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microtubules. The method uses the thermosensitive reversible kinesin-5 cut7.24
ts
to
create monopolar spindles, where asters of individual mitotic microtubules are pre-
sented for imaging and subsequent analysis.
INTRODUCTION
The fission yeast
Schizosaccharomyces pombe
has traditionally been a good genetic
model organism to dissect conserved mechanisms of microtubule organization and
function (
Sawin & Tran, 2006
). In particular, fluorescent protein-tagged tubulin can
be expressed in the fission yeast cell for observing microtubule dynamics throughout
the cell cycle (
Snaith, Anders, Samejima, & Sawin, 2010
). In particular, fission yeast
interphase microtubules are organized as several discrete dynamic bundles parallel to
the long axis of the cell (
Drummond & Cross, 2000; Tran, Marsh, Doye, Inoue, &
Chang, 2001
). Each microtubule bundle in turn is composed of mainly two individual
microtubules bundled at their minus ends by an overlapping region at the cell center
and with their dynamic plus ends facing and interacting with the cell tips (
Hoog et al.,
2007
). Interphase microtubules can reach lengths of
10
m
m, roughly the length
scale of the cell, making imaging and subsequent analysis of microtubule dynamics
relatively straightforward. In contrast, during mitosis, the spindle pole bodies
organize the bipolar spindle. Each spindle pole body initially nucleates about
25
individual microtubules (
Ding, McDonald, & McIntosh, 1993
), which are then
cross-linked by the kinesin-5 cut7p (
Hagan & Yanagida, 1992
), and slide apart to
elongate the spindle to a steady-state metaphase length of
m(
Nabeshima
et al., 1998
). During the early stages of mitosis and spindle formation, individual mi-
crotubules cannot be imaged, due to the fact that they are relatively short and numer-
ous, and the spindle poles and the spindle widths are diffraction-limited structures,
that is, less than 300 nm (
Ding et al., 1993
). Nevertheless, given that microtubule
dynamics are known to dramatically change between interphase and mitosis in an-
imal cells (
Rusan, Fagerstrom, Yvon, & Wadsworth, 2001
), it would be useful to
measure cell cycle differences in microtubule dynamics in fission yeast, especially
in the context of mutations which affect microtubule dynamics.
Here, we present a simple method for imaging individual spindle microtubules dur-
ing early mitosis. The method takes advantage of the thermosensitive mutant cut7.24
ts
(
Hagan & Yanagida, 1992
). cut7p is a kinesin-5 and functions in bipolar spindle for-
mation by cross-linking and sliding apart antiparallel microtubules emanating fromop-
posite spindle poles (
Hagan&Yanagida, 1992
). As a cross-linker and slider, cut7p does
not alter microtubule dynamics per se. In addition, the cut7.24
ts
mutation has been
shown to be quickly reversible between the permissive (25
C) and nonpermissive
(37
C) temperatures, without any noticeable effects on subsequent spindle dynamics
(
Velve Casquillas et al., 2011
). We thus reasoned that by inactivating cut7p via using
the cut7.24
ts
mutant at 37
C, we would create monopolar spindles. These monopolar
spindles could not separate their spindle poles, and their respectivemicrotubules would
appear as asters with protruding microtubules. Some of the protruding microtubules,
3
m
