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Biggins, S.

Publications and source records attributed to Biggins, S..

3 recordsLinked to original sources

A Stu2-mediated intrinsic tension-sensing pathway promotes chromosome biorientation in vivo

Accurate segregation of chromosomes to daughter cells is a critical aspect of cell division. It requires the kinetochores on duplicated chromosomes to biorient, attaching to microtubules from opposite poles of the cell. Bioriented attachments come under tension, while incorrect attachments lack tension and must be destabilized. A well-studied error correction pathway is mediated by the Aurora B kinase, which destabilizes low tension-bearing attachments. We recently discovered that in vitro, kinetochores display an additional intrinsic tension-sensing pathway that utilizes Stu2. This pathways contribution to error correction in cells, however, was unknown. Here, we identify a Stu2 mutant that abolishes its kinetochore function and show that it causes error correction defects in vivo. We also show that this intrinsic tension-sensing pathway functions in concert with the Aurora B-mediated pathway. Together, our work indicates that cells employ at least two pathways to ensure biorientation and the accuracy of chromosome segregation.

cell biology

Tension on kinetochore substrates is insufficient to prevent Aurora-triggered detachment

Chromosome segregation requires kinetochores to attach to microtubules from opposite spindle poles. Proper attachments come under tension and are stabilized, but defective attachments lacking tension are released, giving another chance for correct attachments to form. This error correction process requires the Aurora B kinase, which phosphorylates kinetochores to destabilize microtubule attachments. However, the mechanism by which Aurora B can distinguish kinetochore tension remains unclear because it is difficult to detect kinase-triggered detachment and manipulate kinetochore tension in vivo. To address these challenges, we developed an optical trapping-based flow assay with soluble Aurora B and reconstituted kinetochore-microtubule attachments. Strikingly, we found that tension on these attachments suppressed their Aurora B-triggered release, suggesting that tension-dependent changes in the conformation of kinetochores can regulate Aurora B activity or its outcome. Our work uncovers the basis for a key mechano-regulatory event that ensures accurate segregation and may inform studies of other mechanically regulated enzymes.

cell biology

An assay for de novo kinetochore assembly reveals a key role for the CENP-T pathway in budding yeast

Chromosome segregation depends on the kinetochore, the machine that establishes force-bearing attachments between DNA and spindle microtubules. Kinetochores are formed every cell cycle via a highly regulated process that requires coordinated assembly of multiple subcomplexes on specialized chromatin. To elucidate the underlying mechanisms, we developed an assay to assemble kinetochores de novo using centromeric DNA and budding yeast extracts. Assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein and generates kinetochores capable of binding microtubules. We used this assay to investigate why kinetochores recruit the microtubule-binding Ndc80 complex via two receptors: the Mis12 complex and CENP-T. Although the CENP-T pathway is non-essential in yeast, we demonstrate that it becomes essential for viability and Ndc80c recruitment when the Mis12 pathway is crippled by defects in Dsn1 phosphorylation. Assembling kinetochores de novo in yeast extracts provides a powerful and genetically tractable method to elucidate critical regulatory events in the future.

biochemistry