Cell division is one of nature’s most high-stakes operations. Every new cell inherits a complex genetic blueprint, 6.2 billion base pairs of information in humans, that must be perfectly distributed between the two daughter cells. The process of chromosome segregation ensures that each cell receives an exact copy of the genome. Errors in this process can have catastrophic consequences like cancer or developmental disorders, yet the machinery responsible for this critical task assembles from scratch every time a cell divides.
Before cell division, a burst of construction begins at the centromere, a special centralized region on each chromosome. Dozens of proteins arrive and assemble around the centromere in a precisely choreographed sequence to build the kinetochore. This structure functions as a loading dock for microtubules, the cellular cables that hook on to and pull chromosomes toward opposite ends of the dividing cell. These dynamic attachments ensure that each chromosome reaches the correct destination.
The kinetochore is highly complex, consisting of inner components that assemble around the centromere DNA and outer components that interface with microtubules. The kinetochore must get assembled precisely at the centromere, just when a cell is ready to divide. Any errors in this process can derail accurate chromosome segregation and threaten the integrity of the genome.
For decades, biologists have sought to understand how the kinetochore reliably and accurately assembles in real time. But the kinetochore’s small size and short-lived existence make it notoriously difficult to capture this process.
That changed with a recent study from the Biggins Lab in the Basic Sciences Division, where researchers managed to observe kinetochore assembly, molecule by molecule, in budding yeast, a simple organism whose kinetochore closely mirrors that of humans. Lead author Dr. Changkun Hu used an advanced imaging technique called total internal fluorescence microscopy (TIRFM), which allows individual molecules to be tracked with incredible precision. The team labelled centromere DNA with fluorescent tags, tethered it to a glass surface, and added fluorescently labeled kinetochore components in a systematic manner. By allowing these pieces to assemble for varying lengths of time, they could track the precise order and timing of each component’s incorporation and map the kinetochore assembly process in real time. Dr. Hu highlighted, “Our work for the first time revealed the temporal order of kinetochore assembly in any organism”.
The team found that kinetochore assembly is both hierarchical and interdependent. In the simplest models, pieces of the kinetochore would be expected assemble independently of one another, in a stepwise, straightforward, inside-to-outside sequence. But the study revealed a more intricate reality. While inner pieces generally assemble first, some depend on outer components to incorporate efficiently. Similarly, outer components rely on one another, assembling through a network of coordinated interactions rather than a simple linear sequence.
These findings may help explain how cells minimize errors during division. A tightly regulated and interconnected assembly pathway likely prevents kinetochores from forming too early or in the wrong place, safeguarding accurate chromosome segregation.
Behind every healthy, functioning organism, trillions of cells are dividing faithfully, guided in part by the kinetochore, a crucial quality-control machine. Understanding kinetochore assembly has broad implications because errors in chromosome segregation are linked to cancer, congenital disorders, and infertility. The authors hope the methods developed here can be used to test this idea and uncover new principles of kinetochore assembly, potentially even in mammalian systems.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Members Dr. Charles L. Asbury and Dr. Sue Biggins contributed to this research.
The spotlighted research was funded by the National Cancer Institute, National Institute of General Medical Sciences, Jane Coffin Childs Memorial Fund, and Howard Hughes Medical Institute.
Hu C, Popchock AR, Latino AA, Asbury CL, & Biggins S. 2025. Direct observation of interdependent and hierarchical kinetochore assembly on individual centromeres. Nucleic Acids Research. DOI: 10.1093/nar/gkaf1038
Science Spotlight writer Thamiya Vasanthakumar is a postdoctoral research fellow in the Campbell Lab at Fred Hutch. As a structural biologist, she uses cryogenic electron microscopy (cryoEM) to visualize the molecular structures of receptors found on the surface of immune cells.
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