Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts

Abstract

Extracts from Xenopus laevis eggs provide a powerful system for the study of cell division processes in vitro through biochemical reconstitution and manipulation, and microscopic analysis. We provide protocols for the preparation of metaphase-arrested extracts and in vitro assays to examine the following pathways of spindle assembly: 1) Sperm nuclei added to meiotic extracts, supporting the formation of half-spindles and bipolar spindle structures around unreplicated chromosomes; 2) sperm nuclei added to extracts that cycle through interphase and form spindles that are capable of undergoing anaphase and chromosome segregation; and 3) spindle formation around chromatin-coated beads. Finally, we describe methods to inhibit a specific protein by immunodepletion or addition of an inhibitor such as a dominant-negative construct. These techniques can be used to analyze the mitotic function of a given protein. It takes approximately 1.5 h to prepare the extract, 1-3 h for spindle-assembly experiments and an additional 1-3 h if immunodepletion is performed.

Centrosomal protein of 192 kDa (Cep192) promotes centrosome-driven spindle assembly by engaging in organelle-specific Aurora A activation

“…To study AurA regulation during centrosome-mediated spindle assembly, we used cell-free metaphase-arrested Xenopus egg extract (extract) (21,22). When extract is supplemented with demembranated sperm nuclei, which contain a pair of centrioles, the latter recruit PCM, giving rise to a functional centrosome that acts as a MTOC (22). Centrosomal MT assembly begins 2-3 min after sperm addition to extract, peaking ≈7-8 min later (22) (Fig.…”

“…When extract is supplemented with demembranated sperm nuclei, which contain a pair of centrioles, the latter recruit PCM, giving rise to a functional centrosome that acts as a MTOC (22). Centrosomal MT assembly begins 2-3 min after sperm addition to extract, peaking ≈7-8 min later (22) (Fig. 1A, Upper).…”

Paternal chromosome loss and metabolic crisis contribute to hybrid inviability in Xenopus

“…To examine kinetochore assembly, X. laevis sperm nuclei, prepared as previously described 31 , were used as a source of DNA in both X. laevis and X. tropicalis egg extracts. Cycled chromosomes were prepared and spun-down 30 and then processed for immunofluorescence as previously described 32 . Briefly, the coverslips were incubated for 1 min in cold methanol, washed with PBS+NP40 and blocked overnight in PBS + 5% BSA at 4°C.…”

Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles

“…Cytostatic-factor-arrested (CSF) Xenopus laevis egg extract, which corresponds to the 'active' cytoplasm of oocytes arrested in metaphase II of meiosis, was prepared as described previously 18,19,45 . Microtubules structures were assembled using metaphase extracts in the presence of rhodamin-X-labelled tubulin and Ran-NPs or RCC1-NPs, as indicated.…”

“…As a first step towards in vivo manipulations, we used a functional cytoplasm reconstituted from Xenopus eggs. Such cell extracts have provided a powerful system for dissecting mitotic spindle morphogenesis; they permit biochemical manipulations and cell cycle control, while offering very good optical properties when compared with the difficulties inherent to the imaging of large embryonic cells 18,19 . Moreover, this membrane-free system allows the direct delivery of nanoparticles in the cytoplasm, without relying on endocytosis or requiring microinjection.…”