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14-3-3 Negatively Regulates Actin Filament Formation in the Deep Branching Eukaryote Giardia lamblia

Jana Krtková, Jennifer Xu, Marco Lalle, Melissa Steele-Ogus, Germain C. M. Alas, View ORCID ProfileDavid Sept, View ORCID ProfileAlexander R. Paredez
doi: https://doi.org/10.1101/142505
Jana Krtková
1Department of Biology, University of Washington, 24 Kincaid Hall, Seattle, WA 98195
2Department of Experimental Plant Biology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic
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Jennifer Xu
1Department of Biology, University of Washington, 24 Kincaid Hall, Seattle, WA 98195
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Marco Lalle
3Department of Infectious Diseases, Istituto Superiore di Sanità, viale Regina Elena 299, 00161 Rome, Italy
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Melissa Steele-Ogus
1Department of Biology, University of Washington, 24 Kincaid Hall, Seattle, WA 98195
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Germain C. M. Alas
1Department of Biology, University of Washington, 24 Kincaid Hall, Seattle, WA 98195
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David Sept
4Department of Biomedical Engineering and Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan 48109
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Alexander R. Paredez
1Department of Biology, University of Washington, 24 Kincaid Hall, Seattle, WA 98195
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  • ORCID record for Alexander R. Paredez
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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. The deep branching eukaryote Giardia lamblia lacks cofilin and all other canonical actin-binding proteins (ABPs), and 14-3-3 was identified as an actin-associated protein in Giardia, yet its role in actin regulation was unknown. Gl14-3-3 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, GlRac, modulates Gl14-3-3's association with actin, providing the first connection between GlRac and the actin cytoskeleton in Giardia. Giardia 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 negative regulator of actin filament formation.

Importance Giardia lacks canonical actin binding proteins. 14-3-3 was identified as an actin interactor but the significance of this interaction was unknown. Loss of 14-3-3 results in ectopic short actin filaments, indicating that 14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that 14-3-3 complex formation is in part phospho-regulated. We demonstrate that complex formation is downstream of Giardia’s sole Rho family GTPase, GlRac, this result provides the first mechanistic connection between GlRac and actin in Giardia. Native gels and overlay assays indicate intermediate proteins are required to support the interaction between 14-3-3 and actin suggesting that 14-3-3 is regulating multiple actin complexes. Overall, we find that 14-3-3 is a negative regulator of actin filament formation in Giardia.

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Posted May 26, 2017.
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14-3-3 Negatively Regulates Actin Filament Formation in the Deep Branching Eukaryote Giardia lamblia
Jana Krtková, Jennifer Xu, Marco Lalle, Melissa Steele-Ogus, Germain C. M. Alas, David Sept, Alexander R. Paredez
bioRxiv 142505; doi: https://doi.org/10.1101/142505
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14-3-3 Negatively Regulates Actin Filament Formation in the Deep Branching Eukaryote Giardia lamblia
Jana Krtková, Jennifer Xu, Marco Lalle, Melissa Steele-Ogus, Germain C. M. Alas, David Sept, Alexander R. Paredez
bioRxiv 142505; doi: https://doi.org/10.1101/142505

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