Poleward Tubulin Flux in Spindles: Regulation and Function in Mitotic Cells

Abstract

The poleward flux of tubulin subunits through spindle microtubules is a striking and conserved phenomenon whose function and molecular components remain poorly understood. To screen for novel components of the flux machinery, we utilized RNA interference to deplete regulators of microtubule dynamics, individually and in various combinations, from S2 cells and examined the resulting impact on flux rate. This led to the identification of two previously unknown flux inhibitors, KLP59C and KLP67A, and a flux promoter, Mini-spindles. Furthermore, we find that flux rate is regulated by functional antagonism among microtubule stabilizers and destabilizers specifically at plus ends. Finally, by examining mitosis on spindles in which flux has been up-or down-regulated or restored after the codepletion of antagonistic flux regulators, we show that flux is an integral contributor to anaphase A but is not responsible for chromosome congression, interkinetochore tension, or the establishment of normal spindle length during prometaphase/metaphase.

The perpetual movements of anaphase

“…Taken together, these results suggest that microtubule depolymerization at the poles acts as a governor that limits and regulates spindle length in response to external force producers that slide microtubules poleward [65], similar to what has been proposed for spindle elongation during anaphase B [67]. According to this model, one would predict that inhibition of Klp10A or Kif2a attenuates "flux" even if the force sliding microtubules poleward is still present, consistent with what has been experimentally observed [61,64,65,70].…”

“…The classic examples of the former include plants [55] and insect spermatocytes [56]. Drosophila embryos, S2 cells, Xenopus oocyte extract spindles, newt lung cells, mouse, pig and human cells are all examples of the latter [49,50,[57][58][59][60][61][62][63][64][65]. Indeed, in these systems microtubule minus-end depolymerization is turned off or significantly attenuated during anaphase [54,65,66].…”

“…Because RNAi-mediated depletion of kinesin-8 proteins in living Drosophila and human somatic cells accelerates chromosome movement during anaphase A [64,164], the observations in Drosophila primary spermatocytes may be explained by the presence of merotelic attachments.…”

“…Specifically, KLP59C has been proposed to actively depolymerize microtubules at kinetochores in Drosophila embryos, as antibody injections completely block kinetochore pac-man activity and reduce chromosome-to-pole motion by 60% in this system [70]. However, KLP59C depletion by RNAi in Drosophila culture cells does not affect anaphase A [64,165]. Indeed, KLP59C location to inner centromeres does not permit access to most microtubule plus-ends that terminate at the kinetochore outer plate in Drosophila somatic cells [145].…”

Drosophila S2 Cells as a Model System to Investigate Mitotic Spindle Dynamics, Architecture, and Function

“…This mark, originally generated by photobleaching of rhodaminelabeled tubulin, would move towards one pole and the velocity of the displacement measured relative to a fixed point (Buster et al, 2007;Gorbsky and Borisy, 1989). However, the use of this approach poses some problems:…”