A palette of background-free tame fluorescent probes for intracellular multi-color labelling in live cells

Alamudi, Samira Husen; Su, Dongdong; Lee, Kyung Jin; Lee, Jung Yeol; Belmonte‐Vázquez, José L.; Park, Hee-Sung; Peña‐Cabrera, Eduardo; Chang, Young‐Tae

doi:10.1039/c7sc04716a

Cited by 29 publications

(17 citation statements)

References 44 publications

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“…N 3 -Cho, rhodol-DBCO and rhodamine B-DBCO were prepared as previously described (Tamura et al, 2020). 8AB-DBCO was synthesized according to previous report with minor modifications (Alamudi et al, 2018). Briefly, [2-[(Methylthio)(2H-pyrrol-2-ylidene)methyl]-1H-pyrrole](difluoroborane) (TCI, M2609) was reacted with dibenzocyclooctyne-amine (TCI, A2763).…”

Section: Methodsmentioning

confidence: 99%

Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism

Tsuchiya

Tachibana

Nagao

et al. 2022

Preprint

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Lipids comprise biomembranes and are involved in many crucial cell functions. While cellular lipid synthesis and transport appear to be governed by intricate protein networks, the whole scheme is insufficiently understood. Although functional genome-wide screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain – especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, this work revealed previously uncharacterized roles of FLVCR1 as a new choline transporter; CHEK1 as a post-translational regulator of the PC-synthetic pathway, and TMEM30A as responsible for translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.

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Section: Methodsmentioning

confidence: 99%

Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism

Tsuchiya

Tachibana

Nagao

et al. 2022

Preprint

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“…In 2018, Chang and co-workers reported that 8-amino-BODIPY derivatives containing the azide and cyclooctyne moiety (AzA-1, COA-1, Figure 3) are applicable for copper-free click chemistry [23,24]. The probes, namely "tame probes," show high biocompatibility with no background noise after labeling in live cells.…”

Section: Labelingmentioning

confidence: 99%

Blue-Emitting BODIPY Dyes

Kim¹

2019

BODIPY Dyes - A Privilege Molecular Scaffold With Tunable Properties

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BODIPY which consists of a dipyrromethene complex with disubstituted boron has emerged as a superior fluorophore in various research fields. BODIPY typically shows high quantum yield with environment-insensitive fluorescence emission, sharp excitation and emission peaks, high water solubility and biocompatibility, and photostability. So far, various kinds of BODIPY derivatives have been developed and applied in not only academia such as chemistry, biochemistry, biomedical engineering, and medicine but also industries. BODIPY shows dramatic photophysical property changes upon substitution of functional groups or pi bond elongation on the main core structure. Among them, the blue-emitting BODIPY dyes with their synthesis and photophysical analysis were recently reported. In this chapter, the key information of the blue-emitting BODIPY dyes and their recent cutting-edge applications are summarized.

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“…A BODIPY‐derivative tame probe containing a cyclooctyne functional group, CO‐1 , could label specific intracellular tagged‐biomolecules and show no interference on the cell proliferation [5b] . Through strain promoted alkyne‐azide cycloaddition (SPAAC) reaction, CO‐1 forms covalent bonds with its azide‐containing counterparts without fluorescence background noise.…”

Section: Introductionmentioning

confidence: 99%

ABCB1 Can Actively Pump‐out the Background‐Free Tame Fluorescent Probe CO‐1 From Live Cells

Ciaramicoli

Alamudi³

et al. 2022

Chemistry An Asian Journal

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Cell labelling using a small fluorescent probe is an important technique in biomedical sciences. We previously developed a biocompatible and membrane‐permeable probe, CO‐1, which has low nonspecific binding affinity towards nontarget molecules. Although this background‐free tame probe has been utilized for labelling of various intracellular biomolecules in live cells, the probes’ backgroung‐free staining mechanism was not fully understood. Here, we propose that Gating‐Oriented Live‐cell Distinction (GOLD) mechanism occurs when ABCB1 transporter removes unbound CO‐1 molecules from mammalian cells and, in a minor role, DIRC2 pumps CO‐1 out from lysosomes. We also showed that solute carrier transporters were not involved in carrying CO‐1 inside of cells. The role of reporters in assisting the probes’ influx‐efflux was analyzed by the combination of CRISPR library sceenings and inhibitors test. In summary, tame probe CO‐1 cellular staining occurs in a dual mechanism where the probe moves freely through the cells membrane, but its washable property can be directly related to the action of ABCB1 transporter.

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A palette of background-free tame fluorescent probes for intracellular multi-color labelling in live cells

Abstract: A palette of background-free tame fluorescent probes were designed and applied to intracellular multi-color labelling in live cells.

Cited by 29 publications

References 44 publications

Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism

Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism

Blue-Emitting BODIPY Dyes

ABCB1 Can Actively Pump‐out the Background‐Free Tame Fluorescent Probe CO‐1 From Live Cells

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