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Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts
Abstract:
Microtubule proteins and tubulin have been purified from brain and labeled with dichlorotriazinyl fluorescein (DTAF). This procedure compromises neither the polymerizability of the proteins nor their affinities for unlabeled proteins. Within 15 min after microinjection of either DTAF-microtubule proteins or DTAF-tubulin into cultured gerbil fibroma cells, there was an evolution of a fluorescent fibrillar pattern with a distribution similar to that of the microtubular network seen after staining with fluorescent antitubulin. These filaments were colchicine sensitive and could be seen to elongate with time. DTAF-labeled microtubule accessory proteins from brain were not incorporated into filaments and appeared to label autophagic vacuoles.
Insights
Researchers visualized microtubule dynamics in living cells using fluorescently labeled tubulin. These labeled proteins formed dynamic filaments, demonstrating their polymerization and colchicine sensitivity, offering insights into microtubule assembly.
Area of Science:
- Cell Biology
- Cytoskeleton Dynamics
- Protein Biochemistry
Background:
- Microtubules are essential cytoskeletal components involved in cell structure, division, and transport.
- Understanding microtubule dynamics in living cells requires reliable labeling methods that preserve protein function.
Purpose of the Study:
- To develop and validate a method for visualizing dynamic microtubule assembly in living cells.
- To investigate the behavior and polymerization of labeled microtubule proteins and tubulin after microinjection.
Main Methods:
- Purification and fluorescent labeling of microtubule proteins and tubulin from brain tissue using dichlorotriazinyl fluorescein (DTAF).
- Microinjection of DTAF-labeled proteins into cultured gerbil fibroma cells.
- Live-cell imaging to observe the formation and dynamics of fluorescently labeled structures.
- Assessment of colchicine sensitivity of the observed filaments.
Main Results:
- DTAF labeling did not compromise the polymerizability or binding affinities of microtubule proteins and tubulin.
- Microinjected DTAF-tubulin rapidly formed fluorescent fibrillar patterns resembling the endogenous microtubule network.
- These newly formed filaments exhibited colchicine sensitivity and elongated over time.
- DTAF-labeled microtubule accessory proteins did not incorporate into filaments and were observed in autophagic vacuoles.
Conclusions:
- DTAF labeling is a suitable method for studying microtubule dynamics in living cells.
- The results confirm the polymerization capacity and dynamic behavior of tubulin in vivo.
- Microtubule accessory proteins have distinct intracellular fates compared to tubulin.