Chromosome segregation is the ultimate step in cellular replication. Once genomic DNA is copied, the two sets of chromosomes must separate and each complete set must travel to what will become the two daughter cells. Defects in chromosome segregation can lead to birth defects like Down Syndrome or cancer. The mechanism of chromosome segregation involves concerted action of hundreds of proteins. Each chromosome contains a region of DNA called the centromere that serves as the point of attachment for a multiprotein structure called the kinetochore. The kinetochore, in turn serves as the connector between chromosomal DNA and spindle microtubules, protein cables used to pull chromosomes to their destination in the daughter cell. In a recent publication in the journal eLife, Dr. Sue Biggins and colleagues in the Basic Sciences Division developed a new kinetochore reconstitution assay using yeast Saccharomyces cerevisiae proteins to determine the function of a critical kinetochore component. Dr. Biggins explained “The paper is the first time a kinetochore has been completely assembled de novo so it opens the door to discovering the underlying mechanisms that regulate the process. The kinetochore is a megadalton machine compose of hundreds of proteins so it has been difficult to understand how cells build it every cell cycle.”
There are several advantages to using yeast for these studies. Unlike many other eukaryotes, which use complex regions of DNA as centromeres, yeast use a single 125 base pair stretch of DNA sequence wrapped around a specialized nucleosome as the kinetochore attachment site. Secondly, yeast is a genetically tractable organism with well-established methods for knocking out or mutating genes and growing large numbers of cells in a short time. Using the new kinetochore assembly technique, former graduate student Jackie Lang used the centromeric DNA sequence from yeast chromosome III (CEN3 DNA) and yeast protein extracts to show by Western blot that CEN3 DNA bound to a single CENP-A nucleosome (see Figure) in a specific manner that also required the function of a kinetochore chaperone HJURP-Scm3, thus proving that in vitro kinetochore assembly could mimic the process cells use. Lang also showed that 39 of the 49 known core kinetochore components were identified when the reconstituted complex was analyzed by mass spectrometry.
The authors then turned their attention to the role of the CENP-T complex. Although CENP-T is not essential in yeast, mutant yeast strains lacking this kinetochore component have higher rates of chromosome loss suggesting a role in chromosome segregation. CENP-T was previously shown to serve a redundant function along with Mis12c to recruit the Ndc80 complex, a critical microtubule binding site. By using mutant yeast strains lacking components of the kinetochore as the source of protein lysates used in the reconstitution assays, the authors showed convincingly that CENP-T doesn’t bind centromeric DNA directly, as had been proposed, but rather, requires all of the inner kinetochore components for its function. Most significantly, Lang and her colleagues showed that the CENP-T protein is essential when the parallel Mic12c complex is compromised. Looking forward, Biggins says “The paper raises the question why cells use two pathways to assemble the same microtubule binding complex. We will now test these two pathways biophysically using the de novo assembled kinetochores. We are also setting up the first real time single molecule assays to watch the assembly process. This should help us understand the temporal and spatial order of assembly and identify rate-limiting steps. All of this will be new for our lab and provide the first ways to analyze kinetochore assembly using single molecule assays.”
Research was supported by the National Institutes of Health and the Howard Hughes Medical Institute.
Lang J., Barber A., Biggins S. 2018. An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast. Elife, 7. PMCID: PMC6097842.