Assembly and Micromanipulation of Xenopus In Vitro–Assembled Mitotic Chromosomes

Abstract

Mitotic chromosomes have fascinated scientists for several decades. Despite the numerous microscopy studies, chromosome structure is, however, still poorly understood. This is due to both the high complexity of the mitotic chromosomes and the lack of other appropriate techniques suitable for studying their organization. This unit describes a novel physical approach based on measurements of mitotic chromosome elasticity. The elasticity properties are determined by the underlying structure, and knowledge of them has allowed a description of the organization of the mitotic chromosomes and critical analysis of the available models. In this unit, a detailed protocol for the measurements of the elastic response of in vitro assembled mitotic chromosomes in Xenopus egg extract is presented.

The Power of Xenopus Egg Extract for Reconstitution of Centromere and Kinetochore Function

“…However, tolerance of the host to mutation of kinetochore components is still a limitation of this method. This limitation does not apply in egg extract, where tension can be applied directly to sperm or reconstituted kinetochores (Almagro and Dimitrov 2005; Yan et al 2007). Extract also affords the unique opportunity to understand how cytoplasmic factors or those that do not copurify with kinetochores may contribute to their response to tension.…”

Chromatin under mechanical stress: from single 30 nm fibers to single nucleosomes

“…[20–22]). Mitotic chromosomes have been the subject of several ‘mechanical’ studies [23–29] and some of the stretching experiments were performed long before the invention of optical tweezers [30–33]. These studies have recently been thoroughly reviewed [34] and this review will thus concentrate on reviewing single molecule studies of individual nucleosomes, nucleosomal arrays and 30 nm chromatin fibers.…”