Abstract
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.
Highlights
In cell biology studies, protein localization is crucial to understand the cellular functions of proteins and for understanding the dysfunction of proteins in diseases
When the method is performed for the first time or to troubleshoot the experiment, we recommend optimizing each step beforehand, including interaction between GFP binding protein (GBP)-APEX2 and the GFP tagged proteins of interest (POI), enzymatic activity of APEX2 and quality of the sample preparation
Validating APEX2-GBP and GFP Expressions and Co-localization by Fluorescent Microscopy We first verified that the GBP-APEX2 construction colocalized with the GFP tagged POI
Summary
Protein localization is crucial to understand the cellular functions of proteins and for understanding the dysfunction of proteins in diseases. The technique used for this purpose was immunohistochemistry. It requires specific antibodies directed against each of the proteins of interest (POI). The production of good quality primary antibodies is random and labor-intensive. Once obtained, it remains a resource with limited availability. The advent of genetically targetable fluorescent protein tags has offered a possibility to bypass the requirement of antibody production against each POI. Fluorescent tags have further expanded the field of possibilities to in vivo localization in living tissues or cells.
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