Centrosome reproduction in Vitro: Mammalian centrosomes in Xenopus lysates

Abstract

Publisher Summary This chapter discusses reproduction of centrosomes in vitro . The structural complexity of the animal centrosome, particularly the presence of the centriole pair as a conspicuous feature, suggests that a specific study of the animal centrosome is needed. An alternative approach to decipher the centrosome duplication pathway has been to use marine or amphibian eggs in which early development occurs without cell growth by segmentation, following each doubling of chromosomes and of the sperm-inherited centrosome. Xenopus egg extracts are the most characterized cell-free systems to observe not only centrosome assembly but also cell cycle-dependent regulation of this pathway. The chapter discusses a new strategy for developing a centrosome duplication assay in a Xenopus egg extract. The main feature of the assay is to use in situ centrosomes instead of isolated centrosomes. Triton X-100-extracted cytoplasts prepared from mouse L929 cells are used in this assay. This has several advantages: in the absence of nuclei, cytoplasts are very flat and are attached stably to the cover-slip on which the cells are grown. Their cytoplasmic compartments are distributed about the centrosome, which site at the geometric center of the cytoplast. Thus cytoplasts from early G1 cells provide a favorable starting material, as their centrosome does not initiate the structurally identifiable steps of duplication.