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 V. Verkhusha

Research output: Contribution to journalArticle

146 Scopus citations

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

ASJC Scopus subject areas

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

Fingerprint Dive into the research topics of 'Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells'. Together they form a unique fingerprint.

  • Cite this