Independence of Centromeric and Pericentromeric Chromatin Stability on CCAN Components

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• The centromere's CENP-A chromatin is crucial for kinetochore assembly and chromosome segregation during mitosis.
• The constitutive centromeric-associated network (CCAN) and kinetochore assemble on CENP-A chromatin.
• CENP-A chromatin is flanked by pericentromeric heterochromatin and bound by CENP-B.

Purpose of the Study:

• To investigate the mechanical properties of centromeric chromatin and the role of the CCAN in chromosome stiffness.
• To determine how the loss of CCAN components (CENP-C, CENP-N) affects centromere mechanics.
• To understand the relationship between CENP-A chromatin and pericentromeric chromatin under mechanical stress.

Main Methods:

• Mechanical experiments on mitotic chromosomes with simultaneous tracking of CENP-A and CENP-B.
• Degradation of CENP-C and CENP-N using auxin-inducible degrons to assess CCAN function.
• Chromosome stretching assays and nuclease treatments to evaluate chromatin deformation and structural integrity.

Main Results:

• The CENP-A chromatin domain exhibited remarkable resilience, showing no significant stretching even after CCAN component degradation.
• Pericentromeric chromatin deformed upon force application, stretching approximately 3-fold less than the entire chromosome.
• Loss of CENP-C and/or CENP-N did not affect pericentromere stretching, and nuclease treatments revealed no structural impact on CENP-A.

Conclusions:

• Core centromeric chromatin (CENP-A domain) is mechanically robust and likely disconnected from pericentromeric chromatin.
• Pericentric chromatin is deformable but stiffer than chromosome arms.
• The CCAN plays a role in maintaining centromere integrity, but the core CENP-A chromatin possesses inherent mechanical resilience.