Interplay Between Spindle Architecture and Function - International Review of Cell and Molecular Biology - page 109

5.2. Spindle size
Although manipulation of factors such as TPX2 can alter spindle size ( Bird
and Hyman, 2008; Goshima and Scholey, 2010 ), mechanisms that function
physiologically to adjust spindle size and architecture to cells of different sizes
and shapes have only recently come to light, and thus far are limited to
Xenopus in vitro systems. The first was found in a comparison of Xenopus laevis
and its smaller relative Xenopus tropicalis , which produces smaller eggs. Spin-
dles formed in X. tropicalis egg extracts are also smaller, and mixed extracts
produced spindles of intermediate sizes, indicating that intrinsic, dose-
dependent cytoplasmic activities operate ( Brown et al., 2007 ). This
established a system to investigate mechanisms of spindle scaling by identi-
fying differences in spindle MT behavior in the two extracts and then deter-
mining whether the proteins responsible function as regulatory factors.
Computational simulation of a steady-state spindle (described below)
predicted that changes in MT stability could robustly change spindle length
( Loughlin et al., 2010 ) and greater activity of the MT-severing enzyme
Katanin, a hexameric AAA ATPase was observed in X. tropicalis egg cyto-
plasm compared with X. laevis . Consistent with Katanin functioning as a
spindle scaling factor, greater amounts were observed at X. tropicalis spindle
poles and its inhibition increased spindle length to a greater degree in
X. tropicalis compared with X. laevis . Protein levels in egg extracts and activ-
ity of recombinant catalytic p60 subunit of Katanin were similar for the two
species. Instead, a posttranslational mechanism was found to be responsible
for spindle length scaling by Katanin, as X. tropicalis p60 lacks an inhibitory
Aurora B kinase phosphorylation site that is present in X. laevis p60
( Loughlin et al., 2011 ; Fig. 3.7 A).
The second Xenopus system to address size scaling is the early X. laevis
embryo, which, following fertilization, undergoes rapid cleavages in the
absence of growth or transcription, undergoing an exponential decrease
in cell size. At early developmental stages (between two and several hundred
cells), an upper limit to spindle size is observed ( Wuhr et al., 2008 ). Once cell
diameter decreases to near twice the length of the spindle, linear scaling is
observed between spindle length and cell diameter through the 4000 cell
stage, when zygotic transcription initiates and cell cycles become asynchro-
nous. By preparing extracts from embryos at stage 3 (4 cell) and stage
8( 4000 cell), spindle size differences could be recapitulated in vitro , again
allowing a molecular dissection of underlying mechanisms ( Wilbur and
Heald, 2013 ). As for interspecies spindle scaling, higher MT destabilization