Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by β-mercaptoethanol for specific staining of tetracysteine-tagged proteins
Summary (2 min read)
Introduction
- Investigating the distribution and dynamics of proteins inside living cells by fluorescence microscopy has been greatly simplified by genetically encoded recombinant fusion proteins of green fluorescent protein and its variants (Lippincott-Schwartz et al. 2001) .
- It is based on complex formation between a biarsenical variant of fluorescein (fluorescein arsenical helix binder, FlAsH) or resorufin and a genetically encoded tetracysteine motif of only 6-12 amino acids.
- Furthermore, FlAsH and ReAsH are now commercially available as Lumio Greenä and Lumio Redä from Invitrogen.
- This protein of 47 kDa was discovered in their lab as a protein upregulated in goldfish retinal ganglion cells during axon regeneration after optic nerve injury (Schulte et al. 1997; Lang et al. 1998) .
Reagents
- Lumio Green and Disperse Blue were purchased from Invitrogen (Karlsruhe, Germany) as part of the ''Lumio in cell labeling kitä''.
- Mitotracker Orange and Rhodamine 123 were from Molecular Probes (Leiden, Netherlands).
- B-mercaptoethanol (2-ME), ethanedithiol (EDT), formaldehyde and rotenone were from Sigma (Munich, Germany).
- Oligonucleotides were from Operon Biotechnology (Cologne, Germany).
- Enzymes for molecular biology were from New England Biolabs (Beverly, USA) or Fermentas (St. Leon-Rot, Germany).
Cloning of reggie-1-tetracysteine expression vectors
- By ligation with annealed oligonucleotides (fw: 5¢-GATCCATT-CCTGAACTGTTGTCCCGGCTGCTGCATGGAG-CCTTGA-3¢; rv: 5¢-GGCCTCAAGGCTCCATGCAG.
- CAGCCGGGACAACAGTTCAGGAATG-3¢) the authors then introduced the first tetracysteine sequence coding for FLNCCPGCCMEP*, a tetracysteine sequence with optimized flanking amino acids for particularly high affinity for biarsenical ligands (Martin et al. 2005) .
- To introduce a second tetracysteine sequence, the authors linearized the resulting plasmid using BamHI, dephosphorylated the ends using calf intestine alkaline phosphatase and ligated phosphorylated, annealed oligonucleotides (fw: Phos-5¢-GATCACTCTCTTAACTGCTGCCCGGGG-TGTTGTATGGAACCCGTAGTCCTT-3¢, rv: Phos-5¢-GATCAAGGACTACGGGTTCCATACAACACC-CCGGGCAGCAGTTAAGAGAGT-3¢) encoding SLNC-CPGCCMEP.
- The use of different codons made colony PCR screening with a specific reverse primer (5¢-AT-CAAGGACTACGGGTT-3¢) possible.
- All constructs were finally sequenced for verification.
Cell culture and transfection
- Cells were transfected using Lipofectamine 2000 according to the manufacturers instructions.
- For labeling, transfected cells were plated onto chambered coverslips (Nunc, Rochester, USA) coated with poly-Llysine and laminin (both Sigma) 24 h after transfection and labeled 48 h post transfection.
Fluorescence microscopy
- Where indicated, the Apotome system (Carl Zeiss) was used to acquire confocal images by structured illumination.
- Images were processed using AxioVision 4.4 (Carl Zeiss) and ImageJ (Abramoff et al. 2004 ).
FlAsH/Lumio Green accumulates in active mitochondria
- In the absence of thiol reagents during labeling with FlAsH/Lumio Green, the authors observed in both HeLa and N2a cells a very bright staining of mitochondria, which they verified by co-staining using the mitochondrial marker Mitotracker Orange (Fig. 1a ).
- Several other treatments were reported to reduce background staining while using the tetracysteine-biarsenical system.
- Disperse Blue is a hydrophobic dye which suppresses unspecific staining by blocking hydrophobic binding sites (Griffin et al. 2000) and is part of the labeling kit sold by Invitrogen.
- These side effects might severely compromise the value of this labeling system for live cell imaging.
- Furthermore, addition of low concentrations of thiols during labeling reduced morphological changes of the mitochondria induced by FlAsH/Lumio Green at least to a certain extent, probably by reversing the toxic effect of arsenic on the GSH redox system (Zmuda and Friedenson 1983) .
Specific staining of tetracysteine-tagged reggie in the presence of b-mercaptoethanol
- So far, all publications using the tetracysteine-biarsenical labeling system utilized EDT to suppress unspecific binding of the biarsenical fluorescent staining reagent to cellular thiols.
- This clearly demonstrates that specific labeling using the tetracysteine-biarsenical system can be achieved by replacing EDT with the less toxic, easier to handle 2-ME.
- The authors therefore suggest testing different constructs with variable numbers and localizations of tetracysteine sequences, as not only localization of the tag seems to be crucial (N-terminal tagging of reggie-1/flotillin-2 for example results in a mislocalized fusion protein most probably because myristoylation of Gly2 is inhibited), but staining efficiency and intensity might also be influenced.
- The lower signal to noise ratio was caused by a diffuse FlAsH/Lumio Green staining of the whole cell, which is most probably due to binding of the biarsenical reagent to intracellular cysteine-rich proteins as previously described (Stroffekova et al. 2001 ).
- The fact that the FlAsH/Lumio Green label was subject to photobleaching was another problem.
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Cites background from "Accumulation of FlAsH/Lumio Green i..."
...Moreover, owing to potential chemical toxicity caused by the labeling reaction, such as the accumulation of FlAsH in active mitochondria (Langhorst et al., 2006), this technique might be better suited for short-term live-cell imaging....
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99 citations
Cites methods from "Accumulation of FlAsH/Lumio Green i..."
...N2a and HeLa cells were cultured and transfected as described previously [14]....
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92 citations
Cites background or methods from "Accumulation of FlAsH/Lumio Green i..."
...HeLa, PC12 and Jurkat T cells were cultured and transfected as described previously (Langhorst et al., 2006a, b)....
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...C-terminal EGFP-tagging of the full-length reggie-1/ flotillin-2 protein does not significantly alter its biochemical properties and its subcellular localization (Neumann-Giesen et al., 2004; Langhorst et al., 2006b)....
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...Reggie/ flotillin-dependent signaling complexes were shown to be involved in actin remodeling during T cell activation (Langhorst et al., 2006b) and in Glut4translocation to the plasma membrane after insulin stimulation of adipocytes (Baumann et al., 2000; Kimura et al., 2001)....
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References
609 citations
"Accumulation of FlAsH/Lumio Green i..." refers background in this paper
...The recent description of a rational design of new fluorescein variants with improved properties (Urano et al. 2005) might allow the development of more photostable variants, which would greatly facilitate live cell imaging of FlAsH/Lumio Green labeled proteins....
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416 citations
"Accumulation of FlAsH/Lumio Green i..." refers methods in this paper
...…rv: 5¢-GGCCTCAAGGCTCCATGCAG CAGCCGGGACAACAGTTCAGGAATG-3¢) we then introduced the first tetracysteine sequence coding for FLNCCPGCCMEP*, a tetracysteine sequence with optimized flanking amino acids for particularly high affinity for biarsenical ligands (Martin et al. 2005)....
[...]
346 citations
"Accumulation of FlAsH/Lumio Green i..." refers background in this paper
...Smaller labels may overcome some of these problems by reducing steric hindrance (reviewed by Chen and Ting 2005)....
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304 citations
"Accumulation of FlAsH/Lumio Green i..." refers background in this paper
...Its exact function is still unknown, but it most probably acts as a lipid raft-associated scaffolding protein, defining specialized microdomains for multiprotein complex assembly at cellular membranes (reviewed in Langhorst et al. 2005)....
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251 citations