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SITE-SPECIFIC ORTHOGONAL LABELING OF THE CARBOXY TERMINUS OF α-TUBULIN IN LIVE CELLS
SITE-SPECIFIC ORTHOGONAL LABELING OF THE CARBOXY TERMINUS OF α-TUBULIN IN LIVE CELLS
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机译:活细胞中α-微管蛋白羧基末端的特定位正交标记
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摘要
A technique is provided to visualize microtubules in live cells that does not require genetic manipulation or microinjection. Moreover, this method also avoids perturbation of the endogenous microtubule network that occurs with taxol treatment. This technique exploits tyrosination and detyrosination of tubulin, a posttranslational modification cycle specific to the C-terminus of a- tubulin. Specifically, cells are grown in medium supplemented with a tyrosine derivative possessing a reactive functional group. The cellular enzyme tubulin tyrosine ligase attaches the unnatural amino acid to a single site on tubulin. Addition of fresh medium containing a suitably derivatized fluorophore then yields fluorescent tubulin, which incorporate into cellular microtubules. Importantly, the tubulin labeling approach demonstrated here does not detrimentally affect microtubule network or cell morphology. Thus we present a simple, robust labeling technique that allows microscopic analysis of microtubules in live cells.
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