title: Xenopus egg cytoplasm with intact actin.
abstract: We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA; The Marine Biological Laboratory, Woods Hole, Massachusetts, USA. Electronic address: Christine_field@hms.harvard.edu.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Field
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA; The Marine Biological Laboratory, Woods Hole, Massachusetts, USA.
authors.forename: Phuong A
authors.initials: PA
authors.lastname: Nguyen
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA; The Marine Biological Laboratory, Woods Hole, Massachusetts, USA.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ishihara
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA; The Marine Biological Laboratory, Woods Hole, Massachusetts, USA.
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Groen
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA; The Marine Biological Laboratory, Woods Hole, Massachusetts, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Mitchison
mesh_terms: D000199:Actins / Q000302:isolation & purification* / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D002453:Cell Cycle; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D010063:Ovum / Q000737:chemistry* / Q000378:metabolism; D014982:Xenopus laevis / Q000196:embryology / Q000378:metabolism*
chemical_list: D000199:Actins
keywords: Actin; Cell cycle; Cell-free system; Egg extract; Microtubule; Xenopus