A technique that allows live visualization of microtubules without genetic manipulation or perturbing protein function
The present technology provides a technique that can be employed to visualize microtubules in live cells without genetic manipulation or perturbing the protein function. Specifically, this technique exploits the mechanism of tyrosination/detyrosination cycle, a posttranslational modification specific to the C-terminus of α- tubulin, to covalently attach a chemical probe to it. Cells are first grown in medium supplemented with a bioorthogonal amino acid, such as 3-formyltyrosine (3fY). 3-Acetyl tyrosine or tyrosine hydrazide. These un-natural amino acids readily enter the cell and can be appended to the C-terminus of α-tubulin by endogenous tubulin tyro-sine ligase, but are not generally incorporated into proteins produced by the normal protein synthesis mechanisms, thus providing specificity based on the post-translational modification enzymes. Cells are then treated with a suitably derivatized fluorophore and reacts specifically with a reactive chemical site on the bioorthoginal amino acid.
Permits study of microtubule dynamics—formulation of potential chemotherapeutics & assistance in determining mechanism of microtubule drug action. No genetic manipulation involved. Site-specific labeling. Cell morphology, microtubule network and cell viability re-mains unaltered (24 hours). Variety of commercially available probes can be used for labeling