BioMEMS for Cell Biology - Introduction to BioMEMS - page 353

three diferent solutions, a “vacuum PDMS pumping” scheme (see Figure 3.62 in Section 3.8.4.2)
was chosen. To induce polarization and axon pathinding, they used a membrane-permeable,
luorescently tagged form of cAMP (MP-cAMP), a cytosolic second messenger implicated in
axon guidance and neuronal polarization; MP-cAMP happens to bind or associate with macro-
molecules such as bovine serum albumin (BSA), so it could be coabsorbed on the surface at the
same time as BSA. Because cyclic guanosine monophosphate (cGMP) oten plays antagonistic
roles to cAMP, MP-cGMP was coadsorbed with BSA as a negative control to deter axon forma-
tion. For the same reason, MP-cGMP was coadsorbed with PLL to discourage axon growth
on the cell body patterns. It was necessary to locate the BSA/MP-cAMP and BSA/MP-cGMP
patterns approximately 2 μm away from each PLL/MP-cGMP island to orient the neurites in
the correct direction (even though it requires that the growth cone momentarily contacts the
nonadhesive, plain glass substrate), similarly to Stenger's previous work (see Figure 6.58 ). With
the orientation projections added to the design, approximately 60% of the cells attached to PLL/
MP-cGMP sites grew axons along the intended BSA/MP-cAMP pattern, whereas approximately
31% grew them on the more adhesive PLL/MP-cGMP regions. Axons were rarely observed on
the BSA/MP-cGMP pattern (~8%) and were never observed on plain glass.
6.5.3 Synaptogenesis
A paramount goal in neuroscience is to understand the cellular and molecular mechanisms of
synapse formation (“synaptogenesis”). he nerve-muscle synapse or NMJ, because of its accessi-
bility to experimental manipulation, has provided many insights into the molecular and cellular
mechanisms of synaptogenesis.
NMJ SYNAPTOGENESIS
The sequence of molecular signals leading to NMJ formation is qualitatively
well known. During development, axons grow along large distances before the tip contacts
a muscle cell. Contact occurs at the same developmental stage when myoblasts are fusing
to form myotubes. hree key molecules are secreted by the axon tip: agrin , neuregulin ,
and the neurotransmitter acetylcholine (ACh), which then interact with speciic recep-
tors in a small area of the muscle cell and induce diferent aspects of synapse formation.
Synaptogenesis is initiated by the secretion of agrin (a heparan sulfate proteoglycan) by
the nerve terminal, which is involved in the clustering and stabilization of AChRs at the
future synapse. (Both the nerve terminal and the muscle cell produce agrin, but muscle
agrin does not play a signiicant role in AChR clustering; therefore, we will henceforth
refer to neural agrin only.) Not surprisingly, agrin-deicient mutant mice undergo defec-
tive neuromuscular synaptogenesis. he muscle-speciic tyrosine kinase (MuSK) has been
identiied as an essential component of the agrin receptor complex, if not agrin's receptor
itself.
AChR clustering . Approximately two dozen proteins coaggregate postsynaptically at
the NMJ. Rapsyn, in particular, is essential for the aggregation of AChRs. Embryo imag-
ing experiments have shown that innervated AChR clusters in mice are highly dynamic,
with the AChRs continuously being degraded, inserted, and recycled (receptor recycling
is turned of when synaptic activity is blocked), with extrasynaptic AChRs contributing to
the synaptic AChR pool.
he laminin pathway . Laminin-1, a major component of the muscle cell extracellular
basal lamina, also induces AChR clustering, but through an alternative pathway, possi-
bly associated with the well-known laminin-induced clustering of integrins. Denervation