Biomedical Engineering Reference
In-Depth Information
100%
80
60
40
20
0
Stripes: Ephrin-A2-Fc
+PI-PLC
c
Stripes: Ephrin-A2-Fc
81%
a
b
23%
Ephrin-A2-Fc Ephrin-A2-Fc
+PI-PLC
Temporal
axons
Nasal
axons
Temporal
axons
Nasal
axons
FIGURE 6.42
Biochemical. modulation. of. the. stripe. assay.. (From. Martin. R.. Hornberger,. Dieter.
Dütting,.Thomas.Ciossek,.Tomoko.Yamada,.Claudia.Handwerker,.Susanne.Lang,.Franco.Weth,.Julita.
Huf,.Ralf.Weßel,.Cairine.Logan,.Hideaki.Tanaka,.and.Uwe.Drescher,.“Modulation.of.EphA.recep-
tor.function.by.coexpressed.ephrinA.ligands.on.retinal.ganglion.cell.axons,”.
Neuron
.22,.731-742,.
1999..Reprinted.with.permission.from.Elsevier.)
and methyl-terminated silane SAMs as the nonadhesive areas (see
Figure 2.7
in Section 2.3.1).
Although the mechanism by which axons grew preferentially on the
aminosilane
areas was not
explored, this study demonstrated that microengineering axon growth with cultured cells was
possible, efectively paving the way for others to explore more rational surface chemistries and
even three-dimensional approaches. Aebischer and colleagues, for example, used a focused laser
beam to create three-dimensional patterns of cell-adhesive laminin oligopeptide fragments; the
laser produced a photochemical reaction that cross-linked the oligopeptides to selected locations
of an agarose gel background. Even though the resolution did not allow for guiding single axons
(imaging itself was very challenging, and removal of the unreacted oligopeptides was an issue), it
was encouraging that the somas were seen to adhere preferentially to the oligopeptide-conjugated
areas. It is foreseeable that a variation of this technique could be used to immobilize cell-surface
molecules such as those found in glial cells to simulate the natural paths of axon guidance.
A collaborative team led by Gary Banker (Oregon Health & Science University at Portland,
Oregon) and Harold Craighead (Cornell University at Ithaca, New York) have demonstrated a
versatile protein micropatterning scheme for axon guidance experiments (
Figure 6.43
). he irst
step is the immobilization of
poly-l-lysine (PLL)
on glass, either over the whole substrate from
solution or in a pattern (by microstamping). he second step is the microstamping of protein
A, a protein that binds the Fc fragment of immunoglobulins. A chimeric protein (the extracel-
lular domain of the guidance protein L1 recombinantly linked to the Fc fragment of IgG) is next
applied from solution, which causes the L1-Fc chimera to bind to the protein A pattern. he
L1-FC chimera micropattern on a PLL background selectively guides axon growth whereas the
somas attach preferentially on PLL and the dendrites grow preferentially on PLL. his pattern-
ing scheme could be useful for inducing neuronal polarization.
Even simple micropatterns such as a border between two adhesion signals can be used not
only to investigate guidance cues and signal transduction mechanisms acting at the nerve
growth cone but can also be a very valuable tool to probe the intracellular mechanisms oper-
ating to change the direction of axon outgrowth. Paul Bridgman's laboratory at Washington
University in St. Louis, Missouri, using PDMS blocks as masks to deine the borders of stripes,
has shown that growth cones (either from 13.5-day-old mouse embryo explants or from disso-
ciated cells) integrate myosin II-dependent contraction for rapid, coordinated turning at bor-
ders of stripes of laminin plus poly-l-ornithine (PLO; on a background of PLO) in response to
signals from laminin-activated integrin receptors (
Figure 6.44a
and the time-lapse series in
Figure 6.44d
). However, outgrowth continues across the borders when the neurons are phar-
macologically treated with blebbistatin (which inhibits myosin II activity, see
Figure 6.44b
and
