Throughout our bodies, most of our organs are maintained and repaired through regulated cell division, where new cells replace old ones that are lost or damaged. Before a cell divides, it executes a sequence of events whereby chromosomes are duplicated and lined up in the middle the cell before being pulled apart to opposite poles, ensuring that the two new daughter cells receive the same genetic information. Chromosome segregation errors—when partial or entire chromosomes fail to be equally distributed into the daughter cells— often result in alterations in the relative dosage of genes, which can contribute to cancer. The facilitators that coordinate chromosomal segregation during cell division are the microtubules—protein filaments that “all grow and shorten in near perfect unison” to accomplish this critical cellular task, describes Bonni Leeds, a graduate student in Dr. Charles Asbury's lab at the University of Washington. “How all the microtubules in a bundle are so tightly coordinated with one another is mysterious because individual microtubule growth rates are highly variable,” Bonni states. “People have suggested for a long time that mechanical forces may be able to coordinate microtubules,” although this has never been demonstrated experimentally, Bonni points out. She adds that the Asbury lab was “especially interested in studying this from a mechanical perspective,” and believed that a mechanical response to microtubule tips positioned too far in front or behind other microtubule tips could trigger a rapid change in microtubule dynamics, promoting coordination. The researchers predicted that this would occur on “a faster timescale than the selective binding of biochemical factors regulating microtubule dynamics to faster- or slower-growing microtubules.”
In collaboration with Dr. Sue Biggins’ lab in the Basic Sciences Division at Fred Hutch, Bonni and colleagues aimed to understand how effectively “the mechanical coupling of multiple microtubules to a single shared load can coordinate their growth.” Together, these research groups, affiliated with the Cancer Basic Biology Program of the Cancer Consortium, “focused specifically on pairs of microtubules growing in a parallel configuration, because this arrangement mimics the situation within chromosome-attached bundles (kinetochore-fibers or ‘k-fibers’) in dividing cells.” The research team found that “mechanical coupling, as between microtubules in a kinetochore-fiber bundle, can significantly coordinate microtubule growth,” Bonni states. This work, led by Bonni, was recently published in eLife.
“The idea that mechanical coupling can coordinate microtubule tips within a bundle has been around for more than two decades, but this idea has never been tested experimentally,” Bonni explains. To study the movements of microtubules pairs, the researchers developed a laser-based technique to monitor and measure the coordination of microtubule bundles attached to chromosomes, known as k-fibers. More specifically, the authors developed a dual laser trap assay which “uses two separate laser trapping microscopes, located adjacent to one another in the same room and connected to a single computer. On each of the two instruments, we attach a kinetochore-decorated bead to a dynamic microtubule plus-end. The computer then simultaneously monitors and controls the forces on both microtubules, dynamically adjusting each force to simulate an elastic coupling of both plus-ends to a single shared load,” Bonni explains. “Our dual-trap assay tests the feasibility of mechanical coupling playing a central role in the coordination of microtubule growth during cell division.”
Using this dual-trap assay to measure coordinated microtube dynamics, the team found that “mechanical coupling is very effective at coordinating microtubule growth.” Their kinetic analyses revealed that coordinated microtubule growth can be explained by a simple model which included periods of variable growth speed interrupted by force-driven pauses. More specifically, the research team “discovered that tension applied to growing microtubule tips reduces the likelihood that stochastic pauses will occur during their growth and also reduces the duration of such pauses. A combination of experiments and modeling suggests that these tension-dependent influences on pausing play a major role in the coordination of microtubules by mechanical coupling.” Bonni mentions that she was surprised to find that “the microtubule growth measured in our dual-trap assay was consistently better coordinated by mechanical coupling and more variable with weaker coupling than any of our early models could explain. It was also surprising that tension-dependent pausing behaviors were so important to the success of our best model in recapitulating most of the variability and coordination we see in our dual-trap assay.”
“From a basic biology perspective, this work helps us to understand a fundamental aspect of microtubule behavior during mitosis. From a clinical perspective, this work helps us to understand a way that aneuploidy (chromosome loss) might occur, potentially resulting in cancerous growth.” The researchers are continuing to study “microtubule disassembly and how disassembling tips can be coordinated with assembling tips by mechanical coupling,” in addition to investigating how well their microtubule disassembly or assembly simulations “can explain the behavior of microtubule bundles in real mitotic spindles.” Importantly, collaboration between the Biggins and Asbury labs “made studying microtubule coordination under force possible, as this type of experiment could never even have been envisioned without the huge breakthrough from Sue’s lab of purifying native, active kinetochores for the first time. The ability to study kinetochore activity in vitro has been amazing, and is leading to many new insights into how they function,” Dr. Asbury acknowledges.
This work was supported by the National Institutes of Health, a Packard Fellowship, Howard Hughes Medical Institute and Shared Resources of the Fred Hutch/UW/Seattle Children’s Cancer Consortium.
Fred Hutch/UW/Seattle Children’s Cancer Consortium members Drs. Charles Asbury and Sue Biggins contributed to this study.
Leeds BK, Kostello KF, Liu YY, Nelson CR, Biggins S, Asbury CL. Mechanical coupling coordinates microtubule growth. eLife. 2023.