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Related Concept Videos

The Mitotic Spindle02:27

The Mitotic Spindle

The mitotic spindle—or spindle apparatus—is a eukaryotic, cytoskeletal structure made up of long protein fibers called microtubules. Formed during cell division, the spindle separates sister chromatids and moves them to opposite ends of a parental cell, where the now individual chromosomes are distributed to two daughter cell nuclei.
The bipolar configuration of the mitotic spindle facilitates chromosomal segregation, preparing the cell for division. One mechanism that ensures bipolar mitotic...
Spindle Assembly02:50

Spindle Assembly

Spindle assembly occurs through three, often coexisting, pathways – the centrosome-mediated pathway, the chromatin-mediated pathway, and the microtubule-mediated pathway – collectively contributing to form a robust spindle apparatus.
In most cells, centrosomes are the primary microtubule nucleation centers. In the centrosome-mediated pathway, the G2-prophase transition triggers centrosome maturation and increased microtubule nucleation. Progressive nucleation results in a microtubule array...

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Related Experiment Video

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Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization
05:58

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Published on: June 6, 2021

Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts.

Eva Hannak1, Rebecca Heald

  • 1University of California, Berkeley, 315 Life Sciences Addition, Berkeley, CA 94720-3200, USA.

Nature Protocols
|April 5, 2007
PubMed
Summary

Xenopus laevis egg extracts offer a robust in vitro system for studying cell division. Protocols are detailed for spindle assembly assays and protein inhibition to analyze mitotic functions.

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Area of Science:

  • Cell Biology
  • Developmental Biology
  • Biochemistry

Background:

  • Cell division is a fundamental process crucial for development and organismal health.
  • Understanding the molecular mechanisms of spindle assembly is key to deciphering cell division fidelity.

Purpose of the Study:

  • To provide detailed protocols for utilizing Xenopus laevis egg extracts for in vitro studies of spindle assembly.
  • To enable the biochemical reconstitution and manipulation of cell division processes.
  • To offer methods for analyzing the mitotic function of specific proteins.

Main Methods:

  • Preparation of metaphase-arrested Xenopus laevis egg extracts.
  • In vitro spindle assembly assays using sperm nuclei or chromatin-coated beads.
  • Protein inhibition via immunodepletion or dominant-negative constructs.

Main Results:

  • Demonstrated spindle formation around unreplicated chromosomes (half-spindles and bipolar structures).
  • Established protocols for observing spindle assembly and subsequent anaphase progression with chromosome segregation.
  • Validated methods for inhibiting protein function to assess its role in mitosis.

Conclusions:

  • Xenopus laevis egg extracts are a versatile system for dissecting spindle assembly pathways.
  • The described techniques facilitate the functional analysis of proteins involved in cell division.
  • These protocols enable detailed investigation of mitotic mechanisms through biochemical and microscopic approaches.