Biology Reference
In-Depth Information
FIGURE 18.1
MAPs-tubulin interactions and their consequences on tubulin self-assembly at 10 and 37
C.
This scheme represents tubulin self-assembly in a classical polymerization buffer (in which
tubulin spontaneously polymerizes into MT at 37
C). At 10
C, free tubulin forms T2S
complex in the presence of stathmin and circular protofilaments in the presence of tau. At
37
C, MTs depolymerize in the presence of stathmin and tubulin and stathmin form T2S
complex, whereas MTs are stabilized in the presence of tau.
(
Weingarten, Lockwood, Hwo, & Kirschner, 1975
) and until the identification of
stathmin (
Belmont &Mitchison, 1996
), many MAPs had been discovered and inves-
tigated. Their interaction with tubulin has been and is still being studied, using
numerous biochemical and biophysical methods both
in vitro
(
Devred et al.,
2010; Kiris, Ventimiglia, & Feinstein, 2010; Ross & Dixit, 2010; Wilson &
Correia, 2010
) and
in vivo
(
Drubin & Kirschner, 1986; Konzack, Thies, Marx,
Mandelkow, & Mandelkow, 2007; Samsonov, Yu, Rasenick, & Popov, 2004;
Weissmann et al., 2009
). In this chapter, we focus on the use of isothermal titration
calorimetry (ITC). ITC is a very useful technique that has been extensively used in
other fields, but that is still rarely utilized to study the tubulin cytoskeleton. After
describing briefly the principle of ITC, we focus on the important steps (from sample
preparation to data analysis) when applying ITC to the tubulin cytoskeleton and
MAPs. Taking as examples the case of a destabilizing MAP (stathmin) and stabiliz-
ing MAP (tau), we illustrate the pitfalls that must be avoided and the precautions that
must be taken in order to use this technique efficiently to gain new insights into the
molecular mechanism of action of MAPs on MTs.
