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. doi: 10.1021/ja100906g.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Cell Survival
  • Chlorocebus aethiops
  • Color
  • HeLa Cells
  • Humans
  • Light*
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Membrane Proteins / metabolism
  • Microscopy / methods*
  • Molecular Imaging
  • Molecular Sequence Data
  • Mutagenesis
  • Photochemical Processes
  • Temperature

Substances

  • Luminescent Proteins
  • Membrane Proteins
  • red fluorescent protein