Biology Reference
In-Depth Information
FIGURE 9.4
Kinetic Equilibration Paradigm (KEP). This method was developed originally by
Morris and
Lasek (1982, 1984)
for analysis of monomer-polymer equilibrium of cytoskeletal proteins in
the axoplasm. In this diagram, green dots represent modified tubulin with increased stability
and white dots represent unmodified labile tubulins (
Song et al., 2013
). After placing the
axoplasms in buffer X, unassembled tubulin dimers would be extracted into the buffer by
5 min. Labile microtubules begin to depolymerize by this time and by 45 min, the tubulin from
labile microtubules (soluble polymer) has been extracted. By 120 min, only stable
microtubules remain as an axoplasmic “ghost” and do not change over periods as long as
24 h. These ghosts are enriched in modified tubulins.
care to avoid shearing. Elution of tubulin from the axoplasm can be monitored either
by taking aliquots of the media at suitable intervals or by physically transferring the
axoplasm into a fresh reservoir of buffer (
Fig. 9.4
, adapted from
Song, 2010
).
Alternatively, microtubule behavior can be monitored directly in chambers by im-
aging with video-enhanced contrast differential interference microscopy (
Allen, Allen,
& Travis, 1981; Weiss et al., 1988; Weiss, Langford, Seitz-Tutter, & Maile, 1991
).
This is best done by use of lower ionic strength buffers (i.e., buffer X/2) that promote
separation of cytoskeletal elements from the main axoplasm (
Brown & Lasek, 1993;
Weiss et al., 1988
). Exogenous tubulin can be perfused into the chambers and changes
in microtubule numbers and stability followed (
Weiss et al., 1988
). A variant on this
approach would perfuse tubulin with a fluorescent tag either chemical (Texas red) or a
protein (GFP, mCherry, etc.). Incorporation of fluorescent tubulin could be monitored
and the effects of different experimental manipulations evaluated.
9.3
BIOCHEMISTRY OF AXOPLASMIC MICROTUBULES
As microtubules from extruded axoplasm are purely axonal and represent neuronal
microtubule properties in this
ex vivo
setting, various biochemical experiments can
be carried out to examine different tubulin isotype components, posttranslational
