Biomedical Engineering Reference
In-Depth Information
experiments in vivo have shown that there are components in the basal lamina that are
suicient to trigger AChR clustering on the muscle membrane, but possibly because agrin
is by then already bound to the basal lamina. In addition, unless the muscle basal lamina is
removed, AChRs remain focalized to the former NMJ site ater denervation, and the nerve
terminal continues to release ACh vesicles in the absence of the muscle. It seems that the
irst 130 amino acids from the N-terminus of agrin bind to laminin-1 in the basal lamina;
it is therefore conceivable that the basal lamina's role in the maintenance of the NMJ is to
provide a physicochemical support for agrin.
It was long held that agrin was the
cause
of AChR clustering (the “agrin hypothesis”), which
initiated a decades-long controversy that has only been recently resolved. he observation that
traditional myotube cultures form punctated clusters of AChRs on bath or focal application of
agrin or an agrin fragment contributed to fuel the agrin hypothesis. For example, a team led by
Bruce Wheeler, then at the University of Illinois at Urbana-Champaign, cultured
C2C12
myo-
tubes on microstamped agrin micropatterns and observed that the myotubes formed AChRs
clusters above some of the agrin lines (
Figure 6.60
). In this experiment, however, the myotubes
are exposed to agrin for days, not briely in time like in vivo, and the agrin is presented to them
as an immobilized molecule. he author's group at the University of Washington in Seattle con-
irmed that if the focal agrin stimulus is delivered in soluble form using a microluidic delivery
device (
Figure 6.13
) at the right developmental time, i.e., right ater their fusion into myotubes,
then the myotubes also react forming small AChR clusters.
here is now irm evidence that AChR clustering precedes nerve contact (and thus, agrin
secretion). he midline of the muscle forms clusters of AChRs even in the absence of innerva-
tion. Imaging of embryos (both in mouse and zebraish) has revealed that large “protosynaptic”
AChR aggregates of normal synaptic morphology (similar to a “pretzel”) form on the muscle
membrane before the arrival of the nerve. In myotube cultures, AChR clusters of complex mor-
phologies (strikingly similar to those found in vivo) can, also form in the absence of agrin. What
is, then, the role of agrin, given that it is not the cause of AChR clustering?
To ind out, the author's group used a microluidic device where single myotubes had been
microengineered across the width of the device (see
Figure 6.13
in Section 6.2.3), making sure
that the myotubes expressed AChR clusters before agrin application (myotubes cultured on
laminin or Matrigel stripes work well). Next, agrin was focally delivered to the center of the
device as a laminar stream (
Figure 6.61a
) and the evolution of AChR clusters was followed over
Agrin
AChR
40 µm
FIGURE 6.60
Local. AChR. clustering. induced. by. surface-bound. agrin. micropatterns.. (From. Toby.
Cornish,.Darren.W..Branch,.Bruce.C..Wheeler,.and.James.T..Campanelli,.“Microcontact.printing:.
A.versatile.technique.for.the.study.of.synaptogenic.molecules,”.
Mol. Cell. Neurosci.
.20,.140-153,.
2002..Reprinted.with.permission.from.Elsevier..Figure.contributed.by.Bruce.Wheeler.)
