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Updated: Feb 17, 2026
Directly Measuring Forces Within Reconstituted Active Microtubule Bundles
Published on: May 10, 2022
Stu2 uses a 15-nm parallel coiled coil for kinetochore localization and concomitant regulation of the mitotic spindle
Karen P Haase1,2,2, Jaime C Fox1,3, Amy E Byrnes2,3
1Molecular and Cellular Biophysics Program, University of North Carolina, Chapel Hill, NC 27599.
Abstract:
XMAP215/Dis1 family proteins are potent microtubule polymerases, critical for mitotic spindle structure and dynamics. While microtubule polymerase activity is driven by an N-terminal tumor overexpressed gene (TOG) domain array, proper cellular localization is a requisite for full activity and is mediated by a C-terminal domain. Structural insight into the C-terminal domain's architecture and localization mechanism remain outstanding. We present the crystal structure of the Saccharomyces cerevisiae Stu2 C-terminal domain, revealing a 15-nm parallel homodimeric coiled coil. The parallel architecture of the coiled coil has mechanistic implications for the arrangement of the homodimer's N-terminal TOG domains during microtubule polymerization. The coiled coil has two spatially distinct conserved regions: CRI and CRII. Mutations in CRI and CRII perturb the distribution and localization of Stu2 along the mitotic spindle and yield defects in spindle morphology including increased frequencies of mispositioned and fragmented spindles. Collectively, these data highlight roles for the Stu2 dimerization domain as a scaffold for factor binding that optimally positions Stu2 on the mitotic spindle to promote proper spindle structure and dynamics.
Insights
XMAP215/Dis1 proteins regulate cell division by polymerizing microtubules. Their C-terminal domain, revealed by crystal structure, acts as a scaffold for proper spindle localization and function.
Area of Science:
- Cell Biology
- Structural Biology
- Biochemistry
Background:
- XMAP215/Dis1 proteins are crucial microtubule polymerases essential for mitotic spindle organization.
- Microtubule polymerization activity is governed by N-terminal TOG domains, while C-terminal domains mediate cellular localization.
- The precise structure and localization mechanism of the C-terminal domain remain largely uncharacterized.
Purpose of the Study:
- To elucidate the structural architecture of the Saccharomyces cerevisiae Stu2 C-terminal domain.
- To investigate the role of the C-terminal domain in Stu2 localization and mitotic spindle function.
Main Methods:
- X-ray crystallography was employed to determine the structure of the Stu2 C-terminal domain.
- Site-directed mutagenesis was used to probe the function of conserved regions within the coiled coil.
Main Results:
- The crystal structure revealed a 15-nm parallel homodimeric coiled coil.
- Two distinct conserved regions, CRI and CRII, were identified within the coiled coil.
- Mutations in CRI and CRII disrupted Stu2 localization and led to aberrant spindle morphology, including mispositioned and fragmented spindles.
Conclusions:
- The Stu2 C-terminal domain functions as a parallel homodimeric coiled coil.
- This dimerization domain acts as a scaffold, crucial for positioning Stu2 on the mitotic spindle.
- Optimal Stu2 localization mediated by the C-terminal domain is vital for proper spindle structure and dynamics.
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