Interplay Between Spindle Architecture and Function - International Review of Cell and Molecular Biology - page 95

observed to reform and generate an astral MT array ( Hornick et al., 2011 ).
Separation of the de novo MTOC into two MT asters was required for bipo-
lar spindle assembly and failed in 35% of cells, which then formed monopolar
spindles. This result demonstrates that de novo MTOCs, like centrosomes,
form dominant sites of MT nucleation that strongly influence spindle assem-
bly. When present, centrosomes may promote more robust spindle bipolar-
ity. Although dispensable for much of Drosophila development, centrosomes
are essential for the syncytial nuclear divisions during early embryogenesis
and function in spindle positioning through astral MT interactions with
the cell cortex, which is crucial for asymmetric, polarized divisions in cells
such as neuroblasts ( Basto et al., 2006 ). Thus, centrosomes are not necessarily
required for all cell divisions but appear to be essential in animals.
In addition to promoting bipolarity, centrosomes and associated factors
can regulate spindle length. In the C. elegans embryo, spindle size correlates
with centrosome size and molecular perturbations that altered centrosome
size proportionally changed spindle length ( Greenan et al., 2010 ). How cen-
trosome size is established is not understood, but a spindle-associated TPX2-
like protein (discussed in Section 4.1 )in C. elegans has been implicated and
might provide a molecular link between the centrosome and spindle archi-
tecture. In light of these examples, spindle-pole-associated centrosomes and
their astral MT arrays might be considered an architectural feature of differ-
ent spindles rather than an absolute requirement for spindle assembly
or function.
3.2. Mitotic chromatin-mediated nucleation
Experiments using Xenopus egg extracts demonstrated that MTs nucleated
around chromatin-coated beads could organize into a bipolar spindle struc-
ture in the absence of both centrosomes and kinetochores, illustrating the
self-organization pathway of spindle assembly ( Heald et al., 1996 ). In both
egg extracts and human tissue culture cells, chromatin effects are mediated
primarily by a gradient of GTP-bound Ran, which peaks at the chromo-
somes. Single glass beads coated with RCC1, the guanine nucleotide
exchange factor that loads Ran with GTP, could induce bipolar spindle
assembly in Xenopus egg extracts, indicating that the generation of RanGTP
is sufficient for spindle formation ( Halpin et al., 2011 ). However, RCC1
bead spindles were unstable and displayed abnormal MT density, indicating
that other chromatin factors are required to generate an architecturally nor-
mal spindle. The RanGTP gradient induces spindle formation by triggering