Science Spotlight

Pulling it together: Uncovering a role for chTOG at the kinetochores

From the Biggins Lab, Basic Sciences Division

Cell division is one of the most fundamental processes. Every time a cell divides, the genetic material, DNA, is replicated and folded into structures called chromosomes in a process called mitosis. To ensure equal partitioning of DNA between ‘daughter’ cells, mitosis is tightly regulated by a conserved network of proteins known as the kinetochore which attaches each chromosome to microtubule filaments. As mitosis progresses, microtubules physically pull chromosomes into daughter cells. Kinetochores must stay bound to microtubules, which are constantly assembling, disassembling. Faulty segregation due to a lost connection between the kinetochore and the microtubule results in daughter cells with missing or extra chromosomes. Abnormal chromosome number or aneuploidy is the most common source of genomic instability, a hallmark of many human cancers. A better understanding of the mechanisms underlying chromosome segregation can help us understand how defects in the process may lead to cancer.

Cells have evolved mechanisms that ensure correct biorientation of the mitotic spindle to make sure that each pair of kinetochores bind to microtubules anchored to opposite ends of the cell. Correction of mitotic errors is traditionally thought to be primarily regulated via the enzyme Aurora kinase B (AURKB) and its downstream targets. In most species, AURKB works to destabilize low-tension kinetochore-microtubule attachments and to ensure proper biorientation. Researchers in the Biggins lab (Basic Sciences Division) work to understand the architecture, dynamics and function of the kinetochore. 

An image of multiple microtubules bound to kinetochores
A schematic of how AURKB and chTOG regulate Microtubule-Kinetochore attachments (Left). Sequence alignment of different chTOg proteins across species (top right), and immunofluorescence imaging shows erroneous attachments in a chTOG basic patch mutant (bottom right). Image courtesy of Jake Herman

Over the years, they have developed methods to purify native kinetochores from cells and to assemble kinetochores in test tubes. In a previous study, former postdoc Matthew Miller purified yeast kinetochores and studied them in vitro; he identified a role for the Stu2 protein in mediating mitotic error correction by sensing tension in kinetochores-microtubule attachments. Miller went on to show that in yeast cells, Stu2 regulates kinetochore-microtubule attachments in vivo using a separation-of-function Stu2 mutant that lacks the kinetochore association domain yet supports normal mitotic spindle formation in cells.

The human ortholog of Stu2, chTOG (colonic and hepatic tumor-overexpressed gene), also regulates kinetochore-microtubule attachments suggesting perhaps that the Stu2-dependent error correction process observed in budding yeast is a conserved process in human cells. A recent BioRxiv preprint from the Biggins lab described a chTOG mutant that regulates microtubule dynamics but accumulates erroneous kinetochore-microtubule attachments. The study was led by Jake Herman a postdoc in the lab. Herman reflects on the significance of their findings: “For the last twenty years we have studied how mitotic error correction is regulated through biochemical signaling by the kinase Aurora B. Here we show that human cells also use a biophysical mechanism to sense and correct errors in kinetochore-microtubule attachments that has not been studied until now.”

Herman and colleagues started by demonstrating that chTOG localizes to kinetochores by associating with the conserved microtubule binding factor Hec1, during mitosis using both microscopy and biochemical assays. The researchers then used yeast genetics and bioinformatics to determine the residues essential for chTOG’s function at the kinetochores with the budding yeast being more amenable to genetic manipulation. “Stu2/chTOG do about six different things in the cell, so depletion and knock out studies tend to have pleiotropic effects and this mitotic error correction activity has largely gone unnoticed by researchers,” Herman explained.

Evolutionary and genetic analysis in budding yeast revealed the separation of function mutant that Herman was looking for. “But error correction is exceedingly difficult to assay in yeast,” he said. By finding the homologous region in the human protein, the researchers were finally able to visualize and characterize chTOG’s function at the kinetochores. For both Stu2 and chTOG, two conserved basic patch residues were responsible for the kinetochore function. Indeed, the chTOG basic pair mutant was defective in error correction. In contrast, mutating the basic pair did not alter the dynamics or structure of the microtubule cytoskeleton. 

Since chTOG binds Hec1, and Hec1 is a known Aurora B kinase substrate, the authors asked whether chTOG regulated the Aurora B pathway. Depletion of chTOG did not affect the phosphorylation of Hec1 suggesting that chTOG function is independent of Aurora B phospho-regulation of its key kinetochore substrate, Hec1.

The microtubule cytoskeleton and other mitotic regulators like Mps1 and Aurora B have been targeted by chemotherapies in numerous tumor types with mixed success. Herman is excited about the translational potential of their findings. “From a biomedical perspective, we want to know how chTOG is functioning in a cancer context,” he said. Previous studies suggest complete loss of chTOG will be detrimental to healthy and cancer cells alike. He is excited to see if a larger therapeutic window could be achieved by inhibiting a single chTOG activity like error correction.

Herman JA, Miller MP, Biggins S. 2020. chTOG is a conserved mitotic error correction factor. bioRxiv   doi: 10.1101/2020.08.03.235325

This research was supported by funding from the National Institutes of Health and a Damon Runyon Cancer Research Foundation fellowship. Sue Biggins is an investigator of the Howard Hughes Medical Institute.

Cancer consortium member Sue Biggins contributed to this research.