Skip to main content

Optimized Xanthene Fluorophores Permit Dual-Color Live-Cell Imaging

Single-molecule localization microscopy (SMLM) is an important method for the high resolution 

investigation of microscopic cellular structures and processes. Traditional organic fluorophores used 

in SMLM utilize intense laser irradiation to induce fluorescence, but this can cause undesired 

photobleaching and can be cytotoxic. In this study,1 the Urano group sought to apply their developed 

xanthene-based fluorophores, HMSiR and HEtetTFER, in multi-color SMLM. The HMSiR 

fluorophore, a first-in-class spontaneously blinking fluorophore (near-infrared range, NIR), already 

possesses optimal SMLM characteristics because it does not require laser irradiation or additives to 

perform under physiological conditions.2 Conversely, HEtetTFER is a green-light emitting 

fluorophore that has been used with HMSiR to achieve dual-color SMLM in fixed cells,3 but due to 

 poor membrane permeability and unfavorable subcellular localization, HEtetTFER is not suitable 

for imaging live cells.


Building on their previous investigation of the reactivity of xanthene fluorophores with endogenous 

 glutathione (GSH),4 the group developed structurally optimized SMLM-compatible fluorophores,  

SiP650 and CP550, working in the NIR and green range, respectively (Figure 1). Notably, these 

structures needed to be tuned to possess the appropriate equilibrium constants between their 

fluorescent dissociated form and their non-fluorescent GSH adduct form, and with fast blinking 

kinetics. Key parameters are shown in Table 1.





HaloTag ligands of both of these fluorophores confirmed specific protein labelling and spontaneous blinking in live cells. Using HMSiR in combination with the newly developed CP550, dual-color live-cell SMLM was achieved in mammalian and bacterial cells (Figure 3). This work is important because new fluorophores are needed with various optical properties and blinking kinetics, and this approach will minimize photodamage and image buffer optimizations that are common characteristics of similar tools.


 References

1.      1.     Morozumi, A.; Kamiya, M.; Uno, S.-N.; Umezawa, K.; Kojima, R.; Yoshihara, T.; Tobita, S.; Urano, Y. J. Am. Chem. Soc. 2020, 142 (Just accepted 4/28/2020), doi: 10.1021/jacs.0c00451

2     2.     Uno, S.; Kamiya, M.; Yoshihara, T.; Sugawara, K.; Okabe, K.; Tarhan, M. C.; Fujita, H.; Funatsu, T.; Okada, Y.; Tobita, S.; Urano, Y. Nat. Chem. 2014, 6, 681.

3     3.     Uno, S.; Kamiya, M.; Morozumi, A.; Urano, Y. Chem. Commun. 2018, 54, 102.
4     4.     Umezawa, K.; Yoshida, M.; Kamiya, M.; Yamasoba, T.; Urano, Y. Nat. Chem. 2017, 9, 279.

Comments

Post a Comment

Popular posts from this blog

Lysine-Targeted Covalent Inhibitors Gaining Traction

In the past decade, targeted covalent inhibitors (TCIs) has experienced a resurgence due to its sustained target engagement and improved proteome-wide selectivity. Currently, targeting cysteine residues with acrylamides and other α, β-unsaturated carbonyl compounds is the predominant strategy in TCI development. Although lysine is more prevalent than cysteine and possesses lower mutation rates, lysine has been much less frequently considered and more challenging as a residue targeted by TCIs due to its low nucleophilicity. Recently, Jack Taunton and his colleagues reveled their two related work in JACS .   Based on the co-crystal structure of Hsp90 with a reversible inhibitor PU-H71, they designed two arylsulfonyl fluorides 1 and 2 and co-incubated them with Hsp90 protein (Figure 1a, b). Mass spectrometry detected the formation of a 1:1 covalent adduct. Mutation of Lys58 to arginine abrogated the covalent modification, indicating that the TCI selectively modified Lys58 (Fig...
Post a Comment
Read more

Multiple Postdoc and Technician Positions Available at Baylor College of Medicine

  Multiple postdoc and technician positions are available in Dr. Jin Wang’s group in the Department of Pharmacology and Chemical Biology at Baylor College of Medicine (BCM). BCM is located in the world largest medical center and has access to enormous biomedical resources. The research in the Wang lab is highly interdisciplinary and translational. The Wang lab has both chemistry and biology operations. For chemistry work, we have 17 chemical fume hoods, 4 ISCO combiflash chromatography systems, Agilent analytical LC-MS, Agilent preparative HPLC with auto-purification, and Genevac high throughput solvent evaporation, along with convenient access to 600 and 800 MHz NMR. For biology work, we have common equipment for biochemistry, molecular and cell biology, including tissue culture hoods, RT-PCR, plate reader, and imaging plate reader, and perform animal work to evaluate the efficacy of the experimental therapeutics developed in the group. In addition, we recently acquired a state-...
Post a Comment
Read more

RIPK2 PROTAC Burns up the Bridges between PK and PD

GlaxoSmithKline (GSK) previously reported a series of RIPK2 PROTACs with various E3 ligase binders (Figure 1). These PROTACs can degrade RIPK2 potently and efficiently in a concentration-dependent manner (Figure1). However, their poor solubility and high microsomal clearance limited their in vivo applications. To develop a RIPK2 degrader suitable for  in vivo  applications, the GSK group identified another IAP based RIPK2 PROTAC4 that is significantly different from PROTAC2, possessing a different RIPK2 binder, IAP ligase binder and linker (Figure2). PTROTAC4 can degrade RIPK2 in a time-dependent manner and shows reduced lipophilicity and lower clearance in hepatocytes. However, the modest binding affinity between the warhead of PROTAC4 and RIPK2 requires a high dose to reach efficient RIPK2 degradation. To address this limitation, the researchers performed extensive medicinal chemistry optimization based on PROTAC 4 and identified PROTAC 6 (Figure2), which contains an...
Post a Comment
Read more