Supporting Online Material
Methods.
Most methods have been described previously (1,2). Briefly, frog (Rana pipiens) cutaneous pectoris
neuromuscular junction preparations were dissected and mounted in sylgard-lined chambers. The normal
frog Ringer’s solution contained (in mM): 115 NaCl, 2 KCl, 1.8 CaCl2, 5 HEPES, pH=7.2, osmolarity ~224
mOsM. In the hypertonicity experiments, 500 mM sucrose was added to the solution. FM1-43 (Molecular
Probes, Eugene, Oregon) was used at 3.2 µM. Curare (10 µM) was added to the bathing solution to prevent
muscle movement during experiments that involved imaging of nerve terminals during stimulation to
investigate their fluorescence destaining. Stimulation of the nerve trunk was performed with a suction
electrode.
Fluorescence imaging. We used a Nikon Optiphot 2 upright fluorescence microscope equipped with
a Zeiss 63x, 0.9 NA, water immersion objective, a 100 W mercury lamp, excitation filters (435/10 nm or
480/30 nm), dichroic mirror (505 or 565 nm), and emission filters (535/40 nm or 605/55 nm; Chroma,
Brattleboro, VT). Image acquisition was done with a Photometrics SenSys 0400 camera (Photometrics,
Tucson, AZ) using V for Windows software (Digital Optics, Auckland NZ). Image analysis was performed
using software custom-written in Matlab (The Mathworks, Natick, MA).
Estimating vesicular and non-vesicular contributions to background fluorescence. Following
exhaustive stimulation, residual fluorescence reflects both non-releasable dye in synaptic vesicles, and a
constant, non-vesicular, fluorescence (dye trapped in postsynaptic folds, Schwann cells, and elsewhere in
the synaptic region). We separated these two components of residual background with two methods, which
agreed well. In the first method, we compared optical and electron microscopic results, as follows. We
measured fluorescence of stained terminals before and during stimulation-evoked destaining. Then, in
photoconverted preparations, we measured the fraction of the vesicles that were labeled before and after
destaining. The total fluorescence of the preparation can be expressed as: F = B + cV, where B=background
fluorescence, V=labeled vesicle fraction, and c=constant representing the fluorescence of a labeled vesicle.
Before stimulation, F0=B+cV0 ; after stimulation, F’=B+cV’ .The non-vesicular background B is given by
B= (Vo*F’ - V’*Fo)/(Vo- V’).
The second method to measure background was based solely on fluorescence measurements. We
compared fluorescence of loaded and destained terminals with fluorescence of terminals exposed to FM
dye without stimulation (both types of terminals were in the same muscle, in which some intramuscular
nerves had been cut before dye loading).
Electrophysiology. For both miniature end-plate potential recordings (used to determine the dextran
solution-induced increases in spontaneous activity, see below) and two-electrode voltage clamp recordings
(TEVC, for the hypertonicity experiments) we used an Axoclamp-2A (Axon Instruments) amplifier. The
signal was digitized using a MIO-16E-4 interface (National Instruments). The data were acquired using
WCP for Windows (John Dempster, University of Strathclyde, UK) and analyzed using the same software
or custom-written Matlab routines. Micropipettes (filled with 3M potassium acetate, 15-25 MΩ resistance)
were pulled with a Flaming-Brown micropipette puller (Sutter, Novato, CA). Resting potentials were
generally more negative than –70 mV. In the TEVC recordings the cells were held at potentials equal to
their initial membrane potential; hypertonic solution (gravity-fed) was applied through a large bore pipette
(0.88 mm) placed within ~0.5-1 mm from the electrode impalement site. To determine the number of
quanta released by hypertonic stimulation (the size of the RRP in this experiment), for each fiber, we
divided the hypertonicity-induced current by the integrated current corresponding to an average
spontaneous event. The hypertonicity-induced current was measured from the beginning of hypertonic
sucrose application to the plateau of the current curve (since release during the plateau may represent
reserve pool mobilization). The plateau was reached after 25.4+/-5.6 seconds (n=18). On average, 73,000
+/- 6,000 quanta (n=18) were released by this treatment, which represents about 15% of the vesicles in a
terminal containing ~500,000 vesicles total (3,4), in reasonable agreement with other optical and
electrophysiological estimates of RRP size. Applying hyperosmotic solution for ~25 seconds results in
similar FM dye uptake (at the fluorescence level) and similar numbers of vesicles labeled (at the
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photoconversion level) with our standard readily releasable pool-releasing stimulus (10-second, 30 Hz).
Also, in a separate experiment we investigated the release of vesicles that had been labeled via sucrose
application, comparing the destaining rate with that of 10-second stimulated terminals. The two destaining
curves were similar (with the caveat that the experiment was performed after prolonged washing of the
preparations, and thus some functional mixing of the RRP and reserve pools may have happened; results
not shown). Thus, the sucrose-released pool did not seem to be significantly different from the RRP.
Photoconversion. FM dye-labeled preparations were fixed by plunging into fixative solution cooled
to about –2 ºC. We used two fixation solutions, with similar results: 2% glutaraldehyde in normal frog
Ringer, and 1% glutaraldehyde in 0.1 M phosphate buffered saline (PBS; pH=7.2, osmolarity similar to that
of the frog Ringer). In the experiments investigating clathrin-coated objects we used a high osmolarity
fixative (2% paraformaldehyde plus 2% glutaraldehyde). Fixation was performed at about -2ºC for 40-60
minutes. The preparations were then washed with PBS for 45 minutes (with continuous agitation; three
solution changes), incubated in PBS containing 0.1M ammonium chloride for 10 minutes, and washed
again for 30 minutes (three solution changes). Washing was performed at 4ºC. The preparations were then
incubated in diaminobenzidine (DAB, 1.5mg/ml in 0.1 M PBS) for 30 minutes (4ºC). The preparations
were then illuminated via a Nikon 20X, 0.45 NA objective, using a 100W mercury lamp. The nerve
terminals lost FM dye fluorescence and became brown-black as DAB precipitate accumulated after 30-50
minutes of illumination. The DAB solution bathing the muscle was refreshed every ~30 minutes. The
photoconversion reaction was then stopped and the nerve terminals were processed for electron
microscopy.
Electron Microscopy. The preparations were washed in PBS for 10 minutes, and post-fixed with
osmium for 60 minutes at room temperature (2% OsO4 in 0.1 M PBS). They were then dehydrated through
an ascending series of ethanol solutions and propylene oxide (PO, 100%), incubated for 12-18 hours with a
50% Epon solution in PO, under continuous agitation, incubated with 100% Epon for 6-8 hours in open
vials to allow for PO diffusion out of the tissue, and embedded in Epon (12-20 hours, 60ºC). The blocks
were then cross-sectioned and silver-gold (80-90 nm thickness) sections were collected. Light post-staining
of the sections was then performed (1 minute in presence of 2% Uranyl Acetate in 50% ethanol). Electron
micrographs were acquired using a CM-10 Philips electron microscope, enlarged 2.69-fold by transfer to
photographic paper (Eastman Kodak Company, Rochester, NY), digitally scanned at 300-600 dpi and
analyzed by custom written Matlab routines. For serial reconstructions, we digitized the positions of
presynaptic active zones, surface membranes, cluster edges and vesicles, and reconstructed the terminals
assuming a thickness of 80 nm for each section, using Matlab routines. The reconstructed nerve terminal
segments were obtained from three different animals; two of the 12 segments came from a single nerve
terminal. Three-dimensional measurements were done with the assumption that each element found in a
section was centered within the depth of the section.
Rapid freezing. 10,000 Da dextran was used as an endocytic marker, at 10% concentration (w/v).
Larger concentrations (15-30%) caused strong increases in the spontaneous vesicle release, coupled with
adverse effects on morphology (especially at 20-30%; results not shown). 10% dextran caused only a slight
increase in spontaneous release, which peaked at ~4-fold (5 minutes of incubation), and was not significant
after about 10 minutes of incubation, when the experiments were performed; also, it had no discernable
effect on the morphology of the nerve terminals. The preparations were dissected, incubated in 10%
dextran for 10 minutes and stimulated at 2Hz for 2.5 minutes (300 shocks). For high-pressure freezing,
nerve-muscle portions of ~1 mm2 were cut, placed within copper specimen holders, rapidly loaded into a
high-pressure freezer (BAL-TEC HPM-010, Technotrade International, Manchester, NH), pressurized to
~2050 bar and frozen. They were then collected from the holders under liquid nitrogen, freeze-substituted
at -90 to -80°C with an acetone solution containing osmium tetroxide and finally embedded in Epon. For
plunge-freezing, 2-3 mm2 specimen portions were cut and rapidly immersed in liquid ethane (cooled by a
surrounding bath of liquid nitrogen). The samples were then collected and processed as above. Embedded
specimens were processed as described in the previous section. Dextran labeling was more difficult to
address than photoconversion labeling; we counted as labeled only the vesicles in which we discerned
electron-dense particles that did not appear to be bound to the vesicle membrane. As in the work of
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Ceccarelli and collaborators (4), we were unable to distinguish particles of the size of single dextran
molecules (~3 nm in diameter; 4); aggregates of 6-9 nm were generally observed. Specimen freezing and
freeze substitution were performed at the Laboratory for 3-D Fine Structure, Dept. of MCD Biology,
University of Colorado, Boulder, Colorado. For a more detailed description of the methods, see (5).
Data analysis. The positions of presynaptic active zones (defined as dense regions of the
presynaptic membrane, 6), plasma membrane, vesicle cluster edges and individual vesicle centers were
drawn by hand onto the scanned images (using a high resolution monitor and Matlab software), and the
distances from the center of each vesicle to nearest active zone and surface membrane were determined. To
distinguish labeled and unlabeled vesicles from each other, we used one of two tests: lumenal density or
ratio of lumenal to membrane density. Both tests gave comparable results, producing bimodal distributions
with little (~2%) overlap. For lumenal density, we analyzed all vesicles in 20 photoconverted nerve
terminals sections, and in each section a bi-modal distribution was observed when density histograms were
analyzed (results not shown). Control (unphotoconverted) preparations yielded uni-modal distributions,
similar to the distribution of the unlabeled vesicles in the photoconverted preparations. Since the
background and the dynamic range of the images varied between preparations, we also measured the ratio
between the density of the vesicle lumen and the density of the vesicle membrane for 20 sections from
photoconverted terminals and 23 sections from control terminals. Results are shown in Fig. S1.
“Labeled neighbor” analysis. To account for the fact that adjacent vesicles could be found within
different sections, we collapsed three consecutive serial sections into single planes, and then measured the
inter-vesicle distances in the two-dimensional image obtained. We then counted the number of immediately
adjacent neighbors for each labeled vesicle. As a control, we randomly mixed the labeled vesicles within
the total vesicle pool in each terminal, and repeated the neighbor-counting procedure. We performed the
randomizing process 100 times for each of the 12 reconstructions, averaged the results, and compared them
to the ones obtained with the original data. Vesicles were counted as immediate neighbors if their centers
were within 60 nm of each other (value representing the first peak of a histogram of inter-vesicle distances
obtained from 36 single sections, three from each reconstruction; n=123,122 distances lower than 200 nm
analyzed). Results are shown in Fig. S2.
Statistics. We used t-tests, performed using SigmaPlot (SPSS Inc., Chicago, IL). All values
presented in the text represent mean ± SEM, unless otherwise stated.
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B. Ceccarelli, W.P. Hurlbut, A. Mauro, J. Cell Biol. 57, 499 (1973).
T.H. Jr Giddings et al., Methods Cell Biol. 67, 27 (2001).
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H. Teng, J.C. Cole, R.L. Roberts, R.S. Wilkinson, J. Neurosci. 19, 4855 (1999).
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Fig. S1. Labeled and the unlabeled vesicles are readily distinguishable. Top panel: histogram of ratios between
the lumenal and the membrane densities from all vesicles in 23 non-photoconverted nerve terminal profiles.
Middle panel: histogram of ratios for the vesicles assigned as unlabeled in 20 photoconverted nerve terminal
profiles. Bottom panel: histogram of ratios for the vesicles assigned as labeled in the same 20 nerve terminal
profiles.
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Fig. S2. Labeled vesicles have more labeled neighbors than expected from random mixing. (A) Histogram of
distances between vesicles in 36 single sections (3 obtained from each of 12 reconstructed segments).
The first peak of the distribution shows the most common distance between the centers of vesicles in single
sections (~60 nm). (B) Three serial sections collapsed into one image. Labeled vesicles are colored (red,
green, blue) according to their section of origin. Vesicles of one color (2D analysis) have fewer neighbors than
if color is ignored (3D analysis), as expected. Arrows point to relatively large clusters formed by vesicles from
more than one section. (C and D) Quantitative analysis of single sections (2D analysis, panel C) and serial
sections (3D analysis, panel D). Open circles show results of random vesicle placement (the number of labeled
vesicles were randomly assigned positions of any vesicle, labeled or not, in the terminal profile, and the
number of labeled neighbors was determined; 100 iterations were performed and averaged). Filled circles
show observed data. The two populations are significantly different, and the difference is greater in panel D
(3D analysis) than in panel C (2D analysis). The non-random distribution is consistent with a process in which
vesicles were internalized at endocytic “hot spots” (7; cf. Fig. 3A) and tended to remain close to each other.
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