Eg5 Restricts Anaphase B Spindle Elongation in Mammalian Cells

Abstract

During anaphase, overlapping antiparallel microtubules in the spindle interzone elongate and contribute to chromosome segregation. Kinesin-5 family members are required for spindle elongation in some cells, but in other cases they restrict elongation acting like a brake. To determine how kinesin-5 contributes to spindle elongation in mammalian cells, we treated LLC-Pk1 epithelial cells with small molecule inhibitors of the mammalian kinesin-5, Eg5, at anaphase onset and measured the rate and extent of spindle pole separation using multidimensional tracking of centrosomes in cells expressing GFP-γ-tubulin. Centrosome separation was biphasic, with an initial fast phase followed by a slower phase. Treatment with the small molecule inhibitor, STLC, which weakens the interaction of Eg5 with microtubules, resulted in an increase in the rate of centrosome separation. Conversely, treatment with FCPT, which induces a rigor-like interaction of Eg5 with microtubules, reduced the rate of spindle elongation. In control cells, GFP-Eg5 was localized to spindle microtubules and accumulated in the interzone as anaphase progressed. Spindle fluorescence of GFP-Eg5 was decreased following treatment with STLC and increased in cells treated with FCPT. In anaphase cells, cortical dynein increases and rocking motion of spindle poles was detected consistent with the possibility that dynein mediates spindle elongation. In summary, our results demonstrate that Eg5 is not required for spindle elongation, and in fact, restricts the rate of spindle elongation in mammalian cells.