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The distribution of alpha-dystroglycan (alpha DG) relative to acetylcholine receptors (AChRs) and neural agrin was examined by immunofluorescent staining with mAb IIH6 in cultures of nerve and muscle cells derived from Xenopus embryos. In Western blots probed with mAb IIH6, alpha DG was evident in membrane extracts of Xenopus muscle but not brain. alpha DG immunofluorescence was present at virtually all synaptic clusters of AChRs and neural agrin. Even microclusters of AChRs and agrin at synapses no older than 1-2 h (the earliest examined) had alpha DG associated with them. alpha DG was also colocalized at the submicrometer level with AChRs at nonsynaptic clusters that have little or no agrin. The number of large (> 4 microns) nonsynaptic clusters of alpha DG, like the number of large nonsynaptic clusters of AChRs, was much lower on innervated than on noninnervated cells. When mAb IIH6 was included in the culture medium, the large nonsynaptic clusters appeared fragmented and less compact, but the accumulation of agrin and AChRs along nerve-muscle contacts was not prevented. It is concluded that during nerve-muscle synaptogenesis, alpha DG undergoes the same nerve-induced changes in distribution as AChRs. We propose a diffusion trap model in which the alpha DG-transmembrane complex participates in the anchoring and recruitment of AChRs and alpha DG during the formation of synaptic as well as nonsynaptic AChR clusters.
Figure 1. Western blot analysis
of rabbit and frog a- and BDG.
Nitrocellulose transfers of solubilized
rabbit (R) or Xenopus (F)
muscle heavy microsomes that
had been separated by SDSPAGE
under reducing conditions
were incubated together
with mAb HH6 (11116) or rabbit
antisera to I3(3 (FP-B and FP-D).
mAb HH6 recognizes a glycosylated
region of c~DG (Ervasti and Campbell, 1993). The polyclonal
antisera are to fusion proteins of c¢- and/3DG (Ibraghimov-
Beskrovnaya et al., 1992). Anti-FP-B recognizes a region overlapping
the cleavage point between ct- and/ffDG, and anti-FP-D is
specific for c~DG. All three reagents recognize a band at 156 kD
in rabbit muscle, but only mAb HH6 binds to Xenopus c~DG. In addition,
in rabbit muscle anti-FP-B recognizes a doublet at 43 kD
corresponding to /3DG (Ibraghimov-Beskrovnaya et al., 1992),
which is somewhat larger in Xenopus muscle (51-54 kD). Molecular
mass markers (in kilodaltons) are shown to the left.
Figure 2. ~xDG immunofluorescence at synaptic and nonsynaptic clusters of AChRs. (At) AChR fluorescence along a neurite-muscle contact.
(Ae) Corresponding ~xDG immunofluorescence, located at the synaptic clusters of AChRs. (B~) Large clusters of AChRs on noninnervated
muscle cells. (B2) Corresponding aDG immunofluorescence, located at the same sites as the nonsynaptic AChR clusters. (Q)
AChR fluorescence, (C2) otDG immunofluorescence, prominent near the end of a muscle cell where AChR fluorescence is sparse. The
arrow in Ct and C2 points to the same microcluster. Bar, 10 #m.
Figure 3. Precision of colocalization and specificity of otDG immunofluorescence.
(A~) Intricate pattern of AChR distribution within
a large nonsynaptic AChR cluster. (Az) aDG immunofluorescence
has an almost identical pattern. The arrowhead (also in A~) points
to microclusters of colocalized AChRs and t~DG. Arrows point to
portions of the immunofluorescence that extend beyond the limits
of the AChR cluster. (B~) Another large nonsynaptic cluster of
AChRs. (Be) Corresponding nonspecific immunofluorescence obtained
by substituting a control IgM (TEPC 183) for mAb IIH6.
Bar, 5 #m.
Figure 4. Comparison of agrin and otDG immunofluorescence. (AI) Phase-contrast micrograph of a neurite (N) and muscle cells (M).
(A2) Agrin immunofluorescence at sites of neurite-muscle contact (arrows) and along the path of neurite-substrate contact (arrowhead).
(A3) c~DG immunofluorescence is colocalized with, but more extensive than, the agrin along the neurite-muscle contact (arrows). It is
also seen on the edge of another muscle cell (arrowhead) where there was no neurite or agrin, o~DG immunofluorescence is not associated
with the agrin that was deposited along the path of neurite-substrate contact. This was also the case when the neurites were eliminated
before immunofluorescent staining, as shown in BI (phase-contrast), B2 (agrin), and B3 (aDG). Bar, 10 #m.
Figure 5. ~DG immunofluorescence at young synaptic clusters of
AChRs. (.4) Phase-contrast micrograph of a living neurite-muscle
contact. The arrow points to the end of the neurite and has the same
position in B, (7, and D. (B) Same field after staining and fixing.
The neurite grew several micrometers during an interval of 95 min.
(C) AChR microclusters along the neurite-muscle contact, including
that portion known to be <95 min old. (D) otDG immunofluorescence
is present at most of the AChR microclusters. Some additional
microclusters of otDG are also apparent. Bar, 10/zm.
Pigure 6. Incidence of czDG immunofluorescence at young synaptic
clusters of AChRs (open columns) and agrin (stippled columns).
The data for AChR clusters are from three 7-8-h cocultures. Within
those cultures, some of the identified contacts were known to be
<120 min (see Fig. 5). The data for agrin clusters are from two 8-h
cocultures; some of the contacts within these cultures were <70
rain. The numbers above the columns indicate how many clusters
were counted, n, AChR clusters; @, agrin clusters.
Figure 7. t~DG immunofluorescence at young synaptic clusters of
agrin. (A) Phase-contrast micrograph of a living neurite-muscle
contact. The arrow points to the end of the neurite and has the same
position in B, C, and D. (B) Same field after staining and fixing.
The neurite grew several micrometers during an interval of 36 min.
(C) Agrin immunofluorescence along the neurite-muscle contact,
including the portion known to be <36 rain old. Out-of-focus immunofluorescence
is also apparent where the neurite deposited
agrin on the culture substrate. (D) c~DG immunofluorescence is
present at most of the microdeposits of agrin along the neurite-muscle
contact. There is additional ctDG especially near the end of the
muscle cell. Bar, 10/~m.
Figure 8. Effect of mAb HH6 and other IgM mAbs on nonsynaptic
AChR clusters. On the left are the AChR clusters. On the right is
shown the corresponding immunofluorescence for the mAb that
was included in the culture medium: 1:40 mAb II1-16 (A and B),
1:40 mAb HNK-1 (C), 1:40 mAb HepSS-1 (D), and 1:10 mAb
HepSS-1 (E). Bar, 10/~m.
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