Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

Abstract

Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research, and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies have significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analysing the role of POIs in dynamic cellular processes, such as cell migration or cell division, would benefit from more-direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we have selected a number of short-tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POI binding and manipulation in living cells. Using Drosophila, we also find that single short tags can be used for POI manipulation in vivo.

Keywords: In vivo; Nanobodies; Peptide binders; Protein manipulation; Small tag.

Fig. 1.

Schematic representation of the constructs. The transcriptional elements [enhancer, promoter and poly (A) adenylation] of the different mammalian expression vectors are depicted as grey filled boxes. The different protein-coding modules are represented as coloured block arrows, while the resulting fusion protein is depicted as a solid orange arrow below the modules.

Fig. 2.

Intracellular binding of anti-GCN4 scFv (SunTag system). Confocal images of HeLa cells transiently transfected with (A) anti-GCN4_scFv_GFP alone or with (B-E) the combination of anti-GCN4_scFv_GFP and (B) mito_mCherry_SunTag, (C) mCherry_SunTag_H2B, (D) mCherryCD8_OLLAS_SunTag or (E) vimentin_mCherry_SunTag. The first column represents the GFP channel (green), the second column is the mCherry channel (red), the third column is the overlay of the two channels, showing the colocalization (indicated in yellow) of the anti-GCN4_scFv with the respective mitochondrial (B), nuclear (C), membrane (D) and filament (E) baits; the fourth column represents the nuclear Hoechst staining (blue) and the fifth column is the merge of all three channels. Scale bars: 15 µm. Images were taken 24 h post-transfection. Transfected constructs are indicated at the left of each row and the single and merge channels are indicated at the top of the respective columns. The figures are from a representative experiment, performed at least three times.

Fig. 3.

Intracellular binding of anti-gp41 nanobody (MoonTag system). Confocal images of HeLa cells 24 h after transient transfection with constructs indicated at the left of each row and imaged for the channels indicated at the top of each row. The first column represents the GFP channel (green), the second column is the mCherry channel (red), the third column is the overlay of the two channels, showing the colocalization (indicated in yellow) of the anti-gp41_Nb_GFP with the respective mitochondrial (B), nuclear (C), and filament (D) baits; the fourth column represents the nuclear Hoechst staining (blue) and the fifth column is the merge of all three channels. Scale bars: 15 µm. The figures are from a representative experiment, performed at least three times.

Fig. 4.

Intracellular binding of anti-HA_fb_GFP (HA system). Confocal images of HeLa cells 24 h after transient transfection with constructs indicated at the left of each row and imaged for the channels indicated at the top of each row. The first column represents the GFP channel (green), the second column is the mCherry channel (red), the third column is the overlay of the two channels, showing the colocalization (indicated in yellow) of the anti-HA_fb_GFP with the respective mitochondrial (B), nuclear (C), and filament (D) baits; the fourth column represents the nuclear Hoechst staining (blue) and the fifth column is the merge of all three channels. Scale bars: 15 µm. The figures are from a representative experiment, performed at least three times.

Fig. 5.

Intracellular binding of anti-ALFA_Nb_sfGFP (ALFA tag system). Confocal images of HeLa cells 24 h after transient transfection with constructs indicated at the left of each row and imaged for the channels indicated at the top of each row. The first column represents the GFP channel (green), the second column is the mCherry channel (red), the third column is the overlay of the two channels, showing the colocalization (indicated in yellow) of the anti-ALFA_Nb_sfGFP with the respective mitochondrial (B), nuclear (C) and filament (D) baits; the fourth column represents the nuclear Hoechst staining (blue) and the fifth column is the merge of all three channels. Scale bars: 15 µm. The figures are from a representative experiment, performed at least three times.

Fig. 6.

Intracellular binding of anti-HA_fb_GFP (HA system) in vivo. (A-C) Confocal images of salivary glands from 3rd instar Drosophila larvae expressing the UAS constructs indicated at the left of each row using a brk-GAL4 driver. Single and merge channels are indicated at the top of the respective panel. Nuclei are visualized by Hoechst staining (blue). Scale bars: 50 µm.

Fig. 7.

Manipulation of HA-tagged proteins by deGradHA in vivo. (A-F) Side (top row) or frontal (bottom row) views of Drosophila adult eyes carrying the eye-specific GMR-GAL4 driver alone (A), or in combination with the UAS constructs indicated at the top (B-F). Scale bars: 100 µm.

Fig. 8.

Manipulation of endogenously HA-tagged proteins by deGradHA. (A-K) Distribution of TkvHAeGFP (schematically depicted in A) visualized by immunostaining with a HA antibody (B,D) or GFP-autofluorescence (C,E), and pMad (F-H) and Sal (I-K) immunostaining in 3rd instar Drosophila wing imaginal discs of the indicated genotypes. The expression domain of the ap-GAL4 driver is schematically shown in the inset in E. Plots below each panel depict relative fluorescent intensity of ventral (control, blue) and dorsal (experimental, orange) cells along the AP axis of the wing pouch (dashed lines in B indicate areas used for quantification). Owing to the low expression of Tkv in the medial pouch, effects of the deGrad tools are better visible in lateral regions. All larvae carry the engineered TkvHAeGFP allele over a chromosomal deletion of the tkv locus. Scale bars: 50 µm.