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THE MITOTIC APPARATUS ISOLATED IN GLYCEROL-CONTAINING MEDIUM
Hikoichi Sakai1, Ryoko Kuriyama1
1Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan.
Development, Growth & Differentiation
|June 7, 2023
Summary
High glycerol concentrations stabilize sea urchin mitotic apparatus, preserving spindle integrity. Tubulin, a key protein, was extracted and characterized, revealing its subunits are similar to brain tubulin.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The mitotic apparatus is crucial for cell division.
- Microtubules, stabilized by glycerol, are key components of the spindle.
- Understanding tubulin's role in spindle dynamics is vital for cell biology.
Purpose of the Study:
- To isolate and stabilize the sea urchin egg mitotic apparatus.
- To investigate the properties and extractability of tubulin from the mitotic apparatus.
- To characterize the subunits of sea urchin tubulin and compare them to other sources.
Main Methods:
- Isolation of the mitotic apparatus from sea urchin eggs using a glycerol-based medium.
- Stabilization of the isolated apparatus with high glycerol concentrations.
- Extraction of tubulin using Ca ions, organic mercurials, or low temperature with GTP.
- Analysis of tubulin using colchicine binding assays and DEAE Sephadex chromatography.
- Characterization of tubulin subunits via electrophoresis and molecular weight determination.
Main Results:
- High glycerol concentrations (3-4 M) effectively stabilized the mitotic apparatus, preventing chromosome disjunction and spindle fragility.
- Tubulin was successfully extracted from the glycerol-mitotic apparatus under various conditions.
- The extracted tubulin exhibited colchicine binding, with a molecular weight of 105,000 for the complex.
- DEAE Sephadex chromatography yielded a tubulin fraction that bound colchicine.
- Sea urchin tubulin consists of alpha and beta subunits with molecular weights of approximately 55,000 and 51,000, respectively, similar to brain tubulin.
Conclusions:
- Glycerol is effective in stabilizing the sea urchin mitotic apparatus, crucial for maintaining spindle structure.
- Sea urchin mitotic apparatus tubulin shares biochemical properties and subunit characteristics with tubulin from other organisms.
- This study provides insights into the molecular composition and stability of the mitotic spindle during cell division.
