Centralspindlin is essential for the formation of microtubule bundle structures and the equatorial recruitment of factors critical for cytokinesis. Stable accumulation of centralspindlin at the spindle midzone requires its multimerization into clusters and Aurora B kinase activity, which peaks at the central spindle during anaphase. Although Aurora B phosphorylates centralspindlin directly, how this regulates centralspindlin localization is unknown. Here we identify a novel regulatory mechanism by which Aurora B enables centralspindlin to accumulate stably at the spindle midzone. We show that 14-3-3 protein binds centralspindlin when the kinesin-6 component MKLP1 is phosphorylated at S710. 14-3-3 prevents centralspindlin from clustering in vitro, and an MKLP1 mutant that is unable to bind 14-3-3 forms aberrant clusters in vivo. Interestingly, 14-3-3 binding is inhibited by phosphorylation of S708, a known Aurora B target site that lies within the motif bound by 14-3-3. S708 phosphorylation is required for MKLP1 to stably localize to the central spindle, but it is dispensable in an MKLP1 mutant that does not bind 14-3-3. We propose that 14-3-3 serves as a global inhibitor of centralspindlin that allows Aurora B to locally activate clustering and the stable accumulation of centralspindlin between segregating chromosomes.

Opitz syndrome (OS) is a genetically heterogeneous disorder characterized by defects of the ventral midline, including hypertelorism, cleft lip and palate, heart defects, and mental retardation. We recently identified the gene responsible for X-linked OS. The ubiquitously expressed gene product, MID1, is a member of the RING finger family. These proteins are characterized by an N-terminal tripartite protein-protein interaction domain and a conserved C terminus of unknown function. Unlike other RING finger proteins for which diverse cellular functions have been proposed, the function of MID1 is as yet undefined. By using the green f luorescent protein as a tag, we show here that MID1 is a microtubule-associated protein that inf luences microtubule dynamics in MID1-overexpressing cells. We confirm this observation by demonstrating a colocalization of MID1 and tubulin in subcellular fractions and the association of endogenous MID1 with microtubules after in vitro assembly. Furthermore, overexpressed MID1 proteins harboring mutations described in OS patients lack the capability to associate with microtubules, forming cytoplasmic clumps instead. These data give an idea of the possible molecular pathomechanism underlying the OS phenotype.