Photo-convertible tagging for localization and dynamic analyses of low-expression proteins in filamentous fungi

• Published in: Fungal genetics and biology 2014-09, Vol.70, p.33-41
• Main Authors: Perez-de-Nanclares-Arregi, Elixabet, Etxebeste, Oier
• Format: Article
• Language: English
• Subjects: Asexual development; Aspergillus nidulans; Aspergillus nidulans - growth & development; Aspergillus nidulans - metabolism; Dendra2; Filamentous fungi; Fungal Proteins - genetics; Fungal Proteins - metabolism; Growth; Hyphae - growth & development; Hyphae - metabolism; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Photo-convertible tagging; Protein dynamics; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Transcription Factors - genetics; Transcription Factors - metabolism
• ISSN: 1087-1845; 1096-0937
• DOI: 10.1016/j.fgb.2014.06.006

•Tagging of Aspergillus nidulans proteins with the photoconvertible protein Dendra2.•Protocol adapted to track low expression proteins.•Dendra2 green-to-red conversion achieved using a standard mercury lamp, not a laser.•Validation of the method through localization studies of a suitable control.•Photo-conversion of protein pools from specific regions of interest. Photo-convertible fluorescent proteins (PCFPs) undergo a dramatic change in their excitation and emission spectra upon irradiation at specific wavelengths, thus rendering a different color. Dendra2 is a commercially available PCFP used to track the redistribution of proteins within cellular compartments, their life-time or interactions. Before photo-conversion Dendra2 exhibits green fluorescence, which becomes red after irradiation with either UV or blue lights. Multiple studies including Dendra2 as a molecular tool have been described in eukaryotes but not in filamentous fungi. Here we present a method to tag low-expression proteins from the filamentous fungus Aspergillus nidulans with Dendra2 and track their cellular dynamics. The regulator of asexual development FlbB was selected as control, a transcription factor that is expressed at low levels and can be used as a marker for the tip and nuclei of vegetative hyphae. This control provided us with a visual way to confirm the functionality of our genomic and plasmid constructs, since a non-functional FlbB protein renders a block in development and a characteristic aconidial phenotype. Our protocol combines standardized cloning and transformation procedures with the use of a mercury lamp microscope to convert and follow Dendra2 within cells. Hence, we present a rapid, simple and inexpensive method that makes tracking analysis of proteins that present technical difficulties to be followed feasible in filamentous fungi.