Biology Reference
In-Depth Information
detachment. MCAK, a kinesin-related protein whose abundance is highest during
the early stages of mitosis, has been shown to regulate microtubule detachment.
Abnormal increases or decreases in the frequency of detachment interfere with
spindle function and inhibit cell division. It has been shown that drugs able to pro-
mote microtubule assembly (e.g., paclitaxel, epothilones) prevent cell division by
suppressing microtubule detachment from centrosomes. Conversely, cytotoxic
concentrations of microtubule destabilizing drugs (e.g., vinblastine, nocodazole),
tubulin mutations that cause paclitaxel resistance, and specific
-tubulin isotypes
increase the frequency of microtubule detachment. In this chapter, we describe a
method to calculate the frequency of microtubule detachment by transfecting cells
with EGFP-MAP4 and directly observing detachment by live cell imaging.
b
INTRODUCTION
During interphase, microtubules are nucleated at the centrosome, an organelle located
near the nucleus. Their plus ends grow out toward the cell periphery, while their minus
ends remain embedded in the centrosome, thereby establishing a cytoplasmic micro-
tubule network. Despite their static appearance in photographs, microtubules are ac-
tually very dynamic structures that exhibit stochastic episodes of growth and
shortening of their plus ends. This behavior, referred to as dynamic instability, allows
microtubules to constantly probe their environment and remodel in response to chang-
ing cellular conditions and morphology (
Kirschner &Mitchison, 1986; Mitchison &
Kirschner, 1984
). When cells enter mitosis, there is a dramatic rearrangement of the
microtubules. The pair of centrosomes formed by duplication during interphase split
apart and migrate to opposite sides of the cell to become spindle poles (
Hinchcliffe &
Sluder, 2001
). The cytoplasmicmicrotubule complex disassembles and is reorganized
into the mitotic spindle apparatus by nucleation of new microtubules at the spindle
poles and the capture of some of these new filaments by kinetochores on the condensed
chromosomes. When compared to interphase, spindle microtubules have a shorter av-
erage length and are more dynamic. The more rapid dynamics were initially discov-
ered by measuring the time required for microinjected-labeled tubulin to incorporate
into spindle and interphase microtubules and by fluorescence recovery after photo-
bleaching (
Saxton et al., 1984
). The results were more recently extended by directly
observing the behavior of microtubule plus ends in GFP-tubulin-expressing cells
(
Rusan, Fagerstrom, Yvon, & Wadsworth, 2001
).
Given that they are composed of the same tubulin subunits that are present during
interphase, spindle microtubule structural and behavioral changes are likely to result
from differences in the rate of nucleation at the spindle poles versus the interphase
centrosome (
Piehl, Tulu, Wadsworth, & Cassimeris, 2004
) as well as from the bind-
ing and activity of microtubule-interacting proteins (
Jiang & Akhmanova, 2011
). In
addition, it has been shown that chromosomes are involved in nucleating microtu-
bule assembly in a ran-dependent manner. This process plays a major role in the for-
mation of meiotic spindles and may play a significant role in forming mitotic
