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4',6-Diamidino-2-phenylindole, a fluorescent probe for tubulin and microtubules
Abstract:
A new fluorophor for tubulin which has permitted the monitoring of microtubule assembly in vitro is reported. DAPI (4',6-diamidino-2-phenylindole), a fluorophor already known as a DNA intercalator, was shown to bind specifically to a unique tubulin site as a dimer (KD(app) = 43 +/- 5 microM at 37 degrees C) or to tubulin associated in microtubules (KD(app) = 6 +/- 2 microM at 37 degrees C) with the same maximum enhancement in fluorescence. When tubulin polymerization was induced with GTP, the change in DAPI affinity for tubulin resulted in an enhancement of DAPI binding and, consequently, of fluorescence intensity. DAPI, whose binding site is different from that of colchicine, vinblastine, or taxol, did not interfere greatly with microtubule polymerization. It induced a slight diminution of the critical concentration for tubulin assembly due to a decrease in the depolymerizing rate constant. Moreover, DAPI did not interfere with GTP hydrolysis correlated with tubulin polymerization, but it decreased the GTPase activity at the steady state of tubulin assembly. Even at substoichiometric levels DAPI can be used to follow the kinetics of microtubule assembly.
Insights
A novel fluorescent dye, 4
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Microtubule assembly is crucial for cellular processes.
- Monitoring microtubule dynamics requires specific fluorescent probes.
Purpose of the Study:
- To report a new fluorophore, 4',6-diamidino-2-phenylindole (DAPI), for monitoring microtubule assembly.
- To characterize DAPI's binding to tubulin and its effects on polymerization.
Main Methods:
- Spectrofluorometry to measure DAPI binding affinity to tubulin.
- In vitro microtubule assembly assays.
- Kinetic analysis of tubulin polymerization and GTP hydrolysis.
Main Results:
- DAPI binds specifically to tubulin with different affinities for free tubulin and polymerized microtubules.
- DAPI binding enhances fluorescence, allowing real-time monitoring of microtubule assembly.
- DAPI slightly decreases the critical concentration for tubulin assembly without significantly inhibiting polymerization.
- DAPI affects GTPase activity at steady-state but not GTP hydrolysis during polymerization.
Conclusions:
- DAPI is a valuable tool for studying microtubule assembly kinetics in vitro.
- DAPI's unique binding site and minimal interference make it suitable for various microtubule-related studies.
- DAPI enables real-time monitoring of microtubule dynamics at substoichiometric concentrations.