Abstract
Centromeres must withstand high pulling forces in metaphase, yet allow rapid and complete chromosome segregation in anaphase. Using optical tweezers measurements on isolated human metaphase chromosomes, we find that centromeres are stiffer at low forces and more frequently exhibit pronounced softening compared to whole chromosomes, while displaying a similar stiffening response at high forces. We capture these mechanical properties by establishing a softened hierarchical worm-like chain model. Cohesin cleavage alters the mechanical properties of centromeres but does not lead to chromosome segregation in vitro. Topoisomerase IIα is recruited to centromeric regions under significant tension, likely to resolve the persistent centromeric DNA entanglements that provide additional sister chromatid cohesion. These findings advance our understanding of the mechanical resilience and regulation of centromeres during mitosis.