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, 4 (2)

Proximity Staining Using Enzymatic Protein Tagging in Diplomonads

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Proximity Staining Using Enzymatic Protein Tagging in Diplomonads

Ásgeir Ástvaldsson et al. mSphere.

Abstract

The diplomonads are a group of understudied eukaryotic flagellates whose most prominent member is the human pathogen Giardia intestinalis Methods commonly used in other eukaryotic model systems often require special optimization in diplomonads due to the highly derived character of their cell biology. We have optimized a proximity labeling protocol using pea ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM) to enable the study of ultrastructural cellular details in diplomonads. Currently available TEM-compatible tags require light-induced activation (1, 2) or are inactive in many cellular compartments (3), while ascorbate peroxidase has not been shown to have those limitations. Here, we have optimized the in vivo activities of two versions of pea ascorbate peroxidase (APXW41F and APEX) using the diplomonad fish parasite Spironucleus salmonicida, a relative of G. intestinalis We exploited the well-known peroxidase substrates, Amplex UltraRed and 3,3'-diaminobenzidine (DAB), to validate the activity of the two tags and argue that APEX is the most stable version to use in Spironucleus salmonicida Next, we fused APEX to proteins with established localization to evaluate the activity of APEX in different cellular compartments of the diplomonad cell and used Amplex UltraRed as well as antibodies along with superresolution microscopy to confirm the protein-APEX localization. The ultrastructural details of protein-APEX fusions were determined by TEM, and we observed marker activity in all cellular compartments tested when using the DAB substrate. Finally, we show that the optimized conditions established for S. salmonicida can be used in the related diplomonad G. intestinalis IMPORTANCE The function of many proteins is intrinsically related to their cellular location. Novel methods for ascertainment of the ultrastructural location of proteins have been introduced in recent years, but their implementation in protists has so far not been readily realized. Here, we present an optimized proximity labeling protocol using the APEX system in the salmon pathogen Spironucleus salmonicida This protocol was also applicable to the human pathogen Giardia intestinalis Both organisms required extraneous addition of hemin to the growth medium to enable detectable peroxidase activity. Further, we saw no inherent limitation in labeling efficiency coupled to the cellular compartment, as evident with some other proximity labeling systems. We anticipate that the APEX proximity labeling system might offer a great resource to establish the ultrastructural localization of proteins across genetically tractable protists but might require organism-specific labeling conditions.

Keywords: APEX; DAB; Giardia; Spironucleus salmonicida; proximity labeling.

Figures

FIG 1
FIG 1
H2O2 titrations. S. salmonicida transfectants expressing Anx5-APEX or Anx5-APX were grown in LYI medium supplemented with 100 µM hemin. Transfectants were washed with HBSS-G and spotted on a poly-lysine-coated microscopy slide. (A) Cells were fixed with 2% glutaraldehyde in 100 mM cacodylate buffer with 2 mM CaCl2 before being reacted with 0.5 mg/ml DAB and 0 to 3 mM H2O2 for 15 min. Cells were washed with cacodylate buffer and PBS, mounted with VectaShield, and viewed in a phase-contrast microscope. Cells with white deposits in the anterior of the cells are positive for Anx5-APEX or Axn5-APX staining. (B) Cells were fixed with 2% paraformaldehyde in PBS and reacted with 50 µM Amplex UltraRed and 0 to 8.5 mM H2O2 for 30 min. The cells were washed extensively, mounted with VectaShield with DAPI, and imaged in a fluorescence microscope. Cells with red deposits in the anterior of the cells are positive for Anx5-APEX or Axn5-APX staining. Scale bars = 10 µm.
FIG 2
FIG 2
Superresolution microscopy (SIM) of S. salmonicida (A to H) and G. intestinalis (I and J) transfectants. Transfectants expressing APEX-V5 were grown in LYI medium (S. salmonicida) and TYDK medium (G. intestinalis) supplemented with 100 µM hemin. Cultures were fixed with 2% paraformaldehyde in PBS and treated with 50 µM Amplex UltraRed (Red) and 200 µM H2O2 for 30 min. V5 epitope (green) was detected using a primary monoclonal mouse anti-V5 antibody and a secondary polyclonal goat anti-mouse Alexa Fluor 488-conjugated antibody. The cells were stained with 2 µg/ml DAPI solution (blue) for 10 min and mounted with VectaShield. Imaging was done by a Zeiss LSM710 microscope with a SIM module. Scale bars = 1 µm.
FIG 3
FIG 3
SIM and TEM images of two previously uncharacterized proteins. Transfectants expressing 10316-APEX-V5 and 12178-APEX-V5 were grown in LYI medium supplemented with 100 µM hemin. (A and B) SIM images. Transfectants were fixed with 2% paraformaldehyde in PBS and treated with 50 µM Amplex UltraRed (red) and 200 µM H2O2 for 30 min. V5 epitope (green) was detected using a primary monoclonal mouse anti-V5 antibody and a secondary polyclonal goat anti-mouse Alexa Fluor 488-conjugated antibody. The cells were stained with 2 µg/ml DAPI solution (blue) for 10 min and mounted with VectaShield. Imaging was done by a Zeiss LSM710 microscope with a SIM module. Scale bars = 1 µm. (C to E) TEM images. Transfectants were fixed with 2% glutaraldehyde in 100 mM cacodylate buffer with 2 mM CaCl2 and labeled with 0.5 mg/ml DAB and 300 µM H2O2 for 15 min. Samples are postfixed in 2% osmium tetroxide in 0.1 M phosphate buffer following dehydration in ethanol and acetone. Samples were embedded in LX-112 resin, and 50- to 60-nm sections were cut. Samples were viewed at 80 kV on a Hitachi HT 7700 lens and imaged with a Veleta camera. APEX-catalyzed DAB deposition appears as higher contrast areas at the site of the tagged protein. Some areas with labeling are indicated by arrows. Abbreviation: N nucleus. Scale bars are indicated in the images.
FIG 4
FIG 4
Transmission electron microscopy (TEM) images of DAB-stained S. salmonicida transfectants and WT. (A to C) Acid phosphatase-APEX; (D to F) BiP-APEX; (G to I) fibrillarin-APEX; (J to L) IscU-APEX; (M to O) WT. Cultures were grown in LYI medium containing 100 µM hemin. Cells are fixed with 2% glutaraldehyde in 100 mM cacodylate buffer with 2 mM CaCl2 and treated with 0.5 mg/ml DAB and 200 µM H2O2 for 15 min. Samples were postfixed in 2% osmium tetroxide in 0.1 M phosphate buffer following dehydration in ethanol and acetone. Samples were embedded in LX-112 resin, and 50- to 60-nm sections were cut. Samples were viewed at 80 kV on a Hitachi HT 7700 lens and imaged with a Veleta camera. APEX-catalyzed DAB deposition appears as higher-contrast areas at the site of the tagged protein. Some areas with labeling are indicated by arrows. N, nucleus; SL, striated lamina; fl, flagella; H, hydrogenosome; ER, endoplasmic reticulum. Scale bars are 2 µm in panels B, G, and J; 1 µm in A, D, H, M, and N; 500 nm in C, E, F, I, and O; and 200 nm in K and L.
FIG 5
FIG 5
Transmission electron microscopy (TEM) images of DAB-stained G. intestinalis transfectants and WT. (A to C) Alpha 14-giardin-APEX; (D to E) SALP-1-APEX; (G to I) IscU-APEX; (J to L) WT. Cultures were grown in TYDK medium supplemented with 100 µM hemin. The cells were fixed with 2% glutaraldehyde in 100 mM cacodylate buffer with 2 mM CaCl2 and labeled with 0.5 mg/ml DAB and 300 µM H2O2 for 15 min. Samples were postfixed in 2% osmium tetroxide in 0.1 M phosphate buffer following dehydration in ethanol and acetone. Samples were embedded in LX-112 resin, and 50- to 60-nm sections were cut. Samples were viewed at 80 kV on a Hitachi HT 7700 lens and imaged with a Veleta camera. Scale bars are 2 µm in panels D and K; 1 µm in A, B, C, E, F, H, J, and L; and 500 nm in G and I.

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