Biology Reference
In-Depth Information
Glucose oxidase (Sigma, Ref: G2133)
Glycerol (bidistilled 99.5%; VWR ProLabo, Ref: 24388.295)
GMPCPP (Gena Biosciences, Ref: NU-405L)
GTP (Sigma, Ref: G8877)
Kinesin heavy chain (KHC) motor domain (Cytoskeleton, Inc., KR01)
Methanol (Carlo Erba, Ref: 414819)
NHS ester dyes Cy3 and Cy5 (GE Healthcare, Ref: PA23001 or PA25001)
Nocodazole (Sigma, Ref: M1404)
Paraformaldehyde (EuroMedex, Ref: 15710)
PIPES (EuroMedex, Ref: 1124)
Taxol (Paclitaxel; Enzo Life Sciences, Ref: BML-T104-005)
Triton X-100 (EuroMedex, Ref: 2000-C)
Buffers
PEM: 80 mM PIPES, 1 mM EGTA, 1 mM MgCl
2
,pH6.9
PEM-G: PEM
10% glycerol
Permeabilization buffer: PEM
þ
þ
þ
10% glycerol
0.01% Triton X-100
þ
1
m
M Taxol
PEM-T: PEM
PEM-C: PEM
þ
0.2 mg/mL casein
INTRODUCTION
It is well known that microtubules are highly dynamic polymers, which display
dynamic instability (
Mitchison & Kirschner, 1984
). The role of GTP-tubulin in
the regulation of microtubule dynamics has long been known, and it was shown that
hydrolysis of GTP at the plus ends of growing microtubules is necessary to trigger
depolymerization (
Hyman, Salser, Drechsel, Unwin, &Mitchison, 1992
). Loss of the
GTP cap exposes the unstable GDP-bound tubulin core and leads to depolymeriza-
tion. Many teams have studied the stabilizing microtubule cap, both its nature and
size. Among them,
Bayley, Schilstra, and Martin (1990)
proposed the lateral cap
model based on the hypothesis that the addition of a GTP-tubulin at the end causes
the hydrolysis of the GTP of the previous tubulin dimer, which becomes incorporated
into the microtubule lattice (
Bayley et al., 1990
).
Drechsel and Kirschner (1994)
showed that a minimum of 40 subunits is necessary in the GTP cap to stabilize
microtubules. In 1996, Caplow and Shanks proposed, measuring the minimum dis-
assembly rates using guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP)
microtubules, that a cap composed of only 13 or 14 GTP-tubulin subunits is sufficient
to stabilize microtubules (
Caplow & Shanks, 1996
).
Panda, Miller, and Wilson
(2002)
confirmed this idea and proposed that the stabilizing cap is composed of a
monolayer of Tubulin-GDP-Pi.
Once the important role of the GTP-tubulin cap was understood, studies linking the
presence of a GTP cap to microtubule-associated proteins (MAPs) arose. For example,
in an early study (
Severin, Sorger, & Hyman, 1997
), kinetochores were found to bind
