MacCoss, M. | bioRxiv Science

bioRxiv2018

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↗

bioRxiv2018

Spc110 N-Terminal Domains Act Independently to Mediate Stable γ-Tubulin Small Complex Binding and γ-Tubulin Ring Complex Assembly

Microtubule (MT) nucleation in vivo is regulated by the {gamma}-tubulin ring complex ({gamma}TuRC), an approximately 2-megadalton complex conserved from yeast to humans. In Saccharomyces cerevisiae, {gamma}TuRC assembly is a key point of regulation over the MT cytoskeleton. Budding yeast {gamma}TuRC is composed of seven {gamma}-tubulin small complex ({gamma}TuSC) subassemblies which associate helically to form a template from which microtubules grow. This assembly process requires higher-order oligomers of the coiled-coil protein Spc110 to bind multiple {gamma}TuSCs, thereby stabilizing the otherwise low-affinity interface between {gamma}TuSCs. While Spc110 oligomerization is critical, its N-terminal domain (NTD) also plays a role that is poorly understood both functionally and structurally. In this work, we sought a mechanistic understanding of Spc110 NTD using a combination of structural and biochemical analyses. Through crosslinking-mass spectrometry (XL-MS), we determined that a segment of Spc110 coiled-coil is a major point of contact with {gamma}TuSC. We determined the structure of this coiled-coil segment by X-ray crystallography and used it in combination with our XL-MS dataset to generate an integrative structural model of the {gamma}TuSC-Spc110 complex. This structural model, in combination with biochemical analyses of Spc110 heterodimers lacking one NTD, suggests that the two NTDs within an Spc110 dimer act independently, one stabilizing association between Spc110 and {gamma}TuSC and the other stabilizing the interface between adjacent {gamma}TuSCs.

biochemistry↗