As a cell divides, its DNA -the genetic material- is duplicated and condensed into structures called chromosomes, which are carefully organized and distributed so that each daughter cell acquires a complete and accurate set during cell division. Each pair of chromosomes is positioned at the center of the cell through its centromere, which ensures delivery of the exact number of chromosomes to each to each daughter at cell division. But how do chromosomes know where to go? This is the function of the kinetochore, a conserved network of proteins that attach each chromosome to microtubule filaments during chromosome segregation. The kinetochore consists of two parts: the inner kinetochore, which binds to the centromere, and the outer kinetochore, which binds to the microtubules. If chromosome segregation goes wrong and cells end up with too many or too few chromosomes, they may turn cancerous.
As kinetochores are essential for the proper distribution of chromosomes during cell division and kinetochore defects cause chromosomal abnormalities, it is essential to understand the structure of kinetochores to study their function. Unfortunately, the architecture of the kinetochore has not yet been well characterized. This is precisely what Dr. Sue Biggins, a professor in the Basic Sciences Division, and her team set out to accomplish in their recent publication. Using different structural biology techniques, the team built the architecture of native kinetochores from thermophilic yeast.
“Because the kinetochore has so many components, for a long time the field has been struggling to make a detailed architectural model of complete kinetochores,” commented Daniel Barrero, a graduate student in Dr. Biggins’ lab and leading author of the study. “This is the first time we’ve been able to visualize largely complete, native kinetochores in ice, which provides much more information about how kinetochores are built in their cellular context,” Barrero added.
Seems like an easy task, doesn't it? Express and purify large amounts of the different kinetochore components, reconstitute the complex in vitro, and determine its structure. Unfortunately, mammalian kinetochores have hundreds of different protein components, making it extremely difficult to do so. Therefore, Barrero turned to a simpler model: yeast. Because kinetochores share common components and functions between yeast and mammals, it is an ideal model system for purifying and constructing kinetochores.
To accomplish this, Barrero purified native kinetochores from the thermophilic yeast called Kluyveromyces marxianus. This yeast can live at high temperatures and has more stable kinetochore complexes that facilitate structural biology analyses. Barrero then tested if the purified kinetochores were functional using an optical trapping assay, which measures the strength of kinetochore-microtubule attachments. Excitingly, “K. marxianus kinetochores exhibited robust microtubule attachments with a similar median rupture force to kinetochores purified from S. cerevisiae,” Barrero explained, confirming the functionality of the native K. marxianus kinetochores.
Then, Barrero used different structural biology techniques to elucidate the structure of these K marxianuskinetochores. First, Barrero used electron microscopy and found that, “Kinetochores appeared as large paintbrush-like structures with a flared ‘brush’ end and a more compact hub, which often had a long thin projection, akin to a paintbrush ‘handle’.” Moreover, Barrero found that the “brush” corresponds to the outer kinetochore and can bind to the microtubules while the “handle” of the inner kinetochore binds to the centromere."
Next, Barrero turned to cryo-electron tomography (cryo-ET) to visualize the kinetochores at higher resolution in collaboration with Dr. Arimura Yasuhiro, an Assistant Professor in the Basic Sciences Division. In fact, cryo-ET revealed that “the linkages between the brush tips and the compact region appear as long and flexible fibrils that are easier to distinguish compared to the negative stain [electron microscope] images.” Although Barrero aimed to build structural models of the kinetochores, it could not be done likely due to “kinetochore flexibility and heterogeneity combined with difficulty in maintaining the structural integrity of the kinetochore.” For this reason, Barrero turned to atomic force microscopy (AFM), instead. Barrero saw the same paintbrush-like structure as previously observed, as well as more compact particles where the handle appears to be close to the brush head and particles where the handle is not visible. AFM also revealed that the brush tips and handle were flexible, as well as the absence of rigid connections between the inner and outer kinetochore.
“These kinetochores were much larger and more dynamic than we expected,” Barrero said. “We are now very curious as to what, besides the well-established inner kinetochore proteins, is contributing to the densely packed inner kinetochore we see in our data.” Barrero is also excited to “add microtubules to the mix and attempt to visualize for the first time at high resolution how complete kinetochores engage with them.”
The spotlighted research was funded by the National Institutes of Health, the Howard Hughes Medical Institute, and the Stavros Niarchos Foundation Institute for Global Infectious Disease Research.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium members Dr. Charles “Chip” Asbury and Dr. Sue Biggins contributed to this study.
Barrero DJ, Wijeratne SS, Zhao X, Cunningham GF, Rui Y, Nelson CR, Arimura Y, Funabiki H, Asbury CL, Yu Z, Subramanian R, Biggins S. (2024). Architecture and flexibility of native kinetochores revealed by structural studies utilizing a thermophilic yeast. bioRxiv