Selective Visualization of Caveolae by TEM Using APEX2

Ludwig, Alexander

doi:10.1007/978-1-0716-0732-9_1

Cited by 11 publications

(9 citation statements)

References 22 publications

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“…H 2 O 2 is necessary for the oxidation reaction to occur, however, it has also been reported that long incubation could inhibit the reaction ( Ludwig, 2020 ). Therefore, in case of weak signal, the DAB labeling can not very easily be adjusted by changing its duration.…”

Section: Discussion: Critical Parameters and Troubleshootingmentioning

confidence: 99%

“…Therefore, in case of weak signal, the DAB labeling can not very easily be adjusted by changing its duration. Instead it has been suggested that lowering H 2 O 2 concentration to 0.5 mM greatly enhances APEX2 activity and sensitivity, and results in an increased contrast in TEM ( Ludwig, 2020 ). It is therefore possible that small amounts of APEX protein can be detected by adjusting the H 2 O 2 concentration within the 10–0.5 mM range.…”

Section: Discussion: Critical Parameters and Troubleshootingmentioning

confidence: 99%

“…Notably, proteins either expressed by their endogenous promoters or over-expressed have been detected with the exact same conditions, showing that this protocol does not need too much adaptation from one POI to another. We have tested our protocol with classical TEM, but there have been reports showing that EM volume imaging such as SBF (serial block face) technology could also be successfully combined with APEX approaches (data not shown and Ariotti et al, 2015 ; Ludwig, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

“…The bi-partite detection of APEX also offers the possibility for a reliable technique of correlative light and EM (CLEM) whereby the GFP-tagged POI can be visualized using fluorescence microscopy, and the DAB precipitate generated by APEX can be identified by EM at the place of the GFP-tag (For review, see Ariotti et al, 2015 ; Ludwig, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

“…APEX has the advantage to retain activity after fixation with glutaraldehyde, a fixative that very well preserves the ultrastructure of the sample. APEX has been used as a tag in many studies and has largely proven its efficiency, making it now a tag of choice for the detection of fusion proteins in EM ( Martell et al, 2012 , 2017 ; Ariotti et al, 2015 ; Chen et al, 2015 ; Lee et al, 2016 ; Lin et al, 2016 ; Ludwig, 2020 ; Tan et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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GFP-Tagged Protein Detection by Electron Microscopy Using a GBP-APEX Tool in Drosophila

Bernard

Jouette

Pioche−Durieu

et al. 2021

Front. Cell Dev. Biol.

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In cell biology, detection of protein subcellular localizations is often achieved by optical microscopy techniques and more rarely by electron microscopy (EM) despite the greater resolution offered by EM. One of the possible reasons was that protein detection by EM required specific antibodies whereas this need could be circumvented by using fluorescently-tagged proteins in optical microscopy approaches. Recently, the description of a genetically encodable EM tag, the engineered ascorbate peroxidase (APEX), whose activity can be monitored by electron-dense DAB precipitates, has widened the possibilities of specific protein detection in EM. However, this technique still requires the generation of new molecular constructions. Thus, we decided to develop a versatile method that would take advantage of the numerous GFP-tagged proteins already existing and create a tool combining a nanobody anti-GFP (GBP) with APEX. This GBP-APEX tool allows a simple and efficient detection of any GFP fusion proteins without the needs of specific antibodies nor the generation of additional constructions. We have shown the feasibility and efficiency of this method to detect various proteins in Drosophila ovarian follicles such as nuclear proteins, proteins associated with endocytic vesicles, plasma membranes or nuclear envelopes. Lastly, we expressed this tool in Drosophila with the UAS/GAL4 system that enables spatiotemporal control of the protein detection.

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Section: Discussion: Critical Parameters and Troubleshootingmentioning

confidence: 99%

Section: Discussion: Critical Parameters and Troubleshootingmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

GFP-Tagged Protein Detection by Electron Microscopy Using a GBP-APEX Tool in Drosophila

Bernard

Jouette

Pioche−Durieu

et al. 2021

Front. Cell Dev. Biol.

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Improving endothelial cell junction integrity by diphenylmethanone derivatives at oxidative stress: A dual-action directly targeting caveolar caveolin-1

Zhang

Yuan

Sun

et al. 2022

Toxicology and Applied Pharmacology

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

et al. 2020

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Summary The peroxidase APEX2 has been used widely for proximity biotinylation and subsequent proteomics analyses. However, the poor membrane permeability of the biotin phenol substrate and the inhibitory effect of peroxide on the enzyme’s activity has hampered proximity labeling in certain cell culture systems and tissues. Here, we describe an APEX2 protocol that uses alternative peroxide and biotin phenol concentrations. The protocol permits robust proximity biotinylation in confluent epithelial cell cultures and may be applicable to other cell cultures and tissues. For complete details on the use and execution of this protocol, please refer to Tan et al. (2020) .

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Selective Visualization of Caveolae by TEM Using APEX2

Cited by 11 publications

References 22 publications

GFP-Tagged Protein Detection by Electron Microscopy Using a GBP-APEX Tool in Drosophila

GFP-Tagged Protein Detection by Electron Microscopy Using a GBP-APEX Tool in Drosophila

Improving endothelial cell junction integrity by diphenylmethanone derivatives at oxidative stress: A dual-action directly targeting caveolar caveolin-1

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

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