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inactive GDP bound state. Rho-family molecules are
involved in various actin rearrangements, which are
in turn regulated by a host of effector molecules like
the Guanidine Exchange Factors (GEFs) that exchange
GDP for GTP. The best known Rho-family members
are RhoA, Rac1, and Cdc42. RhoA is involved in actin
mediated neurite retraction, while Rac1 and Cdc42
promote filopodia and lamella formation and thus
neurite extension.
Growth cone filopodia are long, motile, and covered
with receptors, so they are able to sample and compare
different parts of their local environment. They also
have a very high surface-to-volume ratio, which can
help convert membrane signals into large changes in
intracellular messengers such as calcium. Filopodia can
show localized transient elevations of intracellular
calcium. These transients reduce filopodial motility.
By stimulating Ca transients through uncaging in the
filopodia on one side of a growth cone, the growth cone
will turn (Gomez et al., 2001). Experiments in which
calcium is uncaged on one side of a growth cone that is
moving forward generally cause the growth cone to
turn toward the side that has the elevated calcium
(Zheng, 2000). Calcium may also be released from inter-
nal stores in response to a signal from the cell surface or
enter the growth cone through calcium channels, and
may stimulate or inhibit neurite outgrowth. Serotonin,
which stops growth cone advancement in certain
neurons of the snail Heliosoma , appears to work by
increasing calcium levels locally, and stopping behavior
can be elicited using calcium ionophores. In this prepa-
ration, it is possible to cut single filopodia off an active
growth cone and study its behavior in isolation. Such
isolated filopodia react to the application of serotonin
by showing a marked increase in calcium along with a
shortening response (Kater and Mills, 1991), giving an
excellent insight into just how localized sensory and
motor responses in growth cones are. Collapse
responses to some factors are blocked by drugs that
inhibit calcium elevations, whereas in other neurons,
calcium appears to stimulate neurite outgrowth and
growth cones will grow in the direction of agents that
increase intracellular calcium on one side of the neuron
over the other. It is not entirely clear how calcium trig-
gers these responses; a likely possibility is that calcium
stimulates the activity of certain cytoplasmic kinases,
such as CAM kinase II or PKC, which then go on to
affect the cytoskeleton.
GAP-43, a g rowth- a ssociated p rotein in axons, is
highly enriched in growth cones of growing and
regenerating axons (Skene et al., 1986). It is an internal
protein that is associated with the cytoplasmic mem-
brane and various cytoskeletal proteins. The function
of GAP-43 has been tested by overexpressing it in cul-
tured neurons and in transgenic mice and by inhibit-
ing its expression. The results show that extra GAP-43
enhances axon outgrowth, while inhibiting GAP-43
compromises growth and leads to stalling (Fishman,
1996). In GAP-43 knockout mice, retinal axons stall
and then take random courses when they reach the
optic chiasm (Strittmatter et al., 1995). When GAP-43
is overexpressed in transgenic mice, exuberant growth
occurs and pathfinding errors are also made. These
results suggest that regulated levels of GAP-43 are
essential for the normal responses of axons to external
cues. The activity of GAP-43 is regulated, in part, by
phosphorylation through PKC and dephosphorylation
through a phosphatase. The phosphorylated form of
GAP-43 seems to stimulate its activity and promote
outgrowth, while the dephosphorylated form is less
active. It is not yet known how GAP-43 regulates axon
growth, but evidence suggests that it directly links the
cytoskeleton with the protrusive membrane of the
growth cone, thus enhancing motility.
Local protein synthesis within the growth cone is
possible because of the presence of mRNAs, which are
targeted and transported to the growth cone, and the
presence of a full complement of protein synthetic
machinery. Similarly, protein endocytosis and degra-
dation machinery, including the ubiquitinating
enzymes that target proteins to the proteasome for
degradation, are present in the growth cone (Campbell
and Holt, 2001). When a guidance molecule stimulates
protein synthesis or degradation, it does so through
MAP kinase pathways. There are several different
MAP kinases, some of which eventually activate a
protein called Target of Rapamycin, mTOR (rapamycin
being a natural toxin of stimulated protein synthesis in
various cell types). TOR is a kinase that phosphory-
lates a protein called eIF4E-BP, a negative regulator
of the translation initiation factor eIF4E (Campbell
and Holt, 2001). Other MAP kinases activate specific
ubiquitin ligases that attach ubiquitin molecules to
particular proteins targeting them for degradation. In
fact, it seems likely that there are a variety of down-
stream kinases that regulate different aspects of the
growth cone response when stimulated by a single
guidance cue.
We began this chapter by comparing axonal naviga-
tion with human navigation. We mentioned the need
for a motor, and we have seen that the growth cone by
virtue of its dynamic cytoskeleton is able to locomote
forward, turn, stop, and even retract. We mentioned the
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