TY - JOUR
T1 - Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging
AU - Schvartz, Tomer
AU - Aloush, Noa
AU - Goliand, Inna
AU - Segal, Inbar
AU - Nachmias, Dikla
AU - Arbely, Eyal
AU - Elia, Natalie
N1 - Publisher Copyright:
© 2017 Schvartz et al.
PY - 2017/10/15
Y1 - 2017/10/15
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85031291704&partnerID=8YFLogxK
U2 - 10.1091/mbc.E17-03-0161
DO - 10.1091/mbc.E17-03-0161
M3 - Article
C2 - 28835375
AN - SCOPUS:85031291704
SN - 1059-1524
VL - 28
SP - 2747
EP - 2756
JO - Molecular Biology of the Cell
JF - Molecular Biology of the Cell
IS - 21
ER -