Interplay Between Spindle Architecture and Function - International Review of Cell and Molecular Biology - page 97

factors that regulate its activity and targeting: INCENP, Survivin, and Bor-
ealin/Dasra. CPC activity is essential in some cases of acentrosomal spindle
assembly, such as Drosophila meiosis and in Xenopus egg extracts ( Colombie
et al., 2008; Gadea and Ruderman, 2005; Sampath et al., 2004 ). The con-
tribution of the CPC to chromatin-mediated spindle assembly is primarily
through Aurora B-dependent phosphorylation and inhibition of
MT-destabilizing proteins Op18/Stathmin and the kinesin-13 MCAK,
thereby promoting MT stability in the vicinity of chromosomes ( Gadea
and Ruderman, 2006; Ohi et al., 2004 ).

3.3. Kinetochore-driven nucleation
Early studies from Brinkley and McIntosh described MTs polymerizing
from kinetochores, but until more recently this was not demonstrated in live
cells ( Khodjakov et al., 2003; Maiato et al., 2004; Pepper and Brinkley,
1979; Snyder and McIntosh, 1975 ). While the molecular mechanism of
MT nucleation at the kinetochore is unknown, there is evidence that this
process, like chromatin-mediated spindle assembly, requires the small

GTPase Ran ( Torosantucci et al., 2008; Tulu et al., 2006 ).
However, experiments with cells proceeding through mitosis with unre-
plicated genomes (MUG) showed that spindles formed around kinetochores
while the fragmented chromatin was excluded, even though it had
established a gradient of RanGTP ( O'Connell et al., 2009 ). If kinetochore
assembly was inhibited, the mitotic chromatin attracted astral MTs but failed
to form a bipolar spindle. These results suggest that kinetochore MT nucle-
ation is dominant in somatic cells and does not require a RanGTP gradient,
which can direct the growth of MTs from centrosomes, but on its own is
insufficient for spindle formation.
Like RanGTP, Aurora B kinase has been observed to generate a gradient
around chromosomes, but in this case, the gradient consists of phosphory-
lated substrates, which are produced by concentration and activation of the
kinase at centromeres/kinetochores in association with the CPC, followed
by release and diffusion to reach substrates at a distance ( Wang et al., 2011 ).
The relative role of the two gradients and their ability to promote spindle
assembly has been examined in Xenopus egg extracts ( Maresca et al.,
2009 ). Interestingly, by adding an inhibitor of RCC1 together with a
Ran mutant locked in the GTP form, the RanGTP gradient was abolished,
but spindle assembly still occurred in the presence of replicated mitotic chro-
mosomes containing centromeres, but not around chromatin-coated beads,