Biology Reference
In-Depth Information
CHAPTER
9
Yuyu Song
*
,{
and Scott T. Brady
{,{
Analysis of Microtubules in
Isolated Axoplasm from the
Squid Giant Axon
*
Department of Genetics and Howard Hughes Medical Institute, Yale School of Medicine, New
Haven, Connecticut, USA
{
Marine Biological Laboratory, Woods Hole, Massachusetts, USA
{
Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, Illinois, USA
CHAPTER OUTLINE
9.1 Preparation of Axoplasm................................................................................... 126
9.2 Analysis of Axoplasmic Microtubule Dynamics................................................... 131
9.3 Biochemistry of Axoplasmic Microtubules ......................................................... 133
9.4 Immunohistochemistry of Axoplasmic Microtubules............................................ 135
References ............................................................................................................. 136
Abstract
Biochemical specialization of cellular microtubules has emerged as a primary mecha-
nism in specifying microtubule dynamics and function. However, study of specific sub-
cellular populations of cytoplasmic microtubules has been limited, particularly in the
nervous system. The complexity of nervous tissue makes it difficult to distinguish neu-
ronal microtubules fromglial microtubules, and axonal microtubules fromdendritic and
cell body microtubules. The problem is further compounded by the finding that a large
fraction of neuronal tubulin is lost during standard preparations of brain tubulin, and this
population of stable microtubules is enriched in axons. Here, we consider a unique bi-
ological model that provides a unique opportunity to study axonal microtubules both
in situ
and
in vitro
: isolated axoplasm from the squid giant axon. The axoplasm model
represents a powerful system for addressing fundamental questions of microtubule
structure and function in the axon.
Microtubules have been studied extensively since the buffer conditions for
in vitro
polymerization were first described. As a result, the biochemistry and biophysics of
microtubule dynamics are relatively well understood in the test tube. However, our
understanding of microtubules
in situ
is limited at best. In cells, microtubules exhibit
