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Raymond, 1986) (Figure 5.12A). In the amphibian
brain, retinal axons must make a posterior turn in the
diencephalon to get to their targets in the midbrain. If
these growing axons are treated with cytochalasin,
they grow past the turning point (Chien et al., 1993)
(Figure 5.12B). Thus, actin filaments are critical for
growth cone navigation.
The actin filaments in filopodia are bundled and ori-
ented so that their fast-growing barbed or plus ends
are pointing away from the growth cone center out
toward the periphery. Time-lapse observations in
tissue culture show that, as the growth cone advances,
the filopodia move backward from their base and
shorten. The filopodia generate tension against the
bulk of the growth cone, which they thus pull forward.
This tension can be observed in culture, for when a
single filopodium from a growth cone contacts another
axon lying in its path, that filopodium can pull the
axon toward the advancing growth cone like someone
pulling the string on an arrow (Bray, 1979) (Figure
5.13). Similarly, a single filopodium that makes contact
with a more adhesive substrate in tissue culture is able
to steer the entire growth cone by pulling it toward the
attachment point (Letourneau, 1996). The importance
of single filopodia in directing growth cones in vivo
has been demonstrated in the Ti1 pioneer axons of the
grasshopper limb. Here it can be seen that when a
single filopodium makes contact with a guidepost cell,
then it attaches firmly while other filopodia retract
(O'Connor et al., 1990) (Figure 5.13).
How is filopodial tension generated? The clutch
hypothesis, formulated by Mitchison and Kirschner
(1988) suggests that actin filaments become anchored
to the substrate, engaging the clutch across the mem-
brane though adhesion complexes. These actin fila-
ments are then pulled centrally, probably by myosin
molecules located at the base of filopodia (Figure 5.13).
Indeed, when myosin function in growth cones is
blocked, forward progress is slowed, while the filopo-
dia themselves tend to lengthen as if a force that pulled
them rearward into the growth cone was attenuated
(Lin et al., 1996).
In culture, growth cones move straight ahead, and
there are approximately equal numbers of filopodia on
the left and right sides. Some conditions cause more
actin polymerization or depolymerization on one side
than the other, leading to an imbalance of filopodial
number and resulting traction force, causing the
growth cone to turn. In fact, this is suspected to be a
main mechanism of growth cone reorientation. Indeed,
if actin filaments are destabilized on one side of the
growth by using a depolymerizing agent locally, the
axon will turn in the other direction (Figure 5.14A)
(Yuan et al., 2003). When a single filopodium is
Fluorescently labelled microtubules
Fluorescent label is
eliminated locally
New label added distally
bleached area
does not move
labelled microtubules
added distally
t 1
beads at t 1
t 2
distance between
beads at t 2
FIGURE 5.9 Microtubules are added at the growing end. A. A
growing axon is labeled with fluorescent tubulin, and then some of
this fluorescence is bleached by a beam of light (circle) focused on
the axon near the growth cone. As the axon elongates distally, the
bleached spot stays in approximately the same place (bottom panel),
implying that the microtubules along the axon shaft do not move
forward but rather that new microtubules are assembled at the distal
tip. B. Two beads placed on an axon move further apart from each
other as the axon grows, with the front bead moving further forward
than the rear bead.
actin and microtubules to the cell membrane, to each
other, and to other cytoskeletal components such as the
molecular motors that generate force (Figure 5.11).
Drugs such as the actin-depolymerizing agent,
cytochalasin, have been used to investigate the func-
tions of actin in the growth cone. Treatment of growth
cones with such drugs prevents filopodia formation,
and such growth cones slow down dramatically,
showing that actin-rich fibers are important for the
forward progress of a growth cone. Cytochalasin-
treated growth cones do not steer properly and usually
lose their way in the developing organism. Thus, when
the Ti1 pioneer neuron in the grasshopper limb is
treated with cytochalasin, its axon meanders off course
and often does not make the turns necessary to grow
to its targets in the CNS (Bentley and Toroian-
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