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Related Concept Videos

Introduction to Actin01:26

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Actin is a highly conserved cytoskeletal protein found abundantly in eukaryotic cells. It constitutes 10% weight of the total cellular protein in muscle cells, while in non-muscle cells, it is lower and makes up around 1–5 percent of the total cell protein. Actin found in the unicellular amoebae and complex multicellular animals is around 80% similar, demonstrating their conservation over a billion years of evolution.  Actin coding genes are conserved within species and across...
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Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization
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Xenopus egg cytoplasm with intact actin.

Christine M Field1, Phuong A Nguyen1, Keisuke Ishihara1

  • 1Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA; The Marine Biological Laboratory, Woods Hole, Massachusetts, USA.

Methods in Enzymology
|March 18, 2014
PubMed
Summary

Researchers developed actin-intact Xenopus egg extracts, a cell-free system preserving natural cytoplasmic components. This method enables detailed study of actin dynamics and early embryonic development without perturbing the system.

Keywords:
ActinCell cycleCell-free systemEgg extractMicrotubuleXenopus

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Area of Science:

  • Cell Biology
  • Developmental Biology
  • Biochemistry

Background:

  • Xenopus egg extracts are valuable cell-free systems for studying cellular processes.
  • Previous methods often used cytochalasin D, which can perturb actin dynamics.
  • A need exists for cell-free preparations that closely mimic native egg physiology.

Purpose of the Study:

  • To develop and optimize a method for preparing Xenopus egg extracts without cytochalasin D.
  • To characterize the resulting
  • actin-intact egg extract
  • as a minimally perturbed cell-free system.
  • To demonstrate the utility of this extract for studying actin dynamics and early embryonic structures.

Main Methods:

  • Optimized protocols for preparing Xenopus egg extracts, specifically omitting cytochalasin D.
  • Characterization of the extract's composition, including organelles, glycogen, and intact actin.
  • Development of assays to assess cell cycle state and ensure extract quality.

Main Results:

  • Successfully prepared
  • actin-intact egg extracts
  • which are undiluted, contain abundant organelles and glycogen.
  • Demonstrated the extract's suitability for studying cell cycle regulation of actin and myosin-II dynamics.
  • Utilized the extract to reconstitute large, interphase asters crucial for early Xenopus embryo organization.

Conclusions:

  • Actin-intact Xenopus egg extracts provide a superior cell-free system for studying actin dynamics and cytoplasmic interactions.
  • This preparation closely mimics egg physiology, offering new avenues for biochemical and biophysical investigations of early development.
  • Detailed protocols and handling tips are provided for reproducible research.