Biology Reference
In-Depth Information
Abstract
Microtubules play an important role in a numberofvitalcellprocessessuchascell
division, intracellular transport, and cell architecture. The highly dynamic structure
of microtubules is tightly regulated by a number of stabilizing and destabilizing
microtubule-associated proteins (MAPs), such as tau and stathmin. Because of
their importance, tubulin-MAPs interactions have been extensively studied using
various methods that provide researchers with complementary but sometimes con-
tradictory thermodynamic data. Isothermal titration calorimetry (ITC) is the only
direct thermodynamic method that enables a full thermodynamic characterization
(stoichiometry, enthalpy, entropy of binding, and association constant) of the in-
teraction after a single titration experiment. This method has been recently applied
to study tubulin-MAPs interactions in order to bring new insights into molecular
mechanisms of tubulin regulation. In this chapter, we review the technical speci-
ficity of this method and then focus on the use of ITC in the investigation of tubu-
lin-MAPs binding. We describe technical issues which could arise during planning
and carrying out the ITC experiments, in particular with fragile proteins such as
tubulin. Using examples of stathmin and tau, we demonstrate how ITC can be used
to gain major insights into tubulin-MAP interaction.
INTRODUCTION
Microtubules, which consist of polymerized tubulin heterodimers, play key roles in
numerous cell processes, including mitosis, active intracellular transport, and neuro-
nal plasticity. Microtubules are highly dynamic structures that switch from elonga-
tion to shrinking phase and
vice versa,
depending on different regulatory factors
(
Mitchison & Kirschner, 1984
). Under physiological conditions, microtubule dy-
namics is tightly controlled by stabilizing microtubule-associated proteins (MAPs),
such as tau, and destabilizing factors or proteins, such as stathmin (
Fig. 18.1
). These
proteins are in turn regulated by posttransitional modifications, the most studied one
being phosphorylation. Targeting microtubules and their dynamics is also a well-
established area of anticancer research. Indeed, altering microtubule dynamics by
drugs named microtubule targeting agents (MTAs) leads to mitotic block and cell
death. MTAs that can also be divided into two classes, molecules that stabilize mi-
crotubules, like taxanes, and molecules that induce microtubule depolymerization,
like vinca-alkaloids, have been studied using a broad spectrum of techniques (for
review, see
Calligaris et al., 2010
). Deciphering the molecular mechanisms of micro-
tubule regulation by MAPs is fundamental to understanding the biology of the cell.
This is of particular interest in oncology as it was proposed that resistance to anti-
cancer drugs could be linked to the level of expression of MAPs (
Alli, Yang,
Ford, & Hait, 2007; Cucchiarelli et al., 2008
; see
Chapter 5
). A better knowledge
of MAP-MTA interplay would help orient treatments and possibly lead to the
discovery of new therapeutic strategies. Since the discovery of the tau family
