An organelle-directed chemical ligation approach enables dual-color detection of mitophagy

Shi, Yuchen; Zou, Xiaoxue; Wen, Shixiong; Gao, Lei; Li, Jian; Han, Jiahuai; Han, Shoufa

doi:10.1080/15548627.2021.1875597

Cited by 13 publications

(14 citation statements)

References 52 publications

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“…Compound 3 (500 mg, 0.95 mmol) and compound 4 (420 mg, 0.95 mmol) were dissolved in CH 2 Cl 2 (25.0 mL) containing TEA (0.4 mL). The reaction mixture was stirred at room temperature for 4 h, washed with an aqueous solution of sodium hydroxide (1 mol/L), and then dehydrated with Na 2 SO 4 .…”

Section: Experimental Proceduresmentioning

confidence: 99%

“…To a flask containing compound 6 (150.0 mg, 0.12 mmol), compound 7 (33,0 mg, 0.13 mmol), and pyridine (5.0 mL) was added EDC (33.0 mg, 0.17 mmol). The reaction mixture was stirred at ambient temperature overnight and then concentrated.…”

Section: Experimental Proceduresmentioning

confidence: 99%

“…13,14 As such, researchers have coined various methods to immobilize synthetic probes in mitochondria, including covalent probe linking with mitochondrial proteins, 15−20 probe self-assembly, 21−24 and intraorganelle probe ligation. 25 Recently, metabolic conversion of Az choline to azidocontaining phosphatidylcholine was harnessed to immobilize optical probes to biological membranes via the bioorthogonal reaction. 26,27 Based on these advances, we sought to anchor an acidity-reporting probe into the mitochondrial inner membrane via bioorthogonal ligation with Az choline preinstalled in mitochondrial membranes, with the aim to image mitophagy by sensing pH acidification upon delivery of mitochondria into lysosomes (Scheme 1).…”

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confidence: 99%

“…Mitophagy is an evolutionarily conserved degradation pathway by which excess or dysfunctional mitochondria are metabolized in lysosomes. − Critical for cell homeostasis and multiple human disorders, mitophagy has been intensively investigated for disease intervention, − which necessitates optical tracking of mitophagy. , Conventional cationic dyes accumulate in mitochondria, driven by mitochondrial membrane potential (ΔΨm), but are prone to dissipation from mitochondria upon ΔΨm loss. − This limits their use to track stressed mitochondria in mitophagy. , As such, researchers have coined various methods to immobilize synthetic probes in mitochondria, including covalent probe linking with mitochondrial proteins, − probe self-assembly, − and intraorganelle probe ligation …”

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confidence: 99%

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Imaging of Mitophagy Enabled by an Acidity-Reporting Probe Anchored on the Mitochondrial Inner Membrane

Wen

Shi

et al. 2021

Anal. Chem.

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Classical chemical probes are prone to dissipation from stressed organelles, as evidenced by the incapability of mitochondrial dyes to image mitophagy linked to multiple diseases. We herein reported mitophagy imaging via covalent anchoring of a lysosomal probe to the mitochondrial inner membrane (CALM). Utilizing DBCO RC-TPP, an azide-conjugatable probe with acidity-triggered fluorescence, CALM is operated via ΔΨm-promoted probe accumulation in mitochondria and thereby bioorthogonal ligation of the trapped probe with azido-choline (Azcholine) metabolically installed on the mitochondrial membrane. Overcoming the limitation of synthetic probes to dissipate from stressed organelles, CALM enables signal-on fluorescence imaging of mitophagy induced by starvation and is further employed to reveal mitophagy in ferroptosis. These results suggest the potential of CALM as a new tool to study mitophagy.

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Section: Experimental Proceduresmentioning

confidence: 99%

Section: Experimental Proceduresmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Imaging of Mitophagy Enabled by an Acidity-Reporting Probe Anchored on the Mitochondrial Inner Membrane

Wen

Shi

et al. 2021

Anal. Chem.

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“…All other chemicals were purchased from Sigma unless specified. HeLa cells and HT1080 cells were obtained from American Type Culture Collection (ATCC), GFP-Lamp2 + HeLa cells, and RIP 3 + HeLa cells were prepared following reported procedures. , RFP-Lamp2 + HeLa cells were a gift from Professor Jiahuai Han, Xiamen University. The cells were generally cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, l -glutamine (2 mM), penicillin (100 IU), and streptomycin (100 mg/mL) under 5% CO 2 in a humidified incubator at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Galectin Trafficking Pathway-Enabled Color-Switchable Detection of Lysosomal Membrane Permeabilization

Gao

Han

2021

Anal. Chem.

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Lysosomal membrane permeabilization (LMP) engaged in multiple human diseases is accompanied by relocation of cytosolic galectin into LMP+ lysosomes. We herein reported a galectin trafficking-targeted method to image LMP using two kinds of glyco-dendrimers, a sialic acid-terminated dendrimer labeled with pH-inert rhodamine and a lactose-terminated dendrimer labeled with fluorescein that becomes green-emissive in pH-elevated lysosomes. Albeit both accumulated in physiological lysosomes, the former is released from LMP+ lysosomes while the latter binds to galectin accumulated in LMP+ lysosomes and thus trapped in LMP+ lysosomes. Accordingly, LMP+ lysosomes exhibit loss of red fluorescence and turn-on green fluorescence due to loss of lysosomal acidity. This red-to-green color switch enables discernment of LMP+ lysosomes from physiological lysosomes and pH-elevated lysosomes and can be further utilized to detect LMP in distinct cell death pathways. These results suggest the utility of galectin trafficking pathway-integrated synthetic probes for detection of LMP, a key factor for diseased cells.

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A Tetrazine‐Caged Carbon‐Dipyrromethene as a Bioorthogonally Activatable Fluorescent Probe

Tam,

Lo,

Cheung

et al. 2023

Chemistry An Asian Journal

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A water‐soluble 1,2,4,5‐tetrazine‐substituted carbon‐dipyrromethene (C–DIPY) was synthesized from the previously reported carbonyl pyrrole dimer through a two‐step procedure. Owing to the presence of a tetrazine moiety, the fluorescence emission of this compound was largely quenched in phosphate‐buffered saline at pH 7.4. Upon addition of a bicyclo[6.1.0]non‐4‐yne (BCN) derivative, the tetrazine‐based quenching component of the compound was disrupted through the inverse electron‐demand Diels‐Alder reaction to restore the fluorescence in up to 6.6‐fold. This bioorthogonal activation was also demonstrated using U‐87 MG human glioblastoma cells, in which the fluorescence intensity of this C–DIPY could be enhanced by 8.7‐fold upon post‐incubation with the BCN derivative. The results showed that this tetrazine‐caged C–DIPY can serve as a bioorthogonally activatable fluorescent probe for bioimaging. The compound, however, was found to reside preferentially in the lysosomes instead of the mitochondria of the cells as predicted based on its cationic character, which could be attributed to its energy‐dependent endocytic cellular uptake pathway, for which lysosomes are the end station.

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An organelle-directed chemical ligation approach enables dual-color detection of mitophagy

Cited by 13 publications

References 52 publications

Imaging of Mitophagy Enabled by an Acidity-Reporting Probe Anchored on the Mitochondrial Inner Membrane

Imaging of Mitophagy Enabled by an Acidity-Reporting Probe Anchored on the Mitochondrial Inner Membrane

Galectin Trafficking Pathway-Enabled Color-Switchable Detection of Lysosomal Membrane Permeabilization

A Tetrazine‐Caged Carbon‐Dipyrromethene as a Bioorthogonally Activatable Fluorescent Probe

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