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Tracking protein dynamics with photoconvertible Dendra2 on spinning disk confocal systems.
J Microsc 2014; 256(3):197-207JM

Abstract

Understanding the dynamic properties of cellular proteins in live cells and in real time is essential to delineate their function. In this context, we introduce the Fluorescence Recovery After Photobleaching-Photoactivation unit (Andor) combined with the Nikon Eclipse Ti E Spinning Disk (Andor) confocal microscope as an advantageous and robust platform to exploit the properties of the Dendra2 photoconvertible fluorescent protein (Evrogen) and analyse protein subcellular trafficking in living cells. A major advantage of the spinning disk confocal is the rapid acquisition speed, enabling high temporal resolution of cellular processes. Furthermore, photoconversion and imaging are less invasive on the spinning disk confocal as the cell exposition to illumination power is reduced, thereby minimizing photobleaching and increasing cell viability. We have tested this commercially available platform using experimental settings adapted to track the migration of fast trafficking proteins such as UBC9, Fibrillarin and have successfully characterized their differential motion between subnuclear structures. We describe here step-by-step procedures, with emphasis on cellular imaging parameters, to successfully perform the dynamic imaging and photoconversion of Dendra2-fused proteins at high spatial and temporal resolutions necessary to characterize the trafficking pathways of proteins.

Authors+Show Affiliations

Centre for Research in Infectious Diseases, School of Medicine and Biomedical Science, University College Dublin (UCD), Dublin, Ireland.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

25186063

Citation

Woods, Elena, et al. "Tracking Protein Dynamics With Photoconvertible Dendra2 On Spinning Disk Confocal Systems." Journal of Microscopy, vol. 256, no. 3, 2014, pp. 197-207.
Woods E, Courtney J, Scholz D, et al. Tracking protein dynamics with photoconvertible Dendra2 on spinning disk confocal systems. J Microsc. 2014;256(3):197-207.
Woods, E., Courtney, J., Scholz, D., Hall, W. W., & Gautier, V. W. (2014). Tracking protein dynamics with photoconvertible Dendra2 on spinning disk confocal systems. Journal of Microscopy, 256(3), pp. 197-207. doi:10.1111/jmi.12172.
Woods E, et al. Tracking Protein Dynamics With Photoconvertible Dendra2 On Spinning Disk Confocal Systems. J Microsc. 2014;256(3):197-207. PubMed PMID: 25186063.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Tracking protein dynamics with photoconvertible Dendra2 on spinning disk confocal systems. AU - Woods,Elena, AU - Courtney,Jane, AU - Scholz,Dimitri, AU - Hall,William W, AU - Gautier,Virginie W, Y1 - 2014/09/03/ PY - 2014/01/13/received PY - 2014/07/31/accepted PY - 2014/9/5/entrez PY - 2014/9/5/pubmed PY - 2015/9/26/medline KW - Dendra2 KW - live cell imaging KW - photoconversion KW - spinning disk confocal microscopy SP - 197 EP - 207 JF - Journal of microscopy JO - J Microsc VL - 256 IS - 3 N2 - Understanding the dynamic properties of cellular proteins in live cells and in real time is essential to delineate their function. In this context, we introduce the Fluorescence Recovery After Photobleaching-Photoactivation unit (Andor) combined with the Nikon Eclipse Ti E Spinning Disk (Andor) confocal microscope as an advantageous and robust platform to exploit the properties of the Dendra2 photoconvertible fluorescent protein (Evrogen) and analyse protein subcellular trafficking in living cells. A major advantage of the spinning disk confocal is the rapid acquisition speed, enabling high temporal resolution of cellular processes. Furthermore, photoconversion and imaging are less invasive on the spinning disk confocal as the cell exposition to illumination power is reduced, thereby minimizing photobleaching and increasing cell viability. We have tested this commercially available platform using experimental settings adapted to track the migration of fast trafficking proteins such as UBC9, Fibrillarin and have successfully characterized their differential motion between subnuclear structures. We describe here step-by-step procedures, with emphasis on cellular imaging parameters, to successfully perform the dynamic imaging and photoconversion of Dendra2-fused proteins at high spatial and temporal resolutions necessary to characterize the trafficking pathways of proteins. SN - 1365-2818 UR - https://www.unboundmedicine.com/medline/citation/25186063/Tracking_protein_dynamics_with_photoconvertible_Dendra2_on_spinning_disk_confocal_systems_ L2 - https://doi.org/10.1111/jmi.12172 DB - PRIME DP - Unbound Medicine ER -