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Cell tracking using photoconvertible proteins during zebrafish development.
J Vis Exp 2012; (67)JV

Abstract

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede, KikGR, Dendra and EosFP, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed β-elimination and subsequent extension of a π-conjugated system. PCFPs and their monomeric variants are useful tools for tracking cells and studying protein dynamics, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish, chicken and mouse for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Authors+Show Affiliations

Max Delbrück Center for Molecular Medicine.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Video-Audio Media

Language

eng

PubMed ID

23052298

Citation

Lombardo, Verónica A., et al. "Cell Tracking Using Photoconvertible Proteins During Zebrafish Development." Journal of Visualized Experiments : JoVE, 2012.
Lombardo VA, Sporbert A, Abdelilah-Seyfried S. Cell tracking using photoconvertible proteins during zebrafish development. J Vis Exp. 2012.
Lombardo, V. A., Sporbert, A., & Abdelilah-Seyfried, S. (2012). Cell tracking using photoconvertible proteins during zebrafish development. Journal of Visualized Experiments : JoVE, (67), doi:10.3791/4350.
Lombardo VA, Sporbert A, Abdelilah-Seyfried S. Cell Tracking Using Photoconvertible Proteins During Zebrafish Development. J Vis Exp. 2012 Sep 28;(67) PubMed PMID: 23052298.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cell tracking using photoconvertible proteins during zebrafish development. AU - Lombardo,Verónica A, AU - Sporbert,Anje, AU - Abdelilah-Seyfried,Salim, Y1 - 2012/09/28/ PY - 2012/10/12/entrez PY - 2012/10/12/pubmed PY - 2012/12/28/medline JF - Journal of visualized experiments : JoVE JO - J Vis Exp IS - 67 N2 - Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede, KikGR, Dendra and EosFP, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed β-elimination and subsequent extension of a π-conjugated system. PCFPs and their monomeric variants are useful tools for tracking cells and studying protein dynamics, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish, chicken and mouse for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model. SN - 1940-087X UR - https://www.unboundmedicine.com/medline/citation/23052298/Cell_tracking_using_photoconvertible_proteins_during_zebrafish_development_ L2 - https://dx.doi.org/10.3791/4350 DB - PRIME DP - Unbound Medicine ER -