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• W2912386236 abstract "•Systematic comparison of SNAP versus Halo tag labeling by confocal and STED microscopy•Target proteins, fluorophores, and model systems are compared•Large differences in Halo versus SNAP intensity with silicon rhodamine fluorophores•Guidelines for one- and two-color super-resolution imaging are provided Super-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. Organic fluorophores may help here as they can yield more photons—by orders of magnitude—than fluorescent proteins. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAP-tags being the most common. However, how these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. Herein, we compare the two labeling approaches in confocal and STED imaging using various proteins and two model systems. Strikingly, we find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging. Super-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. Organic fluorophores may help here as they can yield more photons—by orders of magnitude—than fluorescent proteins. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAP-tags being the most common. However, how these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. Herein, we compare the two labeling approaches in confocal and STED imaging using various proteins and two model systems. Strikingly, we find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging. Super-resolution fluorescence microscopy, also called “nanoscopy,” enables the visualization of cellular structures beyond the diffraction limit of light (Fornasiero and Opazo, 2015Fornasiero E.F. Opazo F. Super-resolution imaging for cell biologists.BioEssays. 2015; 37: 436-451Crossref PubMed Scopus (85) Google Scholar, Hell, 2007Hell S.W. Far-field optical nanoscopy.Science. 2007; 316: 1153-1158Crossref PubMed Scopus (2312) Google Scholar, Huang et al., 2009Huang B. Bates M. Zhuang X.W. Super-resolution fluorescence microscopy.Annu. Rev. Biochem. 2009; 78: 993-1016Crossref PubMed Scopus (1217) Google Scholar, Toomre and Bewersdorf, 2010Toomre D. Bewersdorf J. A new wave of cellular imaging.Annu. Rev. Cell Dev. Biol. 2010; 26: 285-314Crossref PubMed Scopus (275) Google Scholar, van de Linde et al., 2012van de Linde S. Heilemann M. Sauer M. Live-cell super-resolution imaging with synthetic fluorophores.Annu. Rev. Phys. Chem. 2012; 63: 519-540Crossref PubMed Scopus (211) Google Scholar). However, unlike electron microscopy, whose application is limited to fixed cells, nanoscopy enables live-cell imaging to study cellular dynamics in unprecedented spatial detail. Green fluorescent protein (GFP) and its spectral variants (Uno et al., 2015Uno S.N. Tiwari D.K. Kamiya M. Arai Y. Nagai T. Urano Y. A guide to use photocontrollable fluorescent proteins and synthetic smart fluorophores for nanoscopy.Microscopy (Oxf.). 2015; 64: 263-277Crossref PubMed Scopus (32) Google Scholar) have revolutionized biology, as they allow genetically encoded labeling, but they possess mediocre photophysical properties, generally emitting fewer photons than the best organic dyes by one or two orders of magnitude (Dempsey et al., 2011Dempsey G.T. Vaughan J.C. Chen K.H. Bates M. Zhuang X.W. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.Nat. Methods. 2011; 8: 1027-1035Crossref PubMed Scopus (928) Google Scholar, Fernandez-Suarez and Ting, 2008Fernandez-Suarez M. Ting A.Y. Fluorescent probes for super-resolution imaging in living cells.Nat. Rev. Mol. Cell Biol. 2008; 9: 929-943Crossref PubMed Scopus (1052) Google Scholar). While this deficiency may not be limiting for a single confocal image or even an image stack, the demands of nanoscopy are much greater, as every photon counts to obtain the highest resolution. Similarly, for 3D time-lapse fluorescence microscopy (4D imaging), which involves the acquisition of large datasets, correspondingly brighter and more stable fluorophores are required to study the volumetric dynamics of cells and tissues over longer timescales. For both super-resolution imaging and 4D imaging, organic fluorophores are highly appealing because of their brightness and photostability (Dempsey et al., 2011Dempsey G.T. Vaughan J.C. Chen K.H. Bates M. Zhuang X.W. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging.Nat. Methods. 2011; 8: 1027-1035Crossref PubMed Scopus (928) Google Scholar, Fernandez-Suarez and Ting, 2008Fernandez-Suarez M. Ting A.Y. Fluorescent probes for super-resolution imaging in living cells.Nat. Rev. Mol. Cell Biol. 2008; 9: 929-943Crossref PubMed Scopus (1052) Google Scholar). Organic fluorophores can be attached to proteins by combining click chemistry with unnatural amino acid incorporation (Lang et al., 2012aLang K. Davis L. Torres-Kolbus J. Chou C.J. Deiters A. Chin J.W. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction.Nat. Chem. 2012; 4: 298-304Crossref PubMed Scopus (367) Google Scholar, Lang et al., 2012bLang K. Davis L. Wallace S. Mahesh M. Cox D.J. Blackman M.L. Fox J.M. Chin J.W. Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic diels-alder reactions.J. Am. Chem. Soc. 2012; 134: 10317-10320Crossref PubMed Scopus (387) Google Scholar). A second option is the direct coupling to proteins in live cells by using self-labeling proteins such as SNAP-tags (Keppler et al., 2003Keppler A. Gendreizig S. Gronemeyer T. Pick H. Vogel H. Johnsson K. A general method for the covalent labeling of fusion proteins with small molecules in vivo.Nat. Biotechnol. 2003; 21: 86-89Crossref PubMed Scopus (1359) Google Scholar) (or a variant called CLIP-tag; Gautier et al., 2008Gautier A. Juillerat A. Heinis C. Correa I.R. Kindermann M. Beaufils F. Johnsson K. An engineered protein tag for multiprotein labeling in living cells.Chem. Biol. 2008; 15: 128-136Abstract Full Text Full Text PDF PubMed Scopus (752) Google Scholar) and HaloTags (Los et al., 2008Los G.V. Encell L.P. McDougall M.G. Hartzell D.D. Karassina N. Zimprich C. Wood M.G. Learish R. Ohane R.F. Urh M. et al.HaloTag: a novel protein labeling technology for cell imaging and protein analysis.ACS Chem. Biol. 2008; 3: 373-382Crossref PubMed Scopus (1372) Google Scholar). Alternatively, labeling can be achieved by combining click chemistry and self-labeling proteins (Murrey et al., 2015Murrey H.E. Judkins J.C. am Ende C.W. Ballard T.E. Fang Y. Riccardi K. Di L. Guilmette E.R. Schwartz J.W. Fox J.M. et al.Systematic evaluation of bioorthogonal reactions in live cells with clickable halotag ligands: implications for intracellular imaging.J. Am. Chem. Soc. 2015; 137: 11461-11475Crossref PubMed Scopus (108) Google Scholar). Like GFP, these self-labeling SNAP-tags and HaloTags can be expressed as fusion proteins (Hinner and Johnsson, 2010Hinner M.J. Johnsson K. How to obtain labeled proteins and what to do with them.Curr. Opin. Biotechnol. 2010; 21: 766-776Crossref PubMed Scopus (221) Google Scholar) and selectively reacted with the substrates benzylguanine (BG) and chloralkane (CA), respectively, which are tagged with organic fluorophores. While this labeling strategy is becoming increasingly popular for super-resolution imaging (Bottanelli et al., 2016Bottanelli F. Kromann E.B. Allgeyer E.S. Erdmann R.S. Wood Baguley S. Sirinakis G. Schepartz A. Baddeley D. Toomre D.K. Rothman J.E. et al.Two-colour live-cell nanoscale imaging of intracellular targets.Nat. Commun. 2016; 7: 10778Crossref PubMed Scopus (141) Google Scholar, Bottanelli et al., 2017Bottanelli F. Kilian N. Ernst A.M. Rivera-Molina F. Schroeder L.K. Kromann E.B. Lessard M.D. Erdmann R.S. Schepartz A. Baddeley D. et al.A novel physiological role for ARF1 in the formation of bi-directional tubules from the Golgi.Mol. Biol. Cell. 2017; 28: 1676-1687Crossref PubMed Google Scholar, Grimm et al., 2015Grimm J.B. English B.P. Chen J. Slaughter J.P. Zhang Z. Revyakin A. Patel R. Macklin J.J. Normanno D. Singer R.H. et al.A general method to improve fluorophores for live-cell and single-molecule microscopy.Nat. Methods. 2015; 12: 244-250Crossref PubMed Scopus (823) Google Scholar, Stagge et al., 2013Stagge F. Mitronova G.Y. Belov V.N. Wurm C.A. Jakobs S. Snap-, CLIP- and Halo-tag labelling of budding yeast cells.PLoS One. 2013; 8: e78745Crossref PubMed Scopus (70) Google Scholar, Xue et al., 2015Xue L. Karpenko I.A. Hiblot J. Johnsson K. Imaging and manipulating proteins in live cells through covalent labeling.Nat. Chem. Biol. 2015; 11: 917-923Crossref PubMed Scopus (136) Google Scholar), especially since several commercial fluorescent SNAP and HALO ligands are available, it is unclear if these different tags influence the fluorescence properties of organic dyes, thereby possibly affecting image quality. Herein, by conducting quantitative comparisons of SNAP and Halo tagging, we present strong evidence that the tag, its molecular targeting location, and its environment can have a major impact on the brightness of the introduced fluorophores. The difference in brightness can be striking—by nearly an order of magnitude—indicating that the labeling strategy matters greatly and can have a profound impact on image quality and duration by 4D confocal microscopy and stimulated emission depletion (STED) nanoscopy. We first compared HaloTag and SNAP-tag systems in cells transiently co-expressing mannosidase II (ManII)-GFP (Velasco et al., 1993Velasco A. Hendricks L. Moremen K.W. Tulsiani D.R.P. Touster O. Farquhar M.G. Cell-type dependent variations in the subcellular-distribution of alpha-mannosidase-I and alpha-mannosidase-II.J. Cell Biol. 1993; 122: 39-51Crossref PubMed Scopus (279) Google Scholar) and sialyltransferase (ST; Kweon et al., 2004Kweon H.S. Beznoussenko G.V. Micaroni M. Polishchuk R.S. Trucco A. Martella O. Di Giandomenico D. Marra P. Fusella A. Di Pentima A. et al.Golgi enzymes are enriched in perforated zones of Golgi cisternae but are depleted in COPI vesicles.Mol. Biol. Cell. 2004; 15: 4710-4724Crossref PubMed Scopus (77) Google Scholar) fused to either the HaloTag or the SNAP-tag at its C terminus (Figure S1). Cells were labeled with Halo or SNAP ligands conjugated to the near far-red fluorophore silicon rhodamine (SiR): SiR-CA and SiR-BG for HaloTags and SNAP-tags, respectively (Lukinavicius et al., 2013Lukinavicius G. Umezawa K. Olivier N. Honigmann A. Yang G.Y. Plass T. Mueller V. Reymond L. Correa I.R. Luo Z.G. et al.A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins.Nat. Chem. 2013; 5: 132-139Crossref PubMed Scopus (595) Google Scholar) (Figure 1A). As expected, both Halo- and SNAP-tagged ST colocalized with ManII-GFP at the Golgi apparatus, as visualized by confocal microscopy. However, the fluorescence of SiR was strikingly much brighter for the Halo-tagged protein than for the SNAP-tagged one. A visual inspection showed that 93% of ManII-GFP-expressing cells were co-labeled with SiR for the HaloTag condition, whereas only 32% of GFP-tagged cells showed co-labeling for SNAP-tag (Figure 1B), suggesting that the majority of SNAP-tag cells were unlabeled with SiR. However, a quantitative analysis of hundreds of cells indicated that most SNAP-tag cells were indeed labeled, because they were clearly brighter than control cells lacking SNAP-tags and HaloTags, which could not be labeled with SiR (faint pink distributions in Figure 1C). The SNAP-tag cells were just much more dimly labeled than the HaloTag cells. The mean intensity of SiR with HaloTag was 2.8-fold brighter than with SNAP-tag, with both labeling systems showing expected Gaussian distributions of SiR intensities (Figure 1C). This surprising difference in brightness between the two popular tagging systems was intriguing and warranted further investigation. While potentially interesting, the observed difference between SNAP- and Halo-tagged signals could be due to a number of trivial explanations, including differences in the following: (1) reaction rate between substrate and self-labeling protein, (2) transfection efficiency, (3) cell permeability to the substrates, and (4) expression level of the SNAP and Halo fusion proteins. To exclude the first possibility that the reported difference in reaction rates influenced labeling density, we confirmed that the labeling reaction was complete under the conditions used (Figure S2). To address the other possibilities, we fused a hemagglutinin (HA) tag to the self-labeling proteins as an independent reporter of expression. After labeling with SiR, cells expressing ST-Halo-HA or ST-SNAP-HA were fixed, permeabilized, and incubated with a primary mouse antibody against HA, followed by staining with a secondary goat anti-mouse antibody that was labeled with Alexa 546 (Figure 2A). This allowed us to determine the transfection efficiency independent of SiR labeling. The analysis of the immunolabeled cells showed that 98% of the cells expressing ManII-GFP were positive for ST-Halo-HA, while 91% were positive for ST-SNAP-HA (Figure 2B). Thus, this modest difference in transfection efficiency cannot fully explain the large difference between HaloTag and SNAP-tag labeling. We next investigated whether differences in cell permeability to the substrates could influence the labeling efficiency. To this end, we tested the labeling of ST-Halo-HA and ST-SNAP-HA in fixed and permeabilized cells—a condition that should negate any potential difference in permeability between SiR-CA and SiR-BG. As shown in Figure 2C, fixation and permeabilization had only a small effect on the labeling efficiency (Figure 2C), indicating that the 3-fold labeling difference seen in the live-cell experiments of Figure 1 is not due to restricted permeability of the SNAP substrate SiR-BG. We note that it is also unlikely that permeability could affect labeling as the reaction was performed with a large excess of substrate (2.5 μM) for 1 h and, as shown in Figure S2, was largely complete under these conditions. Another trivial explanation for the difference in labeling brightness could be that the expression levels of SNAP and Halo fusion proteins were different. To address this issue, we quantified the fluorescence intensity of the immunolabeling of the HA tag in all cells used for the experiment shown in Figure 2B. Overall, the cells expressing ST-SNAP-HA exhibited a 37% brighter immunofluorescence signal than cells expressing ST-Halo-HA (p > 0.0001), indicating that the SNAP fusion protein is expressed at a slightly higher level than the Halo fusion protein (Figures 2D and S3), contrary to the possibility that SNAP-tag labeling might be dimmer because of a lower expression level. To further support the above findings, we tagged aPKC endogenously in Drosophila using CRISPR/Cas9 technology with homologous recombination to make doubly tagged Halo-SNAP-aPKC flies. aPKC is a kinase that localizes subapically in the follicle epithelium that surrounds the egg chamber (Wodarz et al., 2000Wodarz A. Ramrath A. Grimm A. Knust E. Drosophila atypical protein kinase C associates with bazooka and controls polarity of epithelia and neuroblasts.J. Cell Biol. 2000; 150: 1361-1374Crossref PubMed Scopus (378) Google Scholar). This experimental approach has two important advantages over the experiments described above using mammalian cells: (1) the endogenous protein is tagged and (2) the double tag ensures the same expression levels for Halo and SNAP tags. To investigate the labeling differences in this system, we incubated dissected, fixed ovaries with 600 nM either SiR-CA or SiR-BG to label Halo-SNAP-aPKC. The tissues were imaged under a confocal microscope (Figure S4). Analysis of the images revealed strikingly different mean intensities of egg chambers labeled with SiR-CA and SiR-BG. The mean intensity with SiR-CA was 4.5-fold higher than that with SiR-BG (p < 0.0001) (Figure 2E). This result is in line with the finding in Figure 1C and unequivocally demonstrates that the difference in intensity is not due to different expression levels of SNAP and Halo fusion proteins. Since we ruled out the above trivial explanations for the difference between HaloTag and SNAP-tag labeling, we hypothesized that the brightness of the labeling might depend on environmental factors. We, and others, have shown that the fluorescence intensity of carboxyl and hydroxymethyl SiRs correlates with the hydrophobicity of their environment (Erdmann et al., 2014Erdmann R.S. Takakura H. Thompson A.D. Rivera-Molina F. Allgeyer E.S. Bewersdorf J. Toomre D. Schepartz A. Super-resolution imaging of the golgi in live cells with a bioorthogonal ceramide probe.Angew. Chem. Int. Ed. 2014; 53: 10242-10246Crossref PubMed Scopus (112) Google Scholar, Lukinavicius et al., 2013Lukinavicius G. Umezawa K. Olivier N. Honigmann A. Yang G.Y. Plass T. Mueller V. Reymond L. Correa I.R. Luo Z.G. et al.A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins.Nat. Chem. 2013; 5: 132-139Crossref PubMed Scopus (595) Google Scholar, 2014; Takakura et al., 2017Takakura H. Zhang Y. Erdmann R.S. Thompson A.D. Lin Y. McNellis B. Rivera-Molina F. Uno S.-n. Kamiya M. Urano Y. et al.Long time-lapse nanoscopy with spontaneously blinking membrane probes.Nat. Biotechnol. 2017; 35: 773-780Crossref PubMed Scopus (105) Google Scholar, Uno et al., 2014Uno S.N. Kamiya M. Yoshihara T. Sugawara K. Okabe K. Tarhan M.C. Fujita H. Funatsu T. Okada Y. Tobita S. et al.A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging.Nat. Chem. 2014; 6: 681-689Crossref PubMed Scopus (278) Google Scholar): the more hydrophobic the environment (i.e., the lower its dielectric constant), the less fluorescent the dye. In contrast, methyl SiRs do not show this environmental sensitivity (Koide et al., 2011Koide Y. Urano Y. Hanaoka K. Terai T. Nagano T. Evolution of group 14 rhodamines as platforms for near-infrared fluorescence probes utilizing photoinduced electron transfer.ACS Chem. Biol. 2011; 6: 600-608Crossref PubMed Scopus (276) Google Scholar, Koide et al., 2012Koide Y. Urano Y. Hanaoka K. Piao W. Kusakabe M. Saito N. Terai T. Okabe T. Nagano T. Development of NIR fluorescent dyes based on Si-rhodamine for in vivo imaging.J. Am. Chem. Soc. 2012; 134: 5029-5031Crossref PubMed Scopus (220) Google Scholar). However, since the methyl SiR SNAP substrate led to considerable nonspecific labeling (Figure S5), we did not further investigate this version of the dye. To investigate whether the labeling brightness depends on the protein of interest and its environment, we tested three more fusion proteins in experiments analogous to those of Figure 1. Using HaloTags and SNAP-tags, we labeled ManII, the mitochondrial matrix protein OMP25 (Nemoto and De Camilli, 1999Nemoto Y. De Camilli P. Recruitment of an alternatively spliced form of synaptojanin 2 to mitochondria by the interaction with the PDZ domain of a mitochondrial outer membrane protein.EMBO J. 1999; 18: 2991-3006Crossref PubMed Scopus (131) Google Scholar), and the vesicle coat protein clathrin light chain (CLC; Gaidarov et al., 1999Gaidarov I. Santini F. Warren R.A. Keen J.H. Spatial control of coated-pit dynamics in living cells.Nat. Cell Biol. 1999; 1: 1-7Crossref PubMed Scopus (358) Google Scholar) with SiR (Figure 3A). For all proteins tested, the SiR signal was noticeably dimmer in the SNAP-tagged cells. This difference was reflected in both the labeling efficiency (Figure 3B), which is useful but can mask smaller differences, and the labeling intensity of individual cells (Figures 3C and S6). These four pairs of different proteins showed that the extent of the labeling effect can be variable; nevertheless, the general trend was an ∼2- to 6-fold higher labeling intensity with Halo tags. Interestingly, the labeling effect appeared to be greater for transmembrane proteins at the Golgi, potentially due to the local membrane environment. Additional investigation of the photophysical properties of SiR conjugated to Halo and SNAP tags in fluorimetry experiments showed a 3-fold difference in the extinction coefficient between the two conjugates (Table S1). Taking the small difference of the reported quantum yield into account (Lukinavicius et al., 2013Lukinavicius G. Umezawa K. Olivier N. Honigmann A. Yang G.Y. Plass T. Mueller V. Reymond L. Correa I.R. Luo Z.G. et al.A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins.Nat. Chem. 2013; 5: 132-139Crossref PubMed Scopus (595) Google Scholar), this would represent a 4-fold difference in the brightness of the conjugates, consistent with the difference in labeling brightness observed in cells. Next, we hypothesized that the dye itself may influence the labeling brightness as different dyes might differentially sense the local environment within HaloTags and SNAP-tags. Thus, we labeled the four SNAP/Halo fusion proteins of ST, ManII, OMP25, and CLC with tetramethylrhodamine (TMR) (Figure S7), which is nearly structurally identical to SiR. TMR substitutes a dimethylsilyl group in SiR with an oxygen, which renders it less electrophilic. As such, TMR is less prone to adopt a nonfluorescent spirolactone, making it less environmentally sensitive (Lukinavicius et al., 2013Lukinavicius G. Umezawa K. Olivier N. Honigmann A. Yang G.Y. Plass T. Mueller V. Reymond L. Correa I.R. Luo Z.G. et al.A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins.Nat. Chem. 2013; 5: 132-139Crossref PubMed Scopus (595) Google Scholar). Indeed, the difference in brightness of cells with TMR-labeled Halo and SNAP fusion proteins was considerably smaller than the difference with SiR-labeled fusion proteins (Figures 3D and S8). We also tested more rhodamine-based dyes in Drosophila, using Halo-SNAP-aPKC. For TMR and its brighter, azetidine-containing analog JF549 (Grimm et al., 2015Grimm J.B. English B.P. Chen J. Slaughter J.P. Zhang Z. Revyakin A. Patel R. Macklin J.J. Normanno D. Singer R.H. et al.A general method to improve fluorophores for live-cell and single-molecule microscopy.Nat. Methods. 2015; 12: 244-250Crossref PubMed Scopus (823) Google Scholar), we did not observe a significant difference in brightness between Halo and SNAP tags when labeling egg chambers with TMR- or JF549-containing CA or BG substrates, respectively. In stark contrast, we observed a 4.5- to 9-fold difference between SNAP and Halo tags when the same system was labeled with SiR and its azetidine-containing analog JF646 (Grimm et al., 2015Grimm J.B. English B.P. Chen J. Slaughter J.P. Zhang Z. Revyakin A. Patel R. Macklin J.J. Normanno D. Singer R.H. et al.A general method to improve fluorophores for live-cell and single-molecule microscopy.Nat. Methods. 2015; 12: 244-250Crossref PubMed Scopus (823) Google Scholar), respectively (Figures 3D and S9). As such the JF549/JF646 azetidine dye pair shows the same trend as the TMR/SiR dimethyl rhodamines dye pair, with the far-red dyes showing brighter labeling with the HaloTag under otherwise similar microscopy conditions (see representative image on Figure 3D, right). We speculate that a combination of several factors might lead to the above observations. The local environment of the tag protein (such as pH) as well as the polarity of its surface can influence the absorption and quantum yield of the dye attached to it. To get a sense of whether the local environments around the dye may differ for HaloTag and SNAP-tag proteins, we surveyed the energy-minimized landscape of SiR tagged to SNAP and Halo proteins, based on the known crystal structures of SNAP (PDB: 3KZZ) and Halo proteins (PDB: 5VNP) (Liu et al., 2017Liu Y. Fares M. Dunham N.P. Gao Z. Miao K. Jiang X. Bollinger S.S. Boal A.K. Zhang X. AgHalo: a facile fluorogenic sensor to detect drug-induced proteome stress.Angew. Chem. Int. Ed. 2017; 56: 8672-8676Crossref PubMed Scopus (56) Google Scholar). After energy minimization, we noted the close proximity of the F143 and M174 residues with the SiR dye in the SiR-CA-Halo protein (Figure S9), which might help stabilize the dye in the open state. Finally, intrinsic dye properties, such as the polarity-dependent fluorescence of SiR-based dyes (Erdmann et al., 2014Erdmann R.S. Takakura H. Thompson A.D. Rivera-Molina F. Allgeyer E.S. Bewersdorf J. Toomre D. Schepartz A. Super-resolution imaging of the golgi in live cells with a bioorthogonal ceramide probe.Angew. Chem. Int. Ed. 2014; 53: 10242-10246Crossref PubMed Scopus (112) Google Scholar, Lukinavicius et al., 2013Lukinavicius G. Umezawa K. Olivier N. Honigmann A. Yang G.Y. Plass T. Mueller V. Reymond L. Correa I.R. Luo Z.G. et al.A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins.Nat. Chem. 2013; 5: 132-139Crossref PubMed Scopus (595) Google Scholar), can lead to a different brightness when tagging various self-labeling proteins. Although the contributions of these factors may be multifactorial, our results nevertheless demonstrate that brighter labeling is generally achieved when labeling Halo fusion proteins with SiR dyes. Importantly, SiR-based dyes (e.g., SiR and JF646) represent a very important dye class for STED nanoscopy due to their brightness and photostability (Bottanelli et al., 2016Bottanelli F. Kromann E.B. Allgeyer E.S. Erdmann R.S. Wood Baguley S. Sirinakis G. Schepartz A. Baddeley D. Toomre D.K. Rothman J.E. et al.Two-colour live-cell nanoscale imaging of intracellular targets.Nat. Commun. 2016; 7: 10778Crossref PubMed Scopus (141) Google Scholar, Bottanelli et al., 2017Bottanelli F. Kilian N. Ernst A.M. Rivera-Molina F. Schroeder L.K. Kromann E.B. Lessard M.D. Erdmann R.S. Schepartz A. Baddeley D. et al.A novel physiological role for ARF1 in the formation of bi-directional tubules from the Golgi.Mol. Biol. Cell. 2017; 28: 1676-1687Crossref PubMed Google Scholar, Erdmann et al., 2014Erdmann R.S. Takakura H. Thompson A.D. Rivera-Molina F. Allgeyer E.S. Bewersdorf J. Toomre D. Schepartz A. Super-resolution imaging of the golgi in live cells with a bioorthogonal ceramide probe.Angew. Chem. Int. Ed. 2014; 53: 10242-10246Crossref PubMed Scopus (112) Google Scholar, Lukinavičius et al., 2015Lukinavičius G. Blaukopf C. Pershagen E. Schena A. Reymond L. Derivery E. Gonzalez-Gaitan M. D’Este E. Hell S.W. Wolfram Gerlich D. et al.SiR–Hoechst is a far-red DNA stain for live-cell nanoscopy.Nat. Commun. 2015; 6: 8497Crossref PubMed Scopus (173) Google Scholar, Lukinavičius et al., 2016Lukinavičius G. Reymond L. Umezawa K. Sallin O. D’Este E. Göttfert F. Ta H. Hell S.W. Urano Y. Johnsson K. Fluorogenic probes for multicolor imaging in living cells.J. Am. Chem. Soc. 2016; 138: 9365-9368Crossref PubMed Scopus (158) Google Scholar, Lukinavičius et al., 2014Lukinavičius G. Reymond L. D'Este E. Masharina A. Gutfert F. Ta H. Guether A. Fournier M. Rizzo S. Waldmann H. et al.Fluorogenic probes for live-cell imaging of the cytoskeleton.Nat. Methods. 2014; 11: 731-733Crossref PubMed Scopus (502) Google Scholar). Near-infrared (IR) dyes avoid cellular green/red autofluorescence, and near-IR light is known to cause much less phototoxicity than green light (Waldchen et al., 2015Waldchen S. Lehmann J. Klein T. van de Linde S. Sauer M. Light-induced cell damage in live-cell super-resolution microscopy.Sci. Rep. 2015; 5: 15348Crossref PubMed Scopus (299) Google Scholar). Most importantly, SiR dyes, unlike dyes of other classes, are compatible with live-cell super-resolution microscopy since they are cell permeative. Thus, we investigated the difference between SNAP-tag and HaloTag labeling with SiR dyes in the context of STED microscopy. First, we imaged the Golgi in HeLa cells transiently expressing ManII-Halo and ManII-SNAP, both labeled with SiR, in confocal and STED mode (Figure 4A and Video S1). As expected, we observed an improvement in resolution in the STED mode compared with the confocal mode. Strikingly, the initial brightness of the Halo-labeled proteins was about 3-fold brighter than that of SNAP-labeled proteins (Figure 4B). The STED kymographs (Figure 4A) and bleaching profile (Figure 4C) show that only the sample labeled using HaloTag was bright for over 100 s. These findings are consistent with a recent single-molecule tracking study, which reported that HaloTag conjugates are more photostable than SNAP-tag conjugates (Presman et al., 2017Presman D.M. Ball D.A. Paakinaho V. Grimm J.B. Lavis L.D. Karpova T.S. Hager G.L. Quantifying transcription factor binding dynamics at the single-molecule level in live cells.Methods. 2017; 123: 76-88Crossref PubMed Scopus (50) Google Scholar). As a second example, we imaged CLC, which labels clathrin-coated endocytic pits. Showing the power of STED, clathrin-coated pits appeared as blurry" @default.
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