Molecular interplay between HURP and Kif18A in mitotic spindle regulation
Juan M Perez-Bertoldi1, Yuanchang Zhao2, Akanksha Thawani3
1Biophysics Graduate Group, University of California, Berkeley, CA, USA.
Abstract:
During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro, we demonstrate that Kif18A motility is regulated by HURP. While sparse decoration of HURP activates the motor, higher concentrations hinder processive motility. To shed light on this behavior, we determined the binding mode of HURP to microtubules using Cryo-EM. The structure reveals that one HURP motif spans laterally across β-tubulin, while a second motif binds between adjacent protofilaments. HURP partially overlaps with the microtubule-binding site of the Kif18A motor domain, indicating that excess HURP inhibits Kif18A motility by steric exclusion. We also observed that HURP and Kif18A function together to suppress dynamics of the microtubule plus-end, providing a mechanistic basis for how they collectively serve in spindle length control.
Insights
Hepatoma-upregulated protein (HURP) regulates Kif18A motor protein activity on microtubules. This interaction is crucial for controlling microtubule dynamics during cell division, ensuring proper chromosome segregation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biophysics
Background:
- Microtubule dynamics are critical for chromosome segregation during mitosis.
- Hepatoma-upregulated protein (HURP) and Kif18A are key regulators of kinetochore-attached microtubule dynamics.
- The precise mechanism of HURP and Kif18A interaction remains unclear.
Purpose of the Study:
- To elucidate the mechanism by which HURP regulates Kif18A motility.
- To determine the structural basis of HURP binding to microtubules.
- To understand the combined role of HURP and Kif18A in spindle length control.
Main Methods:
- Single-molecule imaging in vitro
- Cryo-electron microscopy (Cryo-EM) to determine protein structures
- Biochemical assays to analyze motor protein activity
Main Results:
- HURP's effect on Kif18A motility is concentration-dependent: sparse HURP activates, while high concentrations inhibit Kif18A.
- Cryo-EM revealed HURP's unique binding mode on microtubules, spanning laterally and between protofilaments.
- HURP binding partially overlaps with the Kif18A motor domain binding site, suggesting steric hindrance.
- HURP and Kif18A cooperate to suppress microtubule plus-end dynamics.
Conclusions:
- HURP regulates Kif18A motility through both activation and inhibition via steric hindrance.
- The determined structure provides a mechanistic explanation for Kif18A regulation by HURP.
- Together, HURP and Kif18A play a coordinated role in controlling spindle length during mitosis.
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