title: Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis.
abstract: We report optimized methods for preparing actin-intact Xenopus egg extract. This extract is minimally perturbed, undiluted egg cytoplasm where the cell cycle can be experimentally controlled. It contains abundant organelles and glycogen and supports active metabolism and cytoskeletal dynamics that closely mimic egg physiology. The concentration of the most abundant ∼11,000 proteins is known from mass spectrometry. Actin-intact egg extract can be used for analysis of actin dynamics and interaction of actin with other cytoplasmic systems, as well as microtubule organization. It can be spread as thin layers and naturally depletes oxygen though mitochondrial metabolism, which makes it ideal for fluorescence imaging. When combined with artificial lipid bilayers, it allows reconstitution and analysis of the spatially controlled signaling that positions the cleavage furrow during early cytokinesis. Actin-intact extract is generally useful for probing the biochemistry and biophysics of the large Xenopus egg. Protocols are provided for preparation of actin-intact egg extract, control of the cell cycle, fluorescent probes for cytoskeleton and cytoskeleton-dependent signaling, preparation of glass surfaces for imaging experiments, and immunodepletion to probe the role of specific proteins and protein complexes. We also describe methods for adding supported lipid bilayers to mimic the plasma membrane and for confining in microfluidic droplets to explore size scaling issues.
authors.affiliation: Harvard Medical School, Boston, MA, United States; Marine Biological Laboratory, Woods Hole, MA, United States.
authors.forename: C M
authors.initials: CM
authors.lastname: Field
authors.affiliation: Harvard Medical School, Boston, MA, United States; Marine Biological Laboratory, Woods Hole, MA, United States.
authors.forename: J F
authors.initials: JF
authors.lastname: Pelletier
authors.affiliation: Harvard Medical School, Boston, MA, United States; Marine Biological Laboratory, Woods Hole, MA, United States.
authors.forename: T J
authors.initials: TJ
authors.lastname: Mitchison
mesh_terms: D000199:Actins / Q000737:chemistry; D000818:Animals; D002455:Cell Division / Q000235:genetics; D002457:Cell Extracts / Q000737:chemistry / Q000235:genetics*; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics; D002474:Cell-Free System; D048749:Cytokinesis / Q000235:genetics*; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry; D008870:Microtubules / Q000737:chemistry / Q000235:genetics*; D057054:Molecular Imaging / Q000379:methods*; D010063:Ovum / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D014982:Xenopus laevis / Q000235:genetics*
chemical_list: D000199:Actins; D002457:Cell Extracts; D008051:Lipid Bilayers
keywords: Actin; Cell cycle; Cell-free system; Cytokinesis; Microfluidics; Microtubule; Xenopus; Xenopus egg extract