Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts.
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.
Hannak
Eva
E
University of California, Berkeley, 315 Life Sciences Addition, Berkeley, CA 94720-3200, USA.
Heald
Rebecca
R
D000818:Animals; D002457:Cell Extracts; D002467:Cell Nucleus; D048750:Cell Nucleus Division / Q000502:physiology*; D003584:Cytological Techniques; D005260:Female; D008297:Male; D010063:Ovum; D013094:Spermatozoa; D008941:Spindle Apparatus / Q000502:physiology*; D014982:Xenopus laevis
D002457:Cell Extracts