Quantitative Imaging of Labile Zn2+ in the Golgi Apparatus Using a Localizable Small-Molecule Fluorescent Probe

Kowada, Toshiyuki; Watanabe, Tomomi; Amagai, Yuta; Liu, Rong; Yamada, Momo; Takahashi, Hiroto; Matsui, Toshitaka; Inaba, Kenji; Mizukami, Shin

doi:10.1016/j.chembiol.2020.09.003

Cited by 37 publications

(36 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thereafter, we investigated the fluorescence quenching mechanism of Zn 2+ -free ZnDA probes. In our previous study, density functional theory (DFT) calculations on ZnDA-1H indicated that the fluorescence quenching of Zn 2+ -free ZnDA-1H was not caused by photoinduced electron transfer (PeT) from the pyridine moieties. In this study, ZnDA-2H and ZnDA-3H had one additional aliphatic amino group in their chelator parts, which may have caused fluorescence quenching by PeT in the absence of Zn 2+ .…”

Section: Resultsmentioning

confidence: 99%

Organelle-Level Labile Zn²⁺ Mapping Based on Targetable Fluorescent Sensors

Liu

Kowada

et al. 2022

ACS Sens.

Self Cite

View full text Add to dashboard Cite

Although many Zn 2+ fluorescent probes have been developed, there remains a lack of consensus on the labile Zn 2+ concentrations ([Zn 2+ ]) in several cellular compartments, as the fluorescence properties and zinc affinity of the fluorescent probes are greatly affected by the pH and redox environments specific to organelles. In this study, we developed two turn-on-type Zn 2+ fluorescent probes, namely, ZnDA-2H and ZnDA-3H, with low pH sensitivity and suitable affinity (K d = 5.0 and 0.16 nM) for detecting physiological labile Zn 2+ in various cellular compartments, such as the cytosol, nucleus, ER, and mitochondria. Due to their sufficient membrane permeability, both probes were precisely localized to the target organelles in HeLa cells using HaloTag labeling technology. Using an in situ standard quantification method, we identified the [Zn 2+ ] in the tested organelles, resulting in the subcellular [Zn 2+ ] distribution as [

show abstract

Section: Resultsmentioning

confidence: 99%

Organelle-Level Labile Zn²⁺ Mapping Based on Targetable Fluorescent Sensors

Liu

Kowada

et al. 2022

ACS Sens.

Self Cite

View full text Add to dashboard Cite

show abstract

“…We used the N-terminal transmembrane domain (residues 1-117) of human mannosidase II (MAN2A1 1-117 ) (Velasco et al, 1993) to mediate Golgi-lumen localization of Halo (MAN2A1 1-117 -FLAG-Halo-HiBiT) or FKBP12 F36V (MAN2A1 1-117 -FLAG-FKBP12 F36V -Hi-BiT) proteins in U2OS cells. To confirm that MAN2A1 1-117 -FLAG-Halo/FKBP12 F36V -HiBiT localized at the Golgi, WT U2OS cells or those expressing MAN2A1 1-117 -FLAG-Halo-HiBiT or MAN2A1 1-117 -FLAG-FKBP12 F36V -HiBiT were subjected to anti-FLAG and anti-GM130 (Golgi marker) (Kowada et al, 2020;Zhang et al, 2021) immunofluorescence microscopy (Figure 6A). Both MAN2A1 1-117 -FLAG-Halo-HiBiT and MAN2A1 1-117 -FLAG-FKBP12 F36V -HiBiT staining overlapped with GM130 staining, suggesting that both localize predominantly at the Golgi.…”

Section: Degradation Of Golgi-localized Halo and Dtag Proteins With P...mentioning

confidence: 99%

Target protein localization and its impact on PROTAC-mediated degradation

Simpson

Glennie

Brewer

et al. 2022

Cell Chemical Biology

View full text Add to dashboard Cite

“…Recombinant protein expression and purification. Recombinant eDHFR and HaloTag were expressed and purified aspreviously described 31,52 plated at 5.0 × 10 4 cells per dish and transfected with plasmids encoding eDHFR-miRFP670nano-NES and Halo-mOrange2-CAAX at 1:2 ratio. After 36 h of transfection, the cells were washed twice with HBSS (+) and treated with 5 µM pcDH in DMEM for 1 h. The culture medium was replaced with HBSS (+).…”

Section: In Vitro Pcdh Characterizationmentioning

confidence: 99%

Quantitative and Repetitive Control of Subcellular Protein–Protein Interaction Using a Photochromic Dimerizer

Mashita

Kowada

Yamamoto

et al. 2022

Preprint

View full text Add to dashboard Cite

Artificial control of intracellular protein dynamics with high precision provides deep insight into complicated biomolecular networks. Optogenetics and caged compound-based chemically induced dimerization (CID) systems are emerging as tools for spatiotemporally regulating intracellular protein dynamics. However, both technologies face several challenges for accurate control such as the duration of activation, deactivation rate, and repetition cycles. Herein, we report a photochromic CID system that employs the photoisomerization of a ligand so that both association and dissociation are controlled by light, enabling quick, repetitive, and quantitative regulation of the target protein localization upon violet and green light illumination. We also demonstrated the usability of the photochromic CID system as a potential tool to finely manipulate intracellular protein dynamics to study diverse cellular processes. Utilizing this system to manipulate PINK1/Parkin-mediated mitophagy, we showed that PINK1 recruitment to the mitochondria can promote Parkin recruitment to proceed with mitophagy.

show abstract

Quantitative Imaging of Labile Zn2+ in the Golgi Apparatus Using a Localizable Small-Molecule Fluorescent Probe

Cited by 37 publications

References 65 publications

Organelle-Level Labile Zn²⁺ Mapping Based on Targetable Fluorescent Sensors

Organelle-Level Labile Zn²⁺ Mapping Based on Targetable Fluorescent Sensors

Target protein localization and its impact on PROTAC-mediated degradation

Quantitative and Repetitive Control of Subcellular Protein–Protein Interaction Using a Photochromic Dimerizer

Contact Info

Product

Resources

About

Quantitative Imaging of Labile Zn2+ in the Golgi Apparatus Using a Localizable Small-Molecule Fluorescent Probe

Cited by 37 publications

References 65 publications

Organelle-Level Labile Zn2+ Mapping Based on Targetable Fluorescent Sensors

Organelle-Level Labile Zn2+ Mapping Based on Targetable Fluorescent Sensors

Target protein localization and its impact on PROTAC-mediated degradation

Quantitative and Repetitive Control of Subcellular Protein–Protein Interaction Using a Photochromic Dimerizer

Contact Info

Product

Resources

About

Organelle-Level Labile Zn²⁺ Mapping Based on Targetable Fluorescent Sensors

Organelle-Level Labile Zn²⁺ Mapping Based on Targetable Fluorescent Sensors