Mechanistic Insights into the Microtubule-Mediated Differential Modulations of Aurora B Substrate Structures for Accurate Chromosome Segregation

<p>Faithful chromosome segregation during eukaryotic cell division depends on the ability of sister kinetochores on duplicated chromatids to attach microtubules from opposite spindle poles. Failure in this process causes aneuploidy and drives cancer formation. To maintain genomic integrity, aberrant kinetochore-microtubule attachments must be destabilized, whereas properly attached microtubules must be stabilized. This process is regulated by Aurora B, the kinase subunit of the Chromosomal Passenger Complex (CPC). How Aurora B can distinguish between erroneous and correct attachments and selectively phosphorylate its key substrates in the right contexts remains poorly understood. The Ndc80 complex (Ndc80C) is a key substrate of Aurora B for regulating kinetochore-microtubule attachments. Ndc80C with Hypo-phosphorylated Ndc80 subunit (Hec1 in humans) binds microtubules with high affinity, promoting stable kinetochore-microtubule attachments; Phosphorylation at multiple sites within the Hec1 N-terminal tail weakens this interaction, promoting detachment for error correction. To investigate how phosphorylation of Ndc80C is controlled by microtubule binding, I developed a cryo-EM processing pipeline that resolves key phosphorylation sites of the Ndc80C in its microtubule-bound form. The resolved structure shows the disordered Hec1 tail domain with multiple Aurora B phosphorylation sites, engages in multivalent interactions to support the oligomerizing conformation of Ndc80C on microtubules. This geometry reveals a microtubule-mediated substrate masking mechanism, in which key phosphorylation sites become inaccessible to the kinase, thereby limiting phosphorylation. Combining with functional analyses, I propose that oligomerization of Ndc80 complex enhances its microtubule binding affinity and confers resistance to Aurora B-mediated phosphorylation. This substrate masking mechanism explains how stable kinetochore-microtubule attachments can be resistant to Aurora B triggered detachment. To determine whether substrate masking is a general feature of microtubule-bound Aurora B substrates, I examined mitotic centromere-associated kinesin (MCAK), a microtubule depolymerase whose activity is suppressed by Aurora B phosphorylation to stabilize spindle microtubules. Using the same cryo-EM pipeline, I resolved the structure of microtubule-bound MCAK and found that the key Aurora B phosphorylation site remains accessible when MCAK is bound to microtubules. This indicates that substrate masking is not a universal property of microtubule binding but is instead dependent on the oligomeric conformation specific to Ndc80C. Finally, I resolved the structure of the CPC on microtubules and found that it binds via the single α-helix domain of the INCENP subunit in a conserved manner across species. The CPC and MCAK partially share overlapping microtubule-binding sites, and cryo-EM-based co-decoration assays demonstrate that the CPC competitively displaces MCAK from the microtubule lattice. Given that microtubule binding of the CPC enhances Aurora B kinase activity, these suggest microtubules can promote Aurora B mediated phosphorylation of MCAK on microtubule to inhibit the depolymerization. In summary, the structural study described here demonstrates that microtubules can act as a signaling platform by differentially modulating the substrate accessibility of the Aurora B kinase to orchestrate chromosome dynamics during mitosis for faithful chromosome segregation.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Thesis

Year dc:date.available

2025

Author dc:creator

• Niu, Yiming

Contributors dc:contributor

• Hironori Funabiki

• Aurora B kinase
• kinetochore-microtubule attachments
• Ndc80 complex
• substrate masking
• cryo-electron microscopy (cryo-EM)
• chromosome segregation
• Life Sciences

Repository record dc:identifier

https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/820

OAI identifier oai:identifier

oai:digitalcommons.rockefeller.edu:student_theses_and_dissertations-1824