Target identification of mouse stem cell probe CDy1 as ALDH2 and Abcb1b through live-cell affinity-matrix and ABC CRISPRa library

Miyamoto, Naoki; Go, Young-Hyun; Ciaramicoli, Larissa Miasiro; Kwon, Hyeokjin; Hs, Kim; Bi, Xuezhi; Yu, Young Hyun; Kim, Beomsue; Ha, Hyung‐Ho; Kang, Nam‐Young; Yun, Seong‐Wook; Cha, Hyuk‐Jin; Chang, Young‐Tae

doi:10.1039/d1cb00147g

Cited by 4 publications

(4 citation statements)

References 20 publications

(18 reference statements)

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“…To examine the participation of SLC and/or ABC transporters in the staining‐destaining mechanism of CO‐1 , we constructed a series of CRISPR libraries for target identification by CRISPR screening [7b,9] …”

Section: Resultsmentioning

confidence: 99%

“…ABC‐CRISPR pool preparation was also previously presented, [7b,25] however the procedure was described for CRISPR activation system. In a similar way, for the interference CRISPR, 1530 sgRNA library targeting 153 types of transporters, including 48 types of human ABC transporters were selected.…”

Section: Methodsmentioning

confidence: 99%

“…We previously showed that a fluorescent probe can be a substrate of SLC transporter for the distinction of activated and inactivated macrophages [9] . In addition, we reported that ABC transporters play a role in differentiating mouse embryonic stem cells and mouse embryonic fibroblasts by pumping out CDy‐1 probe [7b] . Despite the distinct nature of MOLD, HOLD and GOLD, they are not independent mechanisms.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…SLC transporters primarily facilitate the uptake of small molecules into cells (pump in), while ABC transporters are responsible for exporting substrates to the extracellular space (pump out). Leveraging transporter-dependent mechanisms, we have developed several selective bioprobes [10][11][12][13][14][15], the mechanisms reliant on transporters uncovered have not fully encompassed all transporter pools yet.…”

mentioning

confidence: 99%

Novel Fluorescent Strategy of B Cell Selective Probe

Cho,

Hong,

Chang

2024

Preprint

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Fluorescent bioprobes are invaluable tools for visualizing live cells and deciphering complex biological processes by targeting intracellular biomarkers without disrupting cellular functions. In addition to protein-binding concepts, fluorescent probes utilize various mechanisms, including membrane, metabolism, and gating-oriented strategies. This study introduces a novel fluorescent mechanism distinct from existing ways. Here, we developed a B-cell selective probe, CDrB, with unique transport mechanisms. Through SLC-CRISPRa screening, we identified two transporters, SLCO1B3 and SLC25A41, by sorting out populations exhibiting higher and lower fluorescence intensities, respectively, demonstrating contrasting activities. We confirmed that SLCO1B3, with comparable expression levels in T and B cells, facilitates the transport of CDrB into cells, while SLC25A41, overexpressed in T lymphocytes, actively exports CDrB. The observation suggests that SLC25A41 plays a crucial role in discriminating between T and B lymphocytes. Furthermore, it reveals the potential for reversible localization of SLC25A41 to demonstrate its distinct activity. This study is the first report to unveil a novel strategy of SLC by exporting the probe. We anticipate that this research will open up new avenues for developing fluorescent probes.

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Live‐Cell‐Selective Probes

2023

Sensors and Probes for Bioimaging

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Target identification of mouse stem cell probe CDy1 as ALDH2 and Abcb1b through live-cell affinity-matrix and ABC CRISPRa library

Abstract: CDy1 is a powerful tool to distingusih embryonic stem cells for reprogramming studies and regeneration medicine.

Cited by 4 publications

References 20 publications

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

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

Novel Fluorescent Strategy of B Cell Selective Probe

Live‐Cell‐Selective Probes

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