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The microtubule-destabilizing kinesin XKCM1 is required for chromosome positioning during spindle assembly
Claire E Walczak1, Eugene C Gan, Arshad Desai
1Medical Sciences, Indiana University, Bloomington, IN 47405, USA. cwalczak@indiana.edu
Abstract:
Xenopus kinesin catastrophe modulator-1 (XKCM1) is a Kin I kinesin family member that uses the energy of ATP hydrolysis to depolymerize microtubules. We demonstrated previously that XKCM1 is essential for mitotic-spindle assembly in vitro and acts by regulating microtubule dynamics as a pure protein, in extracts and in cells. A portion of the XKCM1 pool is specifically localized to centromeres during mitosis and may be important in chromosome movement. To selectively analyze the function of centromere-bound XKCM1, we generated glutathione-S-transferase (GST) fusion proteins containing the N-terminal globular domain (GST-NT), the centrally located catalytic domain (GST-CD), and the C-terminal alpha-helical tail (GST-CT) of XKCM1. The GST-NT protein targeted to centromeres during spindle assembly, suggesting that the N-terminal domain of XKCM1 is sufficient for centromere localization. Addition of GST-NT prior to or after spindle assembly replaced endogenous XKCM1, indicating that centromere targeting is a dynamic process. Loss of endogenous XKCM1 from centromeres caused a misalignment of chromosomes on the metaphase plate without affecting global spindle structure. These results suggest that centromere bound XKCM1 has an important role in chromosome positioning on the spindle.
Insights
Xenopus kinesin catastrophe modulator-1 (XKCM1) is crucial for mitotic spindle assembly. Its N-terminal domain targets centromeres, ensuring proper chromosome alignment during cell division.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Xenopus kinesin catastrophe modulator-1 (XKCM1) is a Kin I kinesin involved in microtubule dynamics.
- XKCM1 is essential for mitotic-spindle assembly and regulates microtubule dynamics.
- A fraction of XKCM1 localizes to centromeres during mitosis, potentially influencing chromosome movement.
Purpose of the Study:
- To investigate the specific function of centromere-bound XKCM1.
- To determine the domain of XKCM1 responsible for centromere localization.
- To analyze the role of centromere-localized XKCM1 in chromosome positioning.
Main Methods:
- Generation of glutathione-S-transferase (GST) fusion proteins of XKCM1 domains (N-terminal, catalytic, C-terminal).
- Assessment of centromere targeting of GST-NT protein during spindle assembly.
- Analysis of chromosome alignment and spindle structure after manipulating endogenous XKCM1 at centromeres.
Main Results:
- The N-terminal domain (GST-NT) of XKCM1 is sufficient for centromere localization.
- Centromere targeting of XKCM1 is a dynamic process.
- Loss of centromere-bound XKCM1 leads to chromosome misalignment without affecting overall spindle structure.
Conclusions:
- Centromere-bound XKCM1 plays a critical role in accurate chromosome positioning during mitosis.
- The N-terminal domain mediates XKCM1's centromere localization.
- Understanding XKCM1's centromeric function provides insights into mitotic regulation and chromosome segregation.