A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure - PubMed (original) (raw)

A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure

Christina T Hauser et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Site-specific fluorescent labeling of proteins in vivo remains one of the most powerful techniques for imaging complex processes in live cells. Although fluorescent proteins in many colors are useful tools for tracking expression and localization of fusion proteins in cells, these relatively large tags (>220 aa) can perturb protein folding, trafficking and function. Much smaller genetically encodable domains (<15 aa) offer complementary advantages. We introduce a small fluorescent chelator whose membrane-impermeant complex with nontoxic Zn(2+) ions binds tightly but reversibly to hexahistidine (His(6)) motifs on surface-exposed proteins. This live-cell label helps to resolve a current controversy concerning externalization of the stromal interaction molecule STIM1 upon depletion of Ca(2+) from the endoplasmic reticulum. Whereas N-terminal fluorescent protein fusions interfere with surface exposure of STIM1, short His(6) tags are accessible to the dye or antibodies, demonstrating externalization.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Structure and spectra of HisZiFiT. (A) Structure of HisZiFiT (red) and proposed mode of Zn2+-mediated binding to a His6 sequence (black), in flattened projection. (B) Normalized fluorescence excitation (solid curves) and emission (dashed) spectra of 1 μM HisZiFiT without Zn2+ (gray curves) and with saturating (10 μM) free Zn2+ (black). (C) FRET response of His6-CFP to HisZiFiT binding (ex420). Emission spectrum of 1 μM His6-CFP with 10 μM buffered free Zn2+ before (black curve) and after (red curve) addition of 1 μM HisZiFiT.

Fig. 2.

Fig. 2.

HisZiFiT labeling is specific for cell-surface His6-tagged proteins. (A–D) HEK293T cells expressing His6-CFP-GPI, labeled with 100 nM HisZiFiT in 1 μM buffered free Zn2+. (A) CFP channel, ex420/em475, shows both surface and internal His6-CFP-GPI. (B) HisZiFiT channel, ex540/em595, of the same cells shows only plasma membrane outlines. (C) Merge of A (displayed in green) and B (red). (D) Differential interference contrast image of the same cells. (E and F) Control HEK293T cells expressing nontagged CFP (CFP-GPI), identically exposed to HisZiFiT. (E) CFP channel. (F) HisZiFit channel shows negligible staining of CFP–GPI protein.

Fig. 3.

Fig. 3.

Orthogonal staining of HEK293T cells expressing two proteins on the PM. (A) Connexin43–4C with an intracellular tetracysteine motif, stained with ReAsH (ex568/55, em653/95). (B) His6 on extracellular PM surface stained and visualized with HisZiFiT (ex495/10, em535/45). (C) Merge of images A and B representing HisZiFiT staining of His6-CFP-GPI (green) and ReAsH staining of Connexin43–4C (red).

Fig. 4.

Fig. 4.

N-terminal tagging with His6 but not CFP allows cell surface exposure of STIM1. (A–L) Images of cells transiently transfected with His6-CFP-STIM1 (A–F) or His6-STIM1-CFP protein (G–L). Cartoons above A–L schematize the expressed polypeptide domains, where SP denotes signal peptide and H6 stands for His6. (A, D, G, and J) Transmitted light images; images in A, D, and J show differential interference contrast. (B, E, H, K) CFP fluorescence (cyan) of the same fields shows that all of the STIM1 fusions are recruited to the plasma membrane after depletion of Ca2+ stores with 2 μM thapsigargin. (C) Lack of specific staining by HisZiFiT (yellow) indicates absence of extracellularly exposed His6-tags on the same cells as in B. (F) Lack of antibody staining for His5 (magenta) confirms absence of extracellularly exposed His6 tags on the same live cell as in E. (I) Surface exposed His6-tags of the same cell as in H are labeled by HisZiFiT (yellow). (L) Live cell His5-Ab staining (detected with Alexa Fluor 568-labeled secondary Ab, magenta) confirms the exposure of N-terminal His6-tags on the same cells as in K. Exposure times with a 5% neutral density filter on excitation were 200, 1,000, and 50 ms for CFP, HisZiFiT, and Alexa Fluor 568, respectively. Cartoons show schematically how His6 remains luminal in His6-CFP-STIM1 (B, C, E, and F) but becomes extracellularly accessible in His6-STIM1-CFP (H, I, K, and L).

Similar articles

Cited by

References

    1. Giepmans BN, Adams SR, Ellisman MH, Tsien RY. Science. 2006;312:217–224. - PubMed
    1. Chen I, Ting AY. Curr Opin Biotechnol. 2005;16:35–40. - PubMed
    1. Griffin BA, Adams SR, Tsien RY. Science. 1998;281:269–272. - PubMed
    1. Kapanidis AN, Ebright YW, Ebright RH. J Am Chem Soc. 2001;123:12123–12125. - PubMed
    1. Guignet EG, Hovius R, Vogel H. Nat Biotechnol. 2004;22:440–444. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources