Full Text].
-
Ceccarelli B,
Grohovaz F,
Hurlbut WP,
Iezzi N
(1979)
Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. II. Effects of electrical stimulation and high potassium.
J Cell Biol
81:178-192[Abstract/Free Full Text].
-
Couteaux R,
Pecot-Dechavassine M
(1970)
Vesicles synaptiques et poches au niveau des "zones activies" de la jonction neuromusculaire.
Comptes Rend Acad Sci Hebd Seances D (Paris)
271:2346-2349.
-
Couteaux R,
Pecot-Dechavassine M
(1974)
Les zones specialisees des membranes presynaptique.
Comptes Rend Acad Sci Hebd Seances D (Paris)
280:299-301.
-
Daly C,
Sugimori M,
Moreira JE,
Ziff EB,
Llin
 s R
(2000)
Synaptophysin regulates clathrin-independent endocytosis of synaptic vesicles.
Proc Natl Acad Sci USA
97:6120-6125[Abstract/Free Full Text]. -
De Camilli P,
Takei K
(1996)
Molecular mechanisms in synaptic vesicle endocytosis and recycling.
Neuron
16:481-486[Web of Science][Medline].
-
Fesce R,
Grohovaz F,
Valtorta F,
Meldolessi J
(1994)
Neurotransmitter release: fusion or "kiss and run"?
Trends Cell Biol
4:1-4[Medline].
-
Harata N,
Buchanan J,
Tsien RW
(1998)
Visualization of endocytosed synaptic vesicles in hippocampal neurons.
Soc Neurosci Abstr
24:77.
-
Heuser J
(1989)
The role of coated vesicles in recycling of synaptic vesicle membrane.
Cell Biol Int Rep
13:1063-1989[Web of Science][Medline].
-
Heuser JE,
Miledi R
(1971)
Effect of lanthanum ions on function and structure of frog neuromuscular junctions.
Proc R Soc Lond [Biol]
179:247-278[Medline].
-
Heuser JE,
Reese TS
(1973)
Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.
J Cell Biol
57:315-344[Abstract/Free Full Text].
-
Illinger D,
Poindron P,
Kuhry J-G
(1991)
Fluid phase endocytosis investigated by fluorescence with trimethylamino-diphenylhexatriene in L929 cells: the influence of temperature and of cytoskeleton depolymerizing drugs.
Biol Cell
73:131-138[Web of Science][Medline].
-
Kadota T,
Mizote M,
Kadota K
(1994)
Dynamics of presynaptic endosomes produced during transmitter release.
J Electron Microsc (Tokyo)
43:62-71[Abstract/Free Full Text].
-
Koenig JH,
Ikeda K
(1996)
Synaptic vesicles have two distinct recycling pathways.
J Cell Biol
135:797-808[Abstract/Free Full Text].
-
Koenig JH,
Ikeda K
(1999)
Contribution of active zone subpopulation of vesicles to evoked and spontaneous release.
J Neurophysiol
81:1495-1505[Abstract/Free Full Text].
-
Koenig JH,
Yamaoka K,
Ikeda K
(1998)
Omega images at the active zone may be endocytotic rather than exocytotic: implications for the vesicle hypothesis of transmitter release.
Proc Natl Acad Sci USA
95:12677-12682[Abstract/Free Full Text].
-
Kuromi H,
Kidokoro Y
(1998)
Two distinct pools of synaptic vesicles in single presynaptic boutons in a temperature-sensitive Drosophila mutant, shibire.
Neuron
20:917-925[Web of Science][Medline].
-
Kuromi H,
Kidokoro Y
(1999)
The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.
J Neurosci
19:1557-1565[Abstract/Free Full Text].
-
Marques MJ,
Conchello JA,
Lichtman JW
(2000)
From plaque to pretzel: fold formation and acetylcholine receptor loss at the developing neuromuscular junction.
J Neurosci
20:3663-3675[Abstract/Free Full Text].
-
Matthews G
(1996)
Synaptic exocytosis and endocytosis: capacitance measurements.
Curr Opin Neurobiol
6:358-364[Web of Science][Medline].
-
Robitaille R
(1998)
Modulation of synaptic efficacy and synaptic depression by glia at the frog neuromuscular junction.
Neuron
21:847-855[Web of Science][Medline].
-
Roos J,
Kelly RB
(1999)
The endocytic machinery in nerve terminals surrounds sites of exocytosis.
Curr Biol
9:1411-1414[Web of Science][Medline].
-
Smith CB,
Betz WJ
(1996)
Simultaneous independent measurement of endocytosis and exocytosis.
Nature
380:531-534[Medline].
-
Takei K,
Mundigl O,
Daniell L,
De Camilli P
(1996)
The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin.
J Cell Biol
133:1237-1250[Abstract/Free Full Text].
-
Teng H,
Wilkinson RS
(1999)
Recently endocytosed vesicles cluster at discrete hot spots in snake neuromuscular boutons.
Soc Neurosci Abstr
25:473.
-
Teng H,
Cole JC,
Roberts RL,
Wilkinson RS
(1999)
Endocytic active zones: hot spots for endocytosis in vertebrate neuromuscular terminals.
J Neurosci
19:4855-4866[Abstract/Free Full Text].
-
von Gersdorff H,
Matthews G
(1994)
Inhibition of endocytosis by elevated internal calcium in a synaptic terminal.
Nature
370:652-655[Medline].
-
Walrond JP,
Reese TS
(1985)
Structure of axon terminals and active zones at synapses on lizard twitch and tonic muscle fibers.
J Neurosci
5:1118-1131[Abstract].
-
Wilkinson RS,
Lichtman JW
(1985)
Regular alternation of fiber types in the transversus abdominis muscle of the garter snake.
J Neurosci
5:2979-2988[Abstract].
-
Wilkinson RS,
Nemeth PM
(1989)
Metabolic fiber types of snake transversus abdominis muscle.
Am J Physiol
256:C1176-C1183[Abstract/Free Full Text].
-
Wilkinson RS,
Son Y-J,
Lunin SD
(1996)
Release properties of isolated neuromuscular boutons of the garter snake.
J Physiol (Lond)
495:503-514[Abstract/Free Full Text].
-
Zefirov A,
Benish T,
Fatkullin N,
Cheranov S,
Khazipov R
(1995)
Localization of active zones.
Nature
376:393-394[Medline].
Copyright © 2000 Society for Neuroscience 0270-6474/00/20217986-08$05.00/0
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Y.-F. Xu, D. Autio, M. B. Rheuben, and W. D. Atchison
Impairment of Synaptic Vesicle Exocytosis and Recycling During Neuromuscular Weakness Produced in Mice by 2,4-Dithiobiuret
J Neurophysiol,
December 1, 2002;
88(6):
3243 - 3258.
[Abstract]
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Y. Sara, M. G. Mozhayeva, X. Liu, and E. T. Kavalali
Fast Vesicle Recycling Supports Neurotransmission during Sustained Stimulation at Hippocampal Synapses
J. Neurosci.,
March 1, 2002;
22(5):
1608 - 1617.
[Abstract]
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N. Harata, T. A. Ryan, S. J Smith, J. Buchanan, and R. W. Tsien
Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion
PNAS,
October 23, 2001;
98(22):
12748 - 12753.
[Abstract]
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ABSTRACT
INTRODUCTION
Compound via MeSH
