Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by β-mercaptoethanol for specific staining of tetracysteine-tagged proteins

Abstract

Recent advances in the field of small molecule labels for live cell imaging promise to overcome some of the limitations set by the size of fluorescent proteins. We tested the tetracysteine-biarsenical labeling system in live cell fluorescence microscopy of reggie-1/flotillin-2 in HeLa and N2a cells. In both cell types, the biarsenical staining reagent FlAsH/Lumio Green accumulated in active mitochondria and led to mitochondrial swelling. This is indicative of toxic side effects caused by arsenic, which should be considered when this labeling system is to be used in live cell imaging. Mitochondrial accumulation of FlAsH/Lumio Green was reversed by addition of low concentrations of thiol-containing reagents during labeling and a subsequent high stringency thiol wash. Both ethanedithiol and beta-mercaptoethanol proved to be effective. We therefore established a staining protocol using beta-mercaptoethanol as thiol binding site competitor resulting in a specific staining of tetracysteine-tagged reggie-1/flotillin-2 of adequate signal to noise ratio, so that the more toxic and inconvenient ethanedithiol could be avoided. Furthermore, we show that staining efficiency was greatly enhanced by introducing a second tetracysteine sequence in tandem.

Actin visualization at a glance

“…The relatively small size of the tetracysteine tag (∼2 kDa) might not interfere dramatically with different functions of actin, although it has been shown that the fission yeast formin Cdc12p, which is required for formation of the contractile ring, excludes tetracysteinetagged fission yeast actin, suggesting very stringent structural requirements for formins to promote the elongation of actin filaments (Chen et al, 2012). 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.…”

Specific Biarsenical Labeling of Cell Surface Proteins Allows Fluorescent- and Biotin-tagging of Amyloid Precursor Protein and Prion Proteins

“…One reason may be background presumably due to FlAsH binding to off-target cysteine-rich proteins (Stroffekova et al, 2001;Berens et al, 2005;Langhorst et al, 2006;Hearps et al, 2007), necessitating a postincubation step to reduce this background. Successful applications of this technology have used protocols similar to that originally reported for FlAsH (Perlman and Resh, 2006;Arhel et al, 2006;Roberti et al, 2007;Tour et al, 2007) but have not addressed the specificity issue to allow one- step labeling of any protein, especially those localized to cell surfaces.…”

“…Initially, we tried labeling cells expressing TC-PrPs with FlAsH by using protocols based on one published for labeling extracellular proteins (Adams et al, 2002). We failed to detect any TC-PrP-specific staining and observed very high background labeling as reported by others (Stroffekova et al, 2001;Berens et al, 2005;Langhorst et al, 2006;Hearps et al, 2007) (see Supplemental Figure 1). …”

“…FlAsH/TC-motif labeling has its own inherent problems, including very high background staining due to FlAsH binding to off-target cysteine-rich proteins (Stroffekova et al, 2001;Berens et al, 2005;Langhorst et al, 2006;Hearps et al, 2007). In addition, extracellular proteins have been poor candidates for FlAsHlabeling because the cysteine residues of the TC-motif are oxidized in extracellular environments and/or during export through the endoplasmic reticulum and consequently lose their affinity for FlAsH.…”

Recent progress in histochemistry

“…Dahan (2006) reviewed the use of quantum dots as Xuorescent probes for live-cell imaging and pointed to their potential, in combination with ultrasensitive imaging techniques, for the analysis of single molecules. Other small molecules can be used in live-cell imaging such as the Xuorescein arsenical helix binder (FlAsH) which has been used for investigating the dynamics of reggie-1/Xotillin-2 protein in cell culture (Langhorst et al 2006). They found that the biarsenical staining reagent FlAsH/Lumio Green accumulated in active mitochondria, resulting in mitochondrial swelling which limits the usefulness of this reagent for livecell imaging.…”