14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote <named-content content-type="genus-species">Giardia lamblia</named-content>

ABSTRACT The phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin; this function has been previously attributed to sequestration of phosphorylated cofilin. 14-3-3 was identified as an actin-associated protein in the deep-branching eukaryote Giardia lamblia; however, Giard...

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Autores principales:	Jana Krtková, Jennifer Xu, Marco Lalle, Melissa Steele-Ogus, Germain C. M. Alas, David Sept, Alexander R. Paredez
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	14-3-3 evolutionary cell biology Rho GTPase actin Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/acf6ec59a212463a8e8c93b3e2d371b0
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spelling	oai:doaj.org-article:acf6ec59a212463a8e8c93b3e2d371b02021-11-15T15:22:05Z14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote <named-content content-type="genus-species">Giardia lamblia</named-content>10.1128/mSphere.00248-172379-5042https://doaj.org/article/acf6ec59a212463a8e8c93b3e2d371b02017-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00248-17https://doaj.org/toc/2379-5042ABSTRACT The phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin; this function has been previously attributed to sequestration of phosphorylated cofilin. 14-3-3 was identified as an actin-associated protein in the deep-branching eukaryote Giardia lamblia; however, Giardia lacks cofilin and all other canonical actin-binding proteins (ABPs). Thus, the role of G. lamblia 14-3-3 (Gl-14-3-3) in actin regulation was unknown. Gl-14-3-3 depletion resulted in an overall disruption of actin organization characterized by ectopically distributed short actin filaments. Using phosphatase and kinase inhibitors, we demonstrated that actin phosphorylation correlated with destabilization of the actin network and increased complex formation with 14-3-3, while blocking actin phosphorylation stabilized actin filaments and attenuated complex formation. Giardia’s sole Rho family GTPase, Gl-Rac, modulates Gl-14-3-3’s association with actin, providing the first connection between Gl-Rac and the actin cytoskeleton in Giardia. Giardia actin (Gl-actin) contains two putative 14-3-3 binding motifs, one of which (S330) is conserved in mammalian actin. Mutation of these sites reduced, but did not completely disrupt, the association with 14-3-3. Native gels and overlay assays indicate that intermediate proteins are required to support complex formation between 14-3-3 and actin. Overall, our results support a role for 14-3-3 as a regulator of actin; however, the presence of multiple 14-3-3–actin complexes suggests a more complex regulatory relationship than might be expected for a minimalistic parasite. IMPORTANCE Giardia lacks canonical actin-binding proteins. Gl-14-3-3 was identified as an actin interactor, but the significance of this interaction was unknown. Loss of Gl-14-3-3 results in ectopic short actin filaments, indicating that Gl-14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that Gl-14-3-3 complex formation is in part phospho-regulated. We demonstrate that complex formation is downstream of Giardia’s sole Rho family GTPase, Gl-Rac. This result provides the first mechanistic connection between Gl-Rac and Gl-actin in Giardia. Native gels and overlay assays indicate intermediate proteins are required to support the interaction between Gl-14-3-3 and Gl-actin, suggesting that Gl-14-3-3 is regulating multiple Gl-actin complexes.Jana KrtkováJennifer XuMarco LalleMelissa Steele-OgusGermain C. M. AlasDavid SeptAlexander R. ParedezAmerican Society for Microbiologyarticle14-3-3evolutionary cell biologyRho GTPaseactinMicrobiologyQR1-502ENmSphere, Vol 2, Iss 5 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	14-3-3 evolutionary cell biology Rho GTPase actin Microbiology QR1-502
spellingShingle	14-3-3 evolutionary cell biology Rho GTPase actin Microbiology QR1-502 Jana Krtková Jennifer Xu Marco Lalle Melissa Steele-Ogus Germain C. M. Alas David Sept Alexander R. Paredez 14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote <named-content content-type="genus-species">Giardia lamblia</named-content>
description	ABSTRACT The phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin; this function has been previously attributed to sequestration of phosphorylated cofilin. 14-3-3 was identified as an actin-associated protein in the deep-branching eukaryote Giardia lamblia; however, Giardia lacks cofilin and all other canonical actin-binding proteins (ABPs). Thus, the role of G. lamblia 14-3-3 (Gl-14-3-3) in actin regulation was unknown. Gl-14-3-3 depletion resulted in an overall disruption of actin organization characterized by ectopically distributed short actin filaments. Using phosphatase and kinase inhibitors, we demonstrated that actin phosphorylation correlated with destabilization of the actin network and increased complex formation with 14-3-3, while blocking actin phosphorylation stabilized actin filaments and attenuated complex formation. Giardia’s sole Rho family GTPase, Gl-Rac, modulates Gl-14-3-3’s association with actin, providing the first connection between Gl-Rac and the actin cytoskeleton in Giardia. Giardia actin (Gl-actin) contains two putative 14-3-3 binding motifs, one of which (S330) is conserved in mammalian actin. Mutation of these sites reduced, but did not completely disrupt, the association with 14-3-3. Native gels and overlay assays indicate that intermediate proteins are required to support complex formation between 14-3-3 and actin. Overall, our results support a role for 14-3-3 as a regulator of actin; however, the presence of multiple 14-3-3–actin complexes suggests a more complex regulatory relationship than might be expected for a minimalistic parasite. IMPORTANCE Giardia lacks canonical actin-binding proteins. Gl-14-3-3 was identified as an actin interactor, but the significance of this interaction was unknown. Loss of Gl-14-3-3 results in ectopic short actin filaments, indicating that Gl-14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that Gl-14-3-3 complex formation is in part phospho-regulated. We demonstrate that complex formation is downstream of Giardia’s sole Rho family GTPase, Gl-Rac. This result provides the first mechanistic connection between Gl-Rac and Gl-actin in Giardia. Native gels and overlay assays indicate intermediate proteins are required to support the interaction between Gl-14-3-3 and Gl-actin, suggesting that Gl-14-3-3 is regulating multiple Gl-actin complexes.
format	article
author	Jana Krtková Jennifer Xu Marco Lalle Melissa Steele-Ogus Germain C. M. Alas David Sept Alexander R. Paredez
author_facet	Jana Krtková Jennifer Xu Marco Lalle Melissa Steele-Ogus Germain C. M. Alas David Sept Alexander R. Paredez
author_sort	Jana Krtková
title	14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote <named-content content-type="genus-species">Giardia lamblia</named-content>
title_short	14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote <named-content content-type="genus-species">Giardia lamblia</named-content>
title_full	14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote <named-content content-type="genus-species">Giardia lamblia</named-content>
title_fullStr	14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote <named-content content-type="genus-species">Giardia lamblia</named-content>
title_full_unstemmed	14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote <named-content content-type="genus-species">Giardia lamblia</named-content>
title_sort	14-3-3 regulates actin filament formation in the deep-branching eukaryote <named-content content-type="genus-species">giardia lamblia</named-content>
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/acf6ec59a212463a8e8c93b3e2d371b0
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14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote <named-content content-type="genus-species">Giardia lamblia</named-content>

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