Biology Reference
In-Depth Information
Abstract
Microtubule-severing enzymes are a novel class of microtubule regulators. They are
enzymes from the ATPases associated with various cellular activities family
(AAA
) that utilize ATP to cut microtubules into smaller filaments. Discovered
over 20 years ago, there are still many open questions about severing enzymes. Both
cellular and biochemical studies need to be pursued to fully understand how these
enzymes function mechanistically in the cell. Here, we present methods to express,
purify, and test the biophysical nature of these proteins
in vitro
to begin to address the
biochemical and biophysical mechanisms of this important and novel group of mi-
crotubule destabilizers.
þ
INTRODUCTION
The microtubule cytoskeleton is a network of microtubule filaments required for cell
morphology and intracellular transport. This network is organized and remodeled
to perform cellular activities such as differentiation and cell division. Various
microtubule-associated proteins (MAPs) interact with microtubules to regulate the
network. Stabilizing and nucleating MAPs are positive regulators. Other important
regulators are required to destabilize the network for the purpose of remodeling the
location and dynamics of the filaments. For instance, maintenance of flagella, axons,
and interphase networks requires remodeling and destabilization of specific micro-
tubules or portions of the microtubule lattice.
Recent cell and biochemical work has begun to highlight the importance and
novel mechanisms behind negative microtubule regulators or destabilizers. Several
microtubule depolymerizing enzymes derived from the kinesin super family pro-
mote disassembly in an adenosine 5
0
-triphosphate (ATP)-dependent manner includ-
ing kinesin-8 (
Gupta, Carvalho, Roof, & Pellman, 2006; Varga et al., 2006
), the
mitotic centromere-associated kinesin or kinesin-13 (
Desai, Verma, Mitchison, &
Walczak, 1999; Helenius, Brouhard, Kalaidzidis, Diez, & Howard, 2006;
Newton, Wagenbach, Ovechkina, Wordeman, & Wilson, 2004
), and kinesin-14
(
Cai, Weaver, Ems-McClung, & Walczak, 2009; Sproul, Anderson, Mackey,
Saunders, & Gilbert, 2005; Troxell et al., 2001
).
Nature developed an exquisite way to remodel microtubules in the form of an-
other class of diffusible factors called microtubule-severing enzymes. These
ATPases negatively regulate microtubules and belong to the meiotic clade of the
AAA
protein superfamily (
Frickey & Lupas, 2004; Ogura & Wilkinson, 2001
).
Amidst these proteins are katanin (
McNally & Vale, 1993
), spastin (
Roll-Mecak
& Vale, 2005
), and fidgetin (
Cox, Mahaffey, Nystuen, Letts, & Frankel, 2000;
Mukherjee et al., 2012a; Zhang, Rogers, Buster, & Sharp, 2007
). These proteins
contain the AAA
þ
þ
segment is highly conserved and contains all the elements involved in ATP binding
and hydrolysis (
Fig. 13.1
A, C, D) (
Hanson & Whiteheart, 2005
).
þ
domain on their C terminal halves. The 230 amino acid AAA
