Proximity Staining Using Enzymatic Protein Tagging in Diplomonads

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 c...

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Autores principales:	Ásgeir Ástvaldsson, Kjell Hultenby, Staffan G. Svärd, Jon Jerlström-Hultqvist
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	APEX DAB Giardia proximity labeling Spironucleus salmonicida Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ba199507900941bea324ea4e85948d7e
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spelling	oai:doaj.org-article:ba199507900941bea324ea4e85948d7e2021-11-15T15:22:22ZProximity Staining Using Enzymatic Protein Tagging in Diplomonads10.1128/mSphereDirect.00153-192379-5042https://doaj.org/article/ba199507900941bea324ea4e85948d7e2019-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphereDirect.00153-19https://doaj.org/toc/2379-5042ABSTRACT 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.Ásgeir ÁstvaldssonKjell HultenbyStaffan G. SvärdJon Jerlström-HultqvistAmerican Society for MicrobiologyarticleAPEXDABGiardiaproximity labelingSpironucleus salmonicidaMicrobiologyQR1-502ENmSphere, Vol 4, Iss 2 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	APEX DAB Giardia proximity labeling Spironucleus salmonicida Microbiology QR1-502
spellingShingle	APEX DAB Giardia proximity labeling Spironucleus salmonicida Microbiology QR1-502 Ásgeir Ástvaldsson Kjell Hultenby Staffan G. Svärd Jon Jerlström-Hultqvist Proximity Staining Using Enzymatic Protein Tagging in Diplomonads
description	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.
format	article
author	Ásgeir Ástvaldsson Kjell Hultenby Staffan G. Svärd Jon Jerlström-Hultqvist
author_facet	Ásgeir Ástvaldsson Kjell Hultenby Staffan G. Svärd Jon Jerlström-Hultqvist
author_sort	Ásgeir Ástvaldsson
title	Proximity Staining Using Enzymatic Protein Tagging in Diplomonads
title_short	Proximity Staining Using Enzymatic Protein Tagging in Diplomonads
title_full	Proximity Staining Using Enzymatic Protein Tagging in Diplomonads
title_fullStr	Proximity Staining Using Enzymatic Protein Tagging in Diplomonads
title_full_unstemmed	Proximity Staining Using Enzymatic Protein Tagging in Diplomonads
title_sort	proximity staining using enzymatic protein tagging in diplomonads
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/ba199507900941bea324ea4e85948d7e
work_keys_str_mv	AT asgeirastvaldsson proximitystainingusingenzymaticproteintaggingindiplomonads AT kjellhultenby proximitystainingusingenzymaticproteintaggingindiplomonads AT staffangsvard proximitystainingusingenzymaticproteintaggingindiplomonads AT jonjerlstromhultqvist proximitystainingusingenzymaticproteintaggingindiplomonads
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Proximity Staining Using Enzymatic Protein Tagging in Diplomonads

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