Initial Separation of Poles to form a Bipolar Spindle is a Rapid and Irreversible Process

Abstract

A critical step in mitosis is the formation of a functional bipolar spindle, a complex molecular machine responsible for accurate chromosome segregation between daughter cells at the end of cell division. The ability of force-generating molecular motors to cross-link and slide anti-parallel microtubules has been implicated in formation of a bi-polar spindle. In the simple eukaryotic spindle of yeast, kinesin-5 (Cin8) and kinesin-14 (Kar3) has long been thought to drive the initial separation of duplicated spindle poles. Here we show that the spindle rapidly and irreversibly transitions from a compacted to extended state, and suggest that the underlying process is powered by the cross-linked bent microtubules as a loaded spring.Using 3-D electron tomography, we model and characterize the initial state of this process, revealing the microtubule overlap architecture on which the Kinesin-5 motor proteins bind. Combining in-vivo imaging with fluorescence labeling of spindle poles and tracking algorithms, we quantify transition characteristics such as spindle pole velocity. Critically, we measure the velocity of pole separation during this transition to be greater than that expected microtubule sliding via Cin8 or Kar3. We observed that the velocity of pole separation in a non-sliding but microtubule cross-linking Cin8 mutant (Cin8-3, Sablin et al., 1996) also supports an alternate process to motor driven anti-parallel sliding. Further characterization of the pole-separation mechanisms will clarify how this basic yet essential aspect of eukaryotic life is accurately completed.