Biology Reference
In-Depth Information
considerable diversity at the molecular level, including tubulin isotypes, posttrans-
lational modifications, and associated proteins. Microtubules in different cell types
or even in different subcellular compartments may exhibit strikingly different prop-
erties with regard to dynamics, composition, and function. This heterogeneity is par-
ticularly striking in neurons, where the bulk of the microtubules are not associated
with the microtubule-organizing center, yet may exhibit exceptional stability. The
answers to questions about the functional diversity of neuronal microtubules may
be critical for understanding many aspects of neuronal development, function,
and pathology.
One obstacle to characterizing specific populations of neuronal microtubules is
the complexity of nervous tissue. Separating neuronal microtubules from glial
microtubules and dendritic microtubules from axonal or cell body microtubules is
effectively impossible when using brain tissue as a source, so any studies on the bio-
chemistry and biophysics of neuronal microtubules from brain reflect the properties
of a mixed pool. The problem is compounded by the fact that a large fraction of neu-
ronal tubulin is lost during standard preparations of brain tubulin, and this population
of stable microtubules has received little attention, despite representing more than
50% of axonal tubulin in mature neurons.
Isolated axoplasm from the squid giant axon provides a unique model system for
studying exclusively axonal microtubules both
in situ
and
in vitro
. Although isolated
axoplasm has not been widely used, experiments using this model have provided novel
insights into the axonal cytoskeleton, and studies on axoplasm have the potential to
produce additional insights. Here, we describe the preparation of isolated axoplasms,
the use of physiological buffers that more accurately reflect intracellular environments,
and examples of experiments that can only be done in this model system (
Fig. 9.1
).
9.1
PREPARATION OF AXOPLASM
The procedures described here are based on our continuing studies using axons from
the Atlantic longfin squid,
Loligo pealeii
and update previous descriptions
(
Brady, Lasek, & Allen, 1985; Brown & Lasek, 1993; Leopold, Lin, Sugimori,
Llinas, & Brady, 1994
). Large to medium squid (0.3-0.5 m in length) are preferred
as they have axons 300-500
m in diameter. Suitable squid are seasonally available
(April-October typically) at the Marine Biological Laboratory in Woods Hole, MA,
but protocols should be readily adapted for other species of squid with suitable axons
(
m
m in diameter). Smaller squid and smaller axons are not suitable as the vis-
coelastic properties of the axoplasmwill lead to disruption of the axoplasm from smal-
ler axons (
>
300
m
m) (unpublished data S. Brady) (
Brady, Richards, &Leopold, 1993
).
A healthy squid with a translucent body is chosen and decapitated to begin the
dissection. The head and tentacles are discarded. The mantle is cut along the dorsal
midline to a sheet of muscle that is placed skin side down on the dissecting light table
with running seawater. The viscera are removed and the clear pen is carefully pulled
away from the mantle, taking care to avoid tearing the nerve fibers that are along each
<
150
m
