Defining the kinetochore’s rules of engagement

D uring mitosis, kinetochores connect chromosomes to micro-tubules (MTs), helping sister chromatids to align on the metaphase spindle and segregate to opposite poles in anaphase. Yeast kinetochores contain a number of MT-associated proteins (MAPs) that together attach to a single MT. Vertebrate kinetochores carry a similar complement of MAPs, but each kinetochore attaches to 20–30 MTs, perhaps by forming a repetitive series of yeast-like attachment sites, with each site operating independently to bind a single MT. Zaytsev et al. reveal that the vertebrate kinetochore–MT interface is better described as a " molecular lawn, " in which individual MAPs can dynamically switch between multiple MTs (1). The Ndc80 complex is an essential MAP at both yeast and vertebrate kinetochores. The Aurora B kinase regulates Ndc80's association with MTs by phosphorylating the complex's Hec1 subunit at up to nine different sites. Jennifer DeLuca's lab at Colorado State University in Fort Collins has previously shown that phosphorylating all nine sites on Hec1 strongly inhibits kinetochore– MT interactions, whereas blocking Hec1 phosphorylation induces hy-perstable attachments to the mitotic spindle (2, 3). The overall level of Hec1 phos-phorylation declines as mito-sis proceeds, although which of the nine sites are phos-phorylated at any one time is unclear. " We wanted to know how differential Hec1 phosphorylation affects kinetochore function, " DeLuca says. DeLuca and her colleagues Lynsie Sun-din and Keith DeLuca therefore analyzed cells whose endogenous Hec1 had been replaced with mutant versions of the protein in which each of the nine phosphorylation sites had been mutated to either nonphos-phorylatable alanine or phosphomimetic as-partate residues (1). None of the mutants supported proper chromosome segregation—in-dicating that Hec1 phosphorylation must be dynamically regulated—but metaphase cells expressing Hec1 mutants with a single phos-phomimetic substitution recapitulated several important properties of wild-type cells. Their kinetochores showed normal movements on the metaphase spindle, and they attached to similar numbers of MTs. " It didn't matter where the substitution was. Any single phosphomimetic substitution could recapit-ulate metaphase chromosome movements, " DeLuca explains. As the number of phos-phomimetic substitutions increased, however , the number of kinetochore–MT attachments gradually decreased and chromosome movements became progressively more erratic. Accordingly, in vitro measurements showed that increasing Hec1 phosphorylation gradually decreased the Ndc80 com-plex's affi nity for MTs. To further investigate how Hec1 phosphoryla-tion infl uences kinetochore function, DeLuca and colleagues turned to Ekaterina Grishchuk and Anatoly Zaytsev at the University of Pennsylvania in Philadelphia, who recently developed …