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Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends
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Published on: March 15, 2014

Microtubules and Ca2+-sequestering membranes in the mitotic apparatus, isolated by a new method

C Petzelt1, M Hafner, D Mazia

  • 1Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg, Federal Republic of Germany.

European Journal of Cell Biology
|February 1, 1988
PubMed

Insights

Sea urchin embryo mitotic apparatus was isolated preserving key structures. Calcium-regulating membranes are centralized, suggesting microtubule regulation by calcium may not require direct proximity.

Area of Science:

  • Cell Biology
  • Developmental Biology
  • Biochemistry

Background:

  • The mitotic apparatus is crucial for cell division.
  • Calcium ions (Ca2+) play a significant role in regulating cellular processes, including microtubule dynamics.
  • Understanding the spatial relationship between calcium stores and microtubules is key to deciphering cell division regulation.

Purpose of the Study:

  • To isolate the sea urchin embryo mitotic apparatus using a method that preserves its structural and functional integrity.
  • To investigate the localization of calcium-sequestering membranes relative to microtubules within the isolated mitotic apparatus.
  • To explore the implications of this spatial organization for calcium-mediated regulation of microtubules.

Main Methods:

  • Isolation of the mitotic apparatus from sea urchin embryos using a polyethylene glycol (PEG)/EGTA-medium.
  • Assessment of the preservation of birefringence and Ca2+ lability.
  • Immunofluorescence microscopy using a monoclonal antibody against a 46-kDa protein to visualize the intracellular Ca2+-transport system.
  • Triple fluorescence staining to map microtubules, Ca2+-sequestering membranes, and chromatin.

Main Results:

  • The isolation procedure effectively preserved the mitotic apparatus, including microtubules and the Ca2+-transport system.
  • Ca2+-sequestering membranes were found to be concentrated in the asters' centers.
  • These membranes did not uniformly distribute along the microtubules within the mitotic apparatus.

Conclusions:

  • The spatial distribution suggests that calcium-mediated regulation of microtubules might not necessitate direct physical contact with Ca2+-regulating sites.
  • This finding offers new insights into the mechanisms governing microtubule dynamics during mitosis.
  • The study highlights the importance of intracellular calcium localization in cell division processes.

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