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Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.
J Am Chem Soc 2010; 132(18):6481-91JA

Abstract

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

Authors+Show Affiliations

Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

20394363

Citation

Subach, Fedor V., et al. "Bright Monomeric Photoactivatable Red Fluorescent Protein for Two-color Super-resolution sptPALM of Live Cells." Journal of the American Chemical Society, vol. 132, no. 18, 2010, pp. 6481-91.
Subach FV, Patterson GH, Renz M, et al. Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells. J Am Chem Soc. 2010;132(18):6481-91.
Subach, F. V., Patterson, G. H., Renz, M., Lippincott-Schwartz, J., & Verkhusha, V. V. (2010). Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells. Journal of the American Chemical Society, 132(18), pp. 6481-91. doi:10.1021/ja100906g.
Subach FV, et al. Bright Monomeric Photoactivatable Red Fluorescent Protein for Two-color Super-resolution sptPALM of Live Cells. J Am Chem Soc. 2010 May 12;132(18):6481-91. PubMed PMID: 20394363.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells. AU - Subach,Fedor V, AU - Patterson,George H, AU - Renz,Malte, AU - Lippincott-Schwartz,Jennifer, AU - Verkhusha,Vladislav V, PY - 2010/4/17/entrez PY - 2010/4/17/pubmed PY - 2010/8/6/medline SP - 6481 EP - 91 JF - Journal of the American Chemical Society JO - J. Am. Chem. Soc. VL - 132 IS - 18 N2 - Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules. SN - 1520-5126 UR - https://www.unboundmedicine.com/medline/citation/20394363/Bright_monomeric_photoactivatable_red_fluorescent_protein_for_two_color_super_resolution_sptPALM_of_live_cells_ L2 - https://dx.doi.org/10.1021/ja100906g DB - PRIME DP - Unbound Medicine ER -