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The in vitro assembly of flagellar outer doublet tubulin
Abstract:
Flagellar outer doublet microtubules were solubilized by use of sonication, and the tubulin was reassembled in vitro into single microtubules containing 14 and 15 protofilaments. The tubulin assembly was dependent on both the KCl and tubulin concentrations, exhibiting a critical concentration of 0.72 mg/ml at optimum solvent conditions. Flagellar tubulin was purified by cycles of temperature-dependent assembly-disassembly and molecular sieve chromatography, and characterized by two-dimensional gel electrophoresis. Although doublet microtubules were not formed in vitro, outer doublet tubulin assembled onto intact A- and B-subfibers of outer doublet microtubules and basal bodies of Chlamydomonas; the rate of assembly from the distal ends of these structures was greater than that from the proximal ends. Microtubule-associated proteins (MAPs) from mammalian brain stimulated outer doublet tubulin assembly, decorating the microtubules with fine filamentous projections.
Insights
Researchers reassembled flagellar tubulin into single microtubules in vitro. Microtubule-associated proteins (MAPs) from mammalian brain stimulated this flagellar tubulin assembly.
Area of Science:
- Cell Biology
- Biochemistry
- Structural Biology
Background:
- Flagellar outer doublet microtubules are essential for motility.
- Understanding tubulin assembly dynamics is crucial for cell biology research.
Purpose of the Study:
- To investigate the in vitro assembly of flagellar outer doublet tubulin.
- To characterize the properties of reassembled flagellar microtubules.
Main Methods:
- Solubilization of flagellar outer doublet microtubules using sonication.
- In vitro reassembly of tubulin under varying KCl and tubulin concentrations.
- Purification via temperature-dependent assembly-disassembly and molecular sieve chromatography.
- Characterization using two-dimensional gel electrophoresis.
Main Results:
- Flagellar tubulin reassembled into single microtubules with 14 and 15 protofilaments.
- Assembly was concentration-dependent, with a critical concentration of 0.72 mg/ml.
- Tubulin assembled onto existing microtubule structures (A- and B-subfibers, basal bodies) with directional preference.
- Mammalian brain Microtubule-Associated Proteins (MAPs) stimulated assembly and altered microtubule structure.
Conclusions:
- Flagellar outer doublet tubulin can be reassembled in vitro into single microtubules.
- Assembly dynamics are influenced by solution conditions and existing microtubule templates.
- MAPs can modulate flagellar tubulin assembly, suggesting conserved regulatory mechanisms.