Encyclopedia of Experiments: Biological Techniques
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Transcript
Actin monomers polymerize in a head-to-tail manner to form thin fibers, called microfilaments. Similarly, tubulin dimers polymerize into longer hollow cylindrical microtubules. These cytoskeleton components maintain cell shape and facilitate cellular motility.
To visualize the actin-microtubule coupling dynamics by total internal reflection microscopy, TIRF, take a customized imaging assembly.
The imaging assembly consists of two coverslips: a top m-PEG silane-coated coverslip and a bottom biotin-PEG silane-coated coverslip. The two coverslips are spaced longitudinally with double-sided adhesive tape and sealed at both ends to create a flow channel.
Pipette BSA into the flow channel to prime the chamber. Add streptavidin solution, and incubate. The streptavidin molecules bind to the biotin molecules attached to the bottom coverslip's PEG-silane.
Flow TIRF buffer into the channel. Add a cytoskeleton mix solution containing unlabeled and green fluorophore-labeled tubulin dimers, along with unlabeled, biotin-labeled, and purple fluorophore-tagged actin monomers.
Next, add a reaction solution containing ATP — actin polymerization-promoting agent, and GTP — tubulin polymerization-promoting agent, and actin-microtubule coupling protein. Incubate.
The tubulin dimers bind to the GTP molecules and polymerize, while the actin monomers bind to the ATP molecules and polymerize to form microtubules and microfilaments, respectively.
Place the imaging assembly under a TIRF microscope. Excite the fluorophores with lasers of appropriate wavelengths.
The excitation light reflects internally, and creates a light field that selectively illuminates the fluorophores in a limited region near the glass-water interface, where there are two media with different refractive indexes. In this region, the microfilaments appear purple, and the microtubules appear green.
