title: A single Drosophila embryo extract for the study of mitosis ex vivo.
abstract: Spindle assembly and chromosome segregation rely on a complex interplay of biochemical and mechanical processes. Analysis of this interplay requires precise manipulation of endogenous cellular components and high-resolution visualization. Here we provide a protocol for generating an extract from individual Drosophila syncytial embryos that supports repeated mitotic nuclear divisions with native characteristics. In contrast to the large-scale, metaphase-arrested Xenopus egg extract system, this assay enables the serial generation of extracts from single embryos of a genetically tractable organism, and each extract contains dozens of autonomously dividing nuclei that can be prepared and imaged within 60-90 min after embryo collection. We describe the microscopy setup and micropipette production that facilitate single-embryo manipulation, the preparation of embryos and the steps for making functional extracts that allow time-lapse microscopy of mitotic divisions ex vivo. The assay enables a unique combination of genetic, biochemical, optical and mechanical manipulations of the mitotic machinery.
authors: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany. ivo.telley@embl.de
authors: Ivo A
authors: IA
authors: Telley
authors: Imre
authors: I
authors: Gáspár
authors: Anne
authors: A
authors: Ephrussi
authors: Thomas
authors: T
authors: Surrey
mesh_terms: D000818:Animals; D002457:Cell Extracts / Q000302:isolation & purification*; D004331:Drosophila melanogaster; D004625:Embryo, Nonmammalian / Q000166:cytology*; D008854:Microscopy, Electron / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008938:Mitosis / Q000502:physiology*; D023421:Models, Animal; D059008:Time-Lapse Imaging / Q000379:methods*; D014981:Xenopus
chemical_list: D002457:Cell Extracts