  The bipolar organization of the microtubule-based mitotic spindle is
essential for the faithful segregation of chromosomes in cell division. Despite
our extensive knowledge of genes and proteins, the physical mechanism of how
the ensemble of microtubules can assemble into a proper bipolar shape remains
elusive. Here, we study the pathways of spindle self-organization using
cell-free Xenopus egg extracts and computer-based automated shape analysis. Our
microscopy assay allows us to simultaneously record the growth of hundreds of
spindles in the bulk cytoplasm and systematically analyze the shape of each
structure over the course of self-organization. We find that spindles that are
maturing into a bipolar shape take a route that is distinct from those ending
up with faulty structures, such as those of a tripolar shape. Moreover, matured
structures are highly stable with little occasions of transformation between
different shape phenotypes. Visualizing the movement of microtubules further
reveals a fraction of microtubules that assemble between extra poles and push
the poles apart, suggesting the presence of active extensile force that
prevents pole coalescence. Together, we propose that a proper control over the
magnitude and location of the extensile, pole-pushing force is crucial for
establishing spindle bipolarity while preventing multipolarity.
