Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells

Fedor V. Subach, George H. Patterson, Malte Renz, Jennifer Lippincott-Schwartz, Vladislav Verkhusha

Research output: Contribution to journalArticle

143 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)6481-6491
Number of pages11
JournalJournal of the American Chemical Society
Volume132
Issue number18
DOIs
StatePublished - May 12 2010

Fingerprint

Color
Proteins
Clathrin Light Chains
Microscopic examination
Molecules
Microscopy
Photochemical Processes
Cells
Imaging techniques
Light
Spectroscopic analysis
Cell membranes
Chromophores
Endocytosis
Photons
red fluorescent protein
Luminance
Diffraction
Fluorescence
Cell Membrane

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells. / Subach, Fedor V.; Patterson, George H.; Renz, Malte; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav.

In: Journal of the American Chemical Society, Vol. 132, No. 18, 12.05.2010, p. 6481-6491.

Research output: Contribution to journalArticle

Subach, Fedor V. ; Patterson, George H. ; Renz, Malte ; Lippincott-Schwartz, Jennifer ; Verkhusha, Vladislav. / Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells. In: Journal of the American Chemical Society. 2010 ; Vol. 132, No. 18. pp. 6481-6491.
@article{82826e265cc249d4ba1be1d6e12be164,
title = "Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells",
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.",
author = "Subach, {Fedor V.} and Patterson, {George H.} and Malte Renz and Jennifer Lippincott-Schwartz and Vladislav Verkhusha",
year = "2010",
month = "5",
day = "12",
doi = "10.1021/ja100906g",
language = "English (US)",
volume = "132",
pages = "6481--6491",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "18",

}

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

PY - 2010/5/12

Y1 - 2010/5/12

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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=77952059303&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77952059303&partnerID=8YFLogxK

U2 - 10.1021/ja100906g

DO - 10.1021/ja100906g

M3 - Article

C2 - 20394363

AN - SCOPUS:77952059303

VL - 132

SP - 6481

EP - 6491

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 18

ER -