Setting a new standard for kinetochores

Setting a new standard for kinetochores T o really understand how a biological structure works, we need to know not just which, but also how many, molecules are present in it. For kine-tochores—the structures that link centro-meric DNA to spindle microtubules— protein components have been tallied by comparing the fluorescence of GFP-tagged kinetochore proteins to fl uorescent versions of the centromeric protein CENP-A, two copies of which are believed to assemble at every budding yeast centromere. Two studies now report that CENP-A is actually much more abundant at yeast centromeres, meaning that kineto-chore protein numbers have previously been underestimated (1, 2). CENP-A—known as Cse4 in budding yeast—is a variant of histone H3 that assembles into specialized nucleosomes that mark the position of centromeric chromatin. S. cerevisiae chromosomes have very small " point " centromeres that are thought to incorporate a single centromeric nucleosome containing two copies of Cse4 (3). This assumption has been used to estimate the numbers of kinetochore proteins that assemble on point centromeres and attach each budding yeast chromosome to a single spindle microtubule (4). Although fi ssion yeast and vertebrates have much larger " regional " centromeres that attach to multiple microtubules, the Cse4 standard has been used to quantify kinetochore proteins in these cell types as well (5, 6). Jian-Qiu Wu, Valerie Coffman, and their colleagues, from The Ohio State University in Columbus, wanted to count the number of proteins in cytokinesis nodes, precursors of the cytokinetic contractile ring. But when they compared fl uorescent node proteins to Cse4-GFP, their results didn't make sense. " For some proteins, there was too few, or even less than one molecule, in each node! " Wu explains. At the same time, Kerry Bloom and Ted Salmon, whose labs at the University of North Carolina, Chapel Hill had developed Cse4 as a standard for counting kinetochore proteins, became aware that several research groups were having similar problems. Both sets of researchers decided to directly count the number of Cse4 molecules at centromeres using several alternative standards, ranging from individual GFP molecules in vitro (1) to an E. coli motor complex that contains 22 copies of a GFP-tagged protein (1, 2). Using these new references , the researchers found that there was more Cse4 in the vicinity of each budding yeast centromere than previously thought—5 or 6 copies according to Bloom, Salmon, and their colleague Josh Lawrimore, or even 8 …