The cell cycle, the biological process underlying how cells grow and multiply, is tightly regulated to ensure a faithful distribution of genetic material to ‘daughter’ cells. This tight regulation of cell division is essential to minimize erroneous partitioning of genetic material which can lead to genomic instability, a common hallmark of cancer.
As such, the mechanisms that guide faithful segregation of chromosomes are orchestrated by hundreds of proteins. This protein network is comprised of large dynamic polymers known as microtubules, which bind the kinetochore complex at the center of the chromosome to separate chromosomes, physically pulling them into daughter cells. As the dynamic movement of microtubules creates tension at the kinetochore attachment sites, the process requires selective stabilization of kinetochore-microtubule attachments.
Tension selectivity favors 'correct', bioriented kinetochore attachments to spindle microtubules while destabilizing 'incorrect', low-tension attachments that would cause mis-segregation. Proteins such as Aurora kinase B ensure that correctly bioriented (aligned) attachments come under tension, while erroneous attachments lacking tension are released. This mechanism is so fundamental for both multicellular and unicellular organisms that the key players are conserved between human and yeast cells. The Biggins lab (Basic Sciences Division) studies cell division mechanisms in yeast and recently published new findings in PLoS Genetics.
Faithful segregation of chromosomes requires selective stabilization of kinetochore-microtubule attachments. Microtubules are dynamic structures, which are constantly moving, lengthening, and shortening. The dynamic movement of microtubules creates tension at the kinetochore attachment sites.
Tension selectivity favors 'correct', bioriented kinetochore attachments to spindle microtubules while destabilizing 'incorrect', low-tension attachments that would cause missegregation. A major question in the field is how cells destabilize the low-tension attachments. The major protein known to be involved is the Aurora B kinase, which phosphorylates kinetochore proteins to destabilize the attachments.
Through a long-standing collaboration with the laboratory of Charles Asbury at the University of Washington Department of Biophysics, the Biggins lab discovered that there is an intrinsic tension-sensing mechanism whereby kinetochores hold on to microtubules for a much longer time at high tension compared to low tension. To do this, they reconstituted kinetochore-microtubule attachments in vitro and put them under tension using an optical trap. There is no phosphorylation in their reconstitution system, indicating that proteins other than the Aurora B kinase are involved in tension-sensing.
Matt Miller, a former postdoc in the Biggins lab, went on to demonstrate that “the Stu2 protein (chTOG in human cells) is required for the intrinsic mechanism using our reconstitution system," as Dr. Sue Biggins explained. "This was a breakthrough because the only known mechanism prior to our work was a phosphorylation mechanism involving the Aurora B kinase,” she added.
"However, we had never shown that the intrinsic mechanism is critical in cells because Stu2 has many cellular functions so we could not specifically probe its kinetochore function,” said Dr. Biggins. To address this, Dr. Miller led another study using budding yeast, an excellent model organism in which to study kinetochore biology. In addition to being genetically tractable, with reliable methods for gene manipulation, budding yeast have a far simpler kinetochore attachment site than many other eukaryotes.
Using yeast as a model, the authors identified a Stu2 mutation that abolishes its kinetochore localization, and showed that it causes biorientation defects in vivo. They reported their findings in a recent issue of the journal PLoS Genetics. Dr. Biggins explained, “This manuscript identifies the first separation of function allele in Stu2 and helped us show that it does regulate kinetochore-microtubule attachments in vivo.”
By mutating the Stu2 protein in yeast cells, the authors demonstrated its role in maintaining kinetochore biorientation. They generated a separation-of-function mutant of Stu2 that lacks the kinetochore association domain yet supports normal mitotic spindle formation in cells. They found that cells with mutant Stu2, albeit proficient in normal spindle formation, exhibited defective biorientation, leading to spindle checkpoint-dependent cell cycle delay.
The authors also showed that Stu2 is required for the establishment of bioriented attachments, but is dispensable for their maintenance. For the group, this was reminiscent of the requirement for Aurora kinase B in the canonical error correction pathway. To test whether there was any cross-talk between the two pathways, they used mutants for both Stu2 and Aurora B and demonstrated that the two pathways acted in concert to ensure faithful segregation. Indeed, they observed additive growth defects in their double mutants that correlated with an additive increase in the rates of chromosome missegregation. The newly published work highlights how cells have evolved multiple pathways to ensure faithful segregation of chromosomes to safeguard genomic integrity.
A fundamental understanding of the mechanisms underlying chromosome segregation can help us understand how defects in the process may lead to cancer. The human ortholog of Stu2, chTOG, stands for Colonic and Hepatic Tumor Overexpressed Gene. The Biggins lab is already studying chTOG in human cells to determine whether its function in chromosomal segregation is conserved in humans. “We have data suggesting it is,” said Dr. Biggins.
Miller MP, Evans RK, Zelter A, Geyer EA, MacCoss MJ, Rice LM, Davis TN, Asbury CL, Biggins S. 2019. “Kinetochore-associated Stu2 promotes chromosome biorientation in vivo”. PLoS Genetics.
This research was supported by a Damon Runyon Cancer Research Fellowship, a David and Lucile Packard Fellowship, an NSF Graduate Research Fellowship and funding from the National Institute of Health and Howard Hughes Medical Institute.
Cancer consortium members Sue Biggins and Charles Asbury contributed to this research.