The best kind of fishing expedition

From the Biggins Lab, Basic Sciences Division

Biologists often take advantage of the sheer multiplicity of life in order to make discoveries—microbiologists cultivate bacteria by the billions, cancer biologists can passage their immortalized cell lines ad infinitum, and biochemists can modify and express arbitrarily large amounts of protein at will. But what if you want to study a decidedly rarer biological entity, one which is present in only a handful of copies per cell? This question is top-of-mind for members of the Biggins Lab in the Basic Sciences Division at Fred Hutch, who study an intricate molecular machine called the kinetochore—a huge, basket-shaped complex of proteins which tethers a dividing cell’s chromosomes to the microtubules that will physically pull the chromosomes to two opposite poles of the cell before cell division. Most cells form only two kinetochores per chromosome during mitosis; a single, diploid yeast cell which the Biggins Lab uses as a model system will thus have only 32 kinetochores come division time (by contrast, a typical yeast will contain roughly 200,000 ribosomes)! Make no mistake, though; despite their rarity, kinetochores are absolutely critical for proper cell division, and kinetochore defects cause chromosomal abnormalities which are hallmarks of cancer.

The rare, complex, and dynamic nature of kinetochores makes them difficult to study using traditional laboratory techniques. Enter Dr. Andrew Popchock, a postdoctoral fellow in the Biggins Lab with a long-standing affinity (no pun intended) for molecular machines. Popchock asks a fundamental question about kinetochores: how are they formed? This question may not appear that difficult, but the nature of kinetochores actually makes it a formidable challenge to observe their formation in living cells. For the astute biochemists in the room, I know what you’re thinking: couldn’t scientists simply express and purify large amounts of the different kinetochore components, reconstitute the complex in vitro, and study it that way? Dr. Popchock is quick in his response. “We have learned a lot about kinetochores from this sort of approach, but since they interface between the cell’s DNA and microtubules, it’s actually really tricky to faithfully replicate these conditions in a tube. Ideally, we’d love a system where we could observe kinetochore formation in conditions which at least approximate those of a cell, with kinetochore components present in physiological quantities and with any cell-specific modifications they’d have in vivo.”

A recent preprint from Popchock and colleagues describes the construction and application of such a system. To accomplish the feat, Popchock leveraged two important things: the fact that the kinetochore-binding regions of yeast chromosomes (called centromere-defining elements, CDEs) are well-characterized and relatively compact, and his expertise in Total Internal Reflection (TIRF) microscopy, a specialized type of fluorescence microscopy which allows for visualization of single fluorescent molecules. To catch kinetochore formation in action, the team went fishing, figuratively speaking—they designed glass coverslips covered in many copies of a short DNA sequence which included the CDEs of yeast chromosome III. Separately, the team engineered yeast strains to express fluorescently tagged versions of different kinetochore components. By putting their slide containing kinetochore ‘bait’ under the microscope and flowing over the lysate from these engineered yeast strains, they are able to watch as their fluorescent components are ‘caught’ by the DNA at single-molecule resolution. The real power of this method, according to Popchock, is that it combines the advantages of single-molecule visualization with the more relevant context of cell lysate containing endogenous kinetochore components. It’s also an ingenious way to surmount the rarity of kinetochores—the assay allows the team to image thousands of kinetochores forming simultaneously! Importantly, the imposing task of computationally identifying and analyzing thousands of these molecules wouldn’t have been possible without the expertise of Dr. Julien Dubrulle, a staff scientist at the Cellular Imaging Shared Resource at the Hutch and contributor to the study.

A schematic depicting the experimental setup described in the article
A schematic depicting the experimental workflow developed by Dr. Popchock and colleagues. Image created by Andrew Popchock

Having created the method and verified that it works as expected, Popchock and colleagues went about applying it—and made a fascinating discovery. They focused on two kinetochore components: CENP-A (a histone variant which binds centromeric DNA and serves as the foundation of kinetochore assembly) and HJURP (a conserved histone chaperone previously shown to be important for CENP-A deposition to the centromere). Popchock generated yeast strains expressing one fluorescently tagged component of CENP-A and HJURP, respectively, and input the lysates into their experimental workflow to track both molecules assembling at centromeric DNA simultaneously. What Popchock saw initially shocked him. Contrary to what was thought before, HJURP wasn’t strictly necessary for CENP-A to bind to DNA; CENP-A can bind with or without HJURP, but the stability of CENP-A binding (measured as ‘residence lifetimes’ in the assay) was much greater with HJURP than without. To overuse the fishing metaphor, it seemed as if plain centromeric DNA represented a ‘hook,’ while HJURP acted as the ‘line and sinker’ to successfully land CENP-A. So, what was HJURP doing to stabilize CENP-A binding? Reasoning that it may help physically wrap the centromeric DNA around CENP-A, the team designed an ingenious experiment made possible by their method: instead of leaving one end of the DNA free-floating, they tethered both ends of the DNA strand to the slide, preventing its wrapping around CENP-A. Consistent with their hypothesis, doing so and tracking CENP-A association led to residence lifetimes equal to those of CENP-A without HJURP.

Impressively, Popchock and colleagues didn’t stop here; they used their new system to report several other findings related to centromeric DNA base pair composition and limiting factors of kinetochore recruitment which beg a close read of the paper. With regards to the utility of their method, Popchock comments that they have only scratched the surface of its capabilities in this study. “Now that the method works, we’re really excited to use it to ask questions about many other kinetochore components and their relationships to each other. Several other groups have recently had some success with solving molecular structures of intact kinetochores, and we’re excited to use those structures as a jumping-off point to form mechanistic hypotheses about kinetochore assembly and test them using our method.” So, if you find yourself with some time in between experiments or emails, give this paper a read—you're guaranteed to be hooked!

The spotlighted research was funded by the National Institutes of Health, the Washington Research Foundation, and the Howard Hughes Medical Institute.

Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium member Dr. Sue Biggins contributed to this study.

Popchock, A. R., Larson, J. D., Dubrulle, J., Asbury, C. L., & Biggins, S. (2023). Single molecule visualization of native centromeric nucleosome formation reveals coordinated deposition by kinetochore proteins and centromere DNA sequence. bioRxiv.