Biology Reference
In-Depth Information
some MAPs, especially small ones, by EM. It is therefore imperative to develop
fluorescence-based assays that enable the direct, real-time observation of microtu-
bule architecture alongside growth, shrinkage, and MAP binding. In this chapter,
we describe our efforts to control microtubule architecture for fluorescence-based
assays. We also describe how microtubule structure can be probed with the help
of GFP-tagged doublecortin, a MAP that binds preferentially to 13-pf microtubules.
INTRODUCTION
In vitro
assays using total internal reflection fluorescence (TIRF) microscopy have
become essential tools to observe the interaction of microtubule-associated proteins
(MAPs) with microtubules. In these assays, microtubules are polymerized
in vitro
from purified tubulin. Single microtubules are adhered to a cover glass surface
and fluorescently labeled MAPs are introduced and visualized. While simple in
concept, TIRF assays have provided insights into the complex mechanisms of
microtubule polymerases (
Brouhard et al., 2008
), depolymerases (
Helenius,
Brouhard, Kalaidzidis, Diez, & Howard, 2006
), severing enzymes (
Diaz-Valencia
et al., 2011
), end-binding proteins (
Bieling et al., 2007
), kinesins (
Vale et al.,
1996
), and dynein (
Reck-Peterson et al., 2006
).
Fluorescence-based assays have not, however, been able to address questions of
microtubule “architecture.” The canonical microtubule is a polymer built from 13 pro-
tofilaments (pfs, see
Fig. 21.1
A). Microtubules are “plastic” polymers, however,
meaning that tubulin forms polymers that differ in their longitudinal and lateral
curvature (
Kueh & Mitchison, 2009
). These polymers include microtubules with dif-
ferent numbers of pfs (
Fig. 21.1
B) (
Sui & Downing, 2010
), as well as open sheets
(
Erickson, 1974
), pf fragments (
Elie-Caille et al., 2007
), pf rings (
Howard &
Timasheff, 1986
), pf spirals (
Erickson, 1975
), inside-out helical ribbons (
Wang,
Long, Finley, & Nogales, 2005
), and zinc-induced “macrotubes” (
Wolf, Mosser, &
Downing, 1993
). The analysis of these diverse polymers by electron crystallography
and electron microscopy (EM) has characterized the tubulin-tubulin bonds that drive
polymerization (
Nogales, Wolf, & Downing, 1998; Wang & Nogales, 2005
). Micro-
tubule growth and shrinkage depend on the ability of tubulin to transition between dif-
ferent structural states; indeed, MAPs that regulate the microtubule cytoskeleton may
recognize different structural states as part of their core mechanism. The microtubule
polymerase Stu2, for example, may bind preferentially to longitudinally curved poly-
mers (
Ayaz, Ye, Huddleston, Brautigam, & Rice, 2012
).
The basis of these diverse structures, especially the distribution of pf numbers, is
the flexibility of interprotofilament bonds in the microtubule lattice. Interprotofila-
ment interactions are mediated by the M-loops and H1
0
-S2 and H2-S3 loops of
a
- and
b
-tubulin. These loops are able to accommodate lateral deformations associated with
changes in pf number and may form lateral bonds with different intrinsic curvatures
(
Wang & Nogales, 2005
). Indeed, the ability to deform laterally without breaking
