Altering membrane topology with Sar1 does not impair spindle assembly in Xenopus egg extracts.

ABSTRACT

Intracellular membrane networks including the endoplasmic reticulum (ER) and the Golgi apparatus experience dramatic reorganization upon entry into mitosis. However, the mechanisms driving these rearrangements and their importance for cell division are poorly understood. The GTPase Sar1 is a component of the secretory pathway and a key activator of anterograde transport of cargo from the ER to the Golgi. Here we show that Sar1 mutant proteins added to metaphase-arrested Xenopus laevis egg extracts cause dramatic effects on membrane organization. Live analysis of membrane structures in egg extract cytoplasm revealed a distinct network of sheets and tubules reflective of the organization of the ER in other systems. Addition of a constitutively active Sar1 GTPase mutant (H79G) increased membrane tubulation, while a dominant negative version Sar1 (T39N) impaired tubule organization. Although microtubule pelleting assays revealed that Sar1 associates with microtubules in the egg extract, and addition of Sar1 (H79G) mutant slightly destabilized spindle poles, bipolar spindle assembly was largely unaffected. Thus, spindles are stable to dramatic changes in mitotic membrane organization at metaphase, suggesting that mitotic membrane is not an upstream regulator of the mitotic spindle apparatus in Xenopus egg extracts.