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Synaptophysin (p38) at the frog neuromuscular junction: its incorporation into the axolemma and recycling after intense quantal secretion.
Valtorta F
,
Jahn R
,
Fesce R
,
Greengard P
,
Ceccarelli B
.
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Recycling of synaptophysin (p38), a synaptic vesicle integral membrane protein, was studied by the use of antisera raised against the protein purified from frog brain. When frog cutaneous pectoris muscles were fixed at rest, a bright, specific immunofluorescent signal was observed in nerve-terminal regions only if their plasma membranes had been previously permeabilized. When muscles were fixed after they had been treated for 1 h with a low dose of alpha-latrotoxin in Ca2+-free medium, an equally intense fluorescence could be observed without previous permeabilization. Under this condition, alpha-latrotoxin depletes nerve terminals of their quantal store of acetylcholine and of synaptic vesicles. These results indicate that fusion of synaptic vesicles leads to the exposure of intravesicular antigenic determinants of synaptophysin on the outer surface of the axolemma, and provide direct support for the vesicle hypothesis of neurotransmitter release. After 1 h treatment with the same dose of alpha-latrotoxin in the presence of 1.8 mM extracellular Ca2+, immunofluorescent images were obtained only after permeabilization with detergents. Under this condition, the vesicle population was maintained by an active process of recycling and more than two times the initial store of quanta were secreted. Thus, despite the active turnover of synaptic vesicles and of quanta of neurotransmitter, no extensive intermixing occurs between components of the vesicle and presynaptic plasma membrane.
Figure 1. Purification of synaptophysin
from frog brain. Lane a,
rat forebrain homogenate (50 Ixg);
lane b, purified rat brain synaptophysin
(2 ~tg); lane c, frog brain
homogenate (50 ~tg); lane d, frog
brain synaptophysin (2 ~tg). Molecular
weight standards: 94,000;
68,000; 45,000; 36,000; 29,000;
and 24,000. The samples were
separated on an SDS-10% polyacrylamide
gel and stained with
Coomassie Brilliant Blue.
Figure 2. Autoradiograph of an immunoblot
showing the specificity of the
antiserum against frog synaptophysin.
The protein samples were separated on
an SDS-t0% polyacrylamide gel and
transferred to a nitrocellulose membrane.
The blot was labeled with anti-frog synaptophysin
antiserum (dilution 1: 3,000),
followed by ~25I-Protein A. (lane a) Frog
brain homogenate, 10 lag; (lane b) purified
frog brain synaptophysin, 0.5 gg;
(lane c) rat forebrain total homogenate,
10 lag; and (lane d) purified rat brain
synaptophysin, 1 lag. Arrow, the single
band of immunoreactivity corresponding
to the synaptophysin band in the frog
brain total homogenate. The upper band
in lane b corresponds to the dimeric
form of synaptophysin.
Figure 3. Light micrographs showing the distribution of immunofluorescence for synaptophysin in two regions of rat brain. Frozen sections
(,~10 lam thick) were stained by immunofluorescence using antiserum against frog brain synaptophysin (1:100) and rhodaminated goat
anti-rabbit IgGs (1:50). (a) CA3-CA4 region of hippocampus; (b) deep mesencephalic nucleus. Immunoreactivity is specifically localized
to regions containing nerve terminals. Bars, 50 lam.
Figure 4. (a) Electron micrograph of a negatively stained crude synaptic
vesicle fraction purified from frog brain and subjected to
pronase treatment for 60 min. (b) Identification of an intravesicular
fragment recognized by antifrog synaptophysin antiserum. A crude
synaptic vesicle fraction purified from frog brain was subjected to
pronase digestion. The digestion was either immediately stopped
(left lane) or continued for 60 min (right lane). Proteins were separated
by SDS-PAGE, transferred to a nitrocellulose membrane,
incubated with antiserum against frog synaptophysin, and the immunoreactive
bands were visualized by immunoperoxidase. The
antiserum recognized both the intact protein and a fragment generated
by proteolytic digestion (arrow). Bar, 0.5 ~m.
Figure 5. Fluorescence micrographs of resting neuromuscular junctions on single muscle fibers teased apart from frog cutaneous pectoris
muscle. The fixed preparation shown in a and b was treated with 0.1% Triton X-IO0 and double stained with (a) fluoresceinated a-bungarotoxin
and with (b) antisynaptophysin antiserum followed by rhodamine-conjugated goat anti-rabbit IgGs. Similar patterns of nerve terminal branching are revealed by the postsynaptic (a) and presynaptic (b) markers. The nerve terminal shown in c and d is from a preparation
double stained as in a and b (c, ~t-bungarotoxin; d, synaptophysin), except that no detergent was used. In this condition, synaptophysin
labeling is undetectable and the nerve terminal region can be identified only by the distribution of a-bungarotoxin labeling. Bar, 50 Ixm.
Figure 6. Effects of a-LTx on the ultrastructure of neuromuscular junctions. (a and b) Electron micrographs of cross-sectioned terminals
treated for 1 h with 0.2 lag/ml a-LTx in Ca2+-free solution. The terminal branches shown in b come from an experiment in which 1.6%
horseradish peroxidase and 0.5 % myoglobin were present. (c) Electron micrograph from a cross-sectioned terminal treated for 1 h with
the same concentration of a-LTx in Ca2÷-containing Ringer's solution. (d and e) Longitudinal sections of terminals treated as in c in the
presence of 1.6% horseradish peroxidase. Notice the normal appearance of the terminal in c and the depletion of synaptic vesicles and
the swelling of the terminal in a and b. In d and e, most of the synaptic vesicles are loaded with horseradish peroxidase reaction product.
Bars, 1 ~'n.
Figure 7. Time course of
m.e.p.p, rate ([]) and cumulative
quantal secretion (x) computed
from fluctuation analysis
of endplate recordings during
exposure to 0.2 p.g/ml ¢t-LTx
in either Ca~+-free or Ca 2÷containing
Ringer's solution.
Each point is the average of
the corresponding values from
three different experiments.
m.e.p.p, rate (s -~) is given on
a logarithmic scale.
Figure 8. Two examples of fluorescence micrographs showing synaptophysin immunoreactivity in neuromuscular junctions treated for 1 h
with 0.2 p.g/ml ct-LTx in Ca2+-free Ringer's solution. No detergent was used. In this condition, no permeabilization was necessary to
reveal synaptophysin immunoreactivity and the nerve terminal branches show a marked increase in their transverse dimension. Bar, 50 p.m.
Figure 9. Fluorescence micrographs of neuromuscular junctions exposed for 1 h to 0.2 I.tg/ml a-LTx in Ca2+-containing Ringer's solution
and double stained as in Fig. 5. (a and b) Neuromuscular junction double stained (a, a-bungarotoxin; b, synaptophysin) after permeabilization
with 0.1% Triton X-100. No obvious difference can be seen between the synaptophysin immunofluorescence pattern of this nerve terminal
and that of a nerve terminal fixed at rest (Fig. 5). (c and d) Neuromuscular junction not treated with detergent. Note that synaptophysin
immunoreactivity (d) is not revealed without permeabilization, whereas et-bungarotoxin labeling (c) shows the normal appearance of the
endplate region. Bar, 50 gtm.
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