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Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins
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CENP-A Is Dispensable for Mitotic Centromere Function after Initial Centromere/Kinetochore Assembly
Sebastian Hoffmann1, Marie Dumont1, Viviana Barra1
1Institut Curie, PSL Research University, CNRS, UMR 144, 26 rue d'Ulm, F-75005, Paris, France.
Cell Reports
|November 24, 2016
Summary
Centromere assembly requires CENP-A initially, but continued binding isn't essential for mitosis after G1/S. CENP-B binding to DNA becomes crucial for kinetochore stability without CENP-A.
Area of Science:
- Cell Biology
- Genetics
- Molecular Biology
Background:
- Human centromeres are epigenetic loci defined by CENP-A chromatin on alphoid DNA.
- Kinetochore-centromere attachment is vital for accurate chromosome segregation during cell division.
Purpose of the Study:
- To investigate the necessity of continuous CENP-A binding for centromere function and kinetochore attachment.
- To elucidate the role of CENP-B in maintaining centromere integrity in the absence of CENP-A.
Main Methods:
- Induction of rapid, complete degradation of endogenous CENP-A in human cells.
- Assessment of kinetochore assembly and protein deposition (CENP-C, CENP-N, CENP-T).
- Analysis of CENP-B binding and its impact on kinetochore-centromere linkage.
Main Results:
- Complete CENP-A degradation after initial assembly does not disrupt kinetochore attachment or centromere function in mitosis.
- Pre-kinetochore assembly CENP-A depletion blocks CENP-C and CENP-N deposition, leading to defective kinetochores.
- CENP-B binding to alphoid DNA is essential for anchoring CENP-C and kinetochores when CENP-A is absent.
Conclusions:
- Continuous CENP-A presence is dispensable for established centromere function in mitosis.
- CENP-B plays a critical role in preserving centromere integrity and kinetochore attachment in the absence of CENP-A.
- A reciprocal dependency exists between CENP-A chromatin and CENP-B-bound DNA for stable chromosome segregation.
Keywords:
CENP-ACENP-BCENP-Cauxincentromerechromosome segregationepigenetickinetochoremitosisprotein degradationMore Related Videos
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