Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging

Tomer Schvartz, Noa Aloush, Inna Goliand, Inbar Segal, Dikla Nachmias, Eyal Arbely, Natalie Elia

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging.

Original languageEnglish
Pages (from-to)2747-2756
Number of pages10
JournalMolecular Biology of the Cell
Volume28
Issue number21
DOIs
StatePublished - 15 Oct 2017

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