A Cell-Free System for Real-Time Analyses of Centriole Disengagement and Centriole-to-Centrosome Conversion

Abstract

Centriole or centrosome number in cycling cells is strictly maintained through coordinated duplication and segregation. Duplication is limited to once only per cell cycle by separating the assembly event that occurs in S/G2 phase from the two licensing events, centriole disengagement and centriole-to-centrosome conversion, both of which occurs in mitosis. In addition to duplication licensing, centriole-to-centrosome conversion also enables centrioles to associate with spindle poles and thereby to segregate equally during cell division. Centriole disengagement and centriole-to-centrosome conversion thus constitute the major regulatory module ensuring centrosome homeostasis in cycling cells. Using Xenopus egg extracts and purified engaged centrioles, we here describe an in vitro assay allowing us to synchronously induce the initiation of centriole disengagement and centrosome formation, pause the reaction anytime during the process, and more importantly, preserve “reaction intermediates” for further analyses.