An Optimized Protocol for Proximity Biotinylation in Confluent Epithelial Cell Cultures Using the Peroxidase APEX2

Tan, Benedict; Peng, Suat; Yatim, Siti Maryam J.M.; Gunaratne, Jayantha; Hunziker, Walter; Ludwig, Alexander

doi:10.1016/j.xpro.2020.100074

Cited by 27 publications

(22 citation statements)

References 19 publications

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“…JIMT-1 cells were transfected with APEX2-TACC3 vector and synchronized for mitosis and interphase as described above. Biotinylation was performed as previously described 52 . Briefly, cells were treated with 2.5 mM biotin phenol (Iris Biotech, Germany) for 1 hr, and 1 mM H 2 O 2 was added at room temperature (RT) for 2 min.…”

Section: Methodsmentioning

confidence: 99%

Targeting TACC3 represents a novel vulnerability in highly aggressive breast cancers with centrosome amplification

Saatçi

Akbulut

Çetįn

et al. 2022

Preprint

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Centrosome amplification (CA) is a hallmark of cancer that is strongly associated with highly aggressive disease and worse clinical outcome. However, there are no effective strategies targeting cancer cells with CA while sparing normal cells. Here, we identified Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) to be overexpressed in tumors with CA, and its high expression is associated with dramatically worse clinical outcome. We demonstrated that TACC3 forms distinct functional interactions in mitotic and non-mitotic cancer cells with CA to facilitate centrosome clustering (CC) and transcriptional repression of tumor suppressors, respectively. We showed, for the first time, that TACC3 interacts with the Kinesin Family Member C1 (KIFC1) via its TACC domain in mitotic cells with CA and inhibition of TACC3 blocks this interaction, leading to apoptosis via multipolar spindle formation and activation of spindle assembly checkpoint (SAC)/CDK1/p-Bcl2 axis. In interphase, TACC3 interacts with the members of the nucleosome remodeling and deacetylase (NuRD) complex (HDAC2 and MBD2) in nucleus, and its inhibition causes p53-independent G1 arrest and apoptosis by blocking these interactions and activating the transcription of key tumor suppressors (e.g., p21, p16 and APAF1). Notably, inducing CA by chemical (cytochalasin D) or genomic (PLK4 overexpression or p53 loss) modulations renders cancer cells highly sensitive to TACC3 inhibition. Targeting TACC3 by small molecule inhibitors or guide RNAs strongly inhibits growth of organoids and breast cancer cell line- and patient-derived xenografts with CA. Altogether our results pave the way towards therapeutic targeting of TACC3 in highly aggressive cancers.

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Section: Methodsmentioning

confidence: 99%

Targeting TACC3 represents a novel vulnerability in highly aggressive breast cancers with centrosome amplification

Saatçi

Akbulut

Çetįn

et al. 2022

Preprint

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“…The APEX2 proximity labeling assays were performed as described previously [31]. Briefly, biotin-phenol labeling was initiated by cell incubation at 37 °C with a 5% CO 2 for 30 min in cell culture medium supplemented with 2.5 mM biotin-phenol.…”

Section: Apex2 Proximity Labeling Assaymentioning

confidence: 99%

Orai1α, but not Orai1β, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells

Sánchez-Collado

López

Jardín

et al. 2022

Cell. Mol. Life Sci.

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The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1β, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1β differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1β are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α–TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively.

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“…To settle this issue, we focused on the proximity labeling enzymes and the biotin-streptavidin reaction [10][11][12][13][14][15] . The proximity labeling methods using engineered ascorbate peroxidases and biotin ligases are mainly developed to analyze protein-protein interactions in living cells 16,17 .…”

Section: Introductionmentioning

confidence: 99%

“…Based on this reaction and biotin-avidin interaction, APEX2 in the cells was detected by a uorescence microscope with uorophore-conjugated streptavidin and detected by electron microscopy using an enzymatic reaction with 3,3 -diaminobenzidine converting into an insoluble osmiophilic polymer 11,12 . One problem for labeling living cells using engineered ascorbate peroxidases is the treatment of living cells with hydrogen peroxide 13 . Until recently, however, because no study has reported the application of engineered ascorbate peroxidases for in-resin CLEM of Eponembedded cells.…”

Section: Introductionmentioning

confidence: 99%

“…Biotin ligase mediates the conjugation of proximity proteins and itself with biotin in living cells [12][13][14][15] . Some engineered biotin ligases including BASU, TurboID, miniTurbo, and AirID are able to label proximal proteins with biotin within 10 min without cell toxicity, whereas the other biotin ligases (BirA, its mutant, BioID, and BioID2) require more than 10 h for proximity labeling 14,15,18,19 .…”

Section: Introductionmentioning

confidence: 99%

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In-Resin CLEM of Epon-Embedded Cells Using Proximity Labeling

Sanada

Yamaguchi

Furuta

et al. 2022

Preprint

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Biotin ligases have been developed as proximity biotinylation enzymes for analyses of the interactome. However, there has been no report on the application of proximity labeling using biotin ligase for in-resin correlative light-electron microscopy of Epon-embedded cells. In this study, we established a proximity-labeled in-resin CLEM of Epon-embedded cells using miniTurbo, a biotin ligase. Biotinylation by miniTurbo was observed in cells within 10 mins following the addition of biotin to the medium. Using fluorophore-conjugated streptavidin, intracellular biotinylated proteins were labeled after fixation of cells with a mixture of paraformaldehyde and glutaraldehyde. Fluorescence of these proteins was resistant to osmium tetroxide staining and was detected in 100-nm ultrathin sections of Epon-embedded cells. Ultrastructures of organelles including mitochondria and endoplasmic reticulum were preserved well in the same sections. Fluorescence in sections was about 14-fold brighter than that in the sections of Epon-embedded cells expressing mCherry2 and was detectable for 14 days. When mitochondria-localized miniTurbo was expressed in the cells, mitochondria-like fluorescent signals were detected in the ultrathin sections, and ultrastructures of mitochondria were observed as fluorescence-positive structures in the same sections by scanning electron microscopy. These results indicated that in-resin correlative light-electron microscopy of Epon-embedded cells using proximity labeling was achieved.

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An Optimized Protocol for Proximity Biotinylation in Confluent Epithelial Cell Cultures Using the Peroxidase APEX2

Cited by 27 publications

References 19 publications

Targeting TACC3 represents a novel vulnerability in highly aggressive breast cancers with centrosome amplification

Targeting TACC3 represents a novel vulnerability in highly aggressive breast cancers with centrosome amplification

Orai1α, but not Orai1β, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells

In-Resin CLEM of Epon-Embedded Cells Using Proximity Labeling

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