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Three-headed outer arm dynein from Chlamydomonas that can functionally combine with outer-arm-missing axonemes
S Takada1, H Sakakibara, R Kamiya
1Department of Molecular Biology, School of Science, Nagoya University, Aichi.
Journal of Biochemistry
|June 1, 1992
Summary
A new method isolates functional outer-arm dynein from Chlamydomonas, crucial for flagellar motility. This purified 23S dynein restores movement in a mutant lacking this motor protein.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Motors
Background:
- Outer-arm dynein is essential for flagellar motility in Chlamydomonas.
- Previous methods resulted in dissociated dynein components (18S and 12S particles).
- These dissociated components lacked the ability to restore motility in outer-arm-missing mutants.
Purpose of the Study:
- To develop a procedure for isolating functional outer-arm dynein.
- To characterize the intact 23S dynein particle.
- To assess the functional recovery of motility in an outer-arm-missing mutant.
Main Methods:
- Developed a high-salt extraction protocol in the presence of Mg2+.
- Utilized ultracentrifugation to isolate the 23S dynein complex.
- Electron microscopy was used to visualize the dynein structure.
- Assessed motility restoration in detergent-extracted cell models of the oda1 mutant.
Main Results:
- A stable 23S outer-arm dynein complex containing alpha, beta, and gamma heavy chains was isolated.
- The 23S dynein exhibited a three-headed bouquet structure.
- Addition of 23S dynein to oda1 axonemes significantly increased beat frequency (28 Hz to 53 Hz).
- Neither the 18S nor 12S dynein particles restored motility.
Conclusions:
- The developed protocol successfully purifies intact and functional outer-arm dynein.
- The 23S dynein is the active form required for restoring flagellar motility.
- This method provides a valuable tool for studying dynein motor function.