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

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          Abstract

          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.

          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.

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          NIH Image to ImageJ: 25 years of image analysis

          For the past twenty five years the NIH family of imaging software, NIH Image and ImageJ have been pioneers as open tools for scientific image analysis. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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            NIH Image to ImageJ: 25 years of image analysis.

            For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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              Phosphate-binding tag, a new tool to visualize phosphorylated proteins.

              We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn(2+) or Mn(2+)) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn(2+)- and Mn(2+)-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn(2+)-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn(2+)-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn(2+)-Phos-tag SDS-PAGE).
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                Author and article information

                Contributors
                Role: Editor
                Journal
                mSphere
                mSphere
                msph
                msph
                mSphere
                mSphere
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                2379-5042
                13 September 2017
                Sep-Oct 2017
                : 2
                : 5
                : e00248-17
                Affiliations
                [a ]Department of Biology, University of Washington, Seattle, Washington, USA
                [b ]Department of Experimental Plant Biology, Faculty of Science, Charles University, Prague, Czech Republic
                [c ]Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy
                [d ]Department of Biomedical Engineering and Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, USA
                University at Buffalo
                Author notes
                Address correspondence to Alexander R. Paredez, aparedez@ 123456uw.edu .

                Citation Krtková J, Xu J, Lalle M, Steele-Ogus M, Alas GCM, Sept D, Paredez AR. 2017. 14-3-3 regulates actin filament formation in the deep-branching eukaryote Giardia lamblia. mSphere 2:e00248-17. https://doi.org/10.1128/mSphere.00248-17.

                Author information
                http://orcid.org/0000-0002-9197-9730
                http://orcid.org/0000-0001-5168-2150
                http://orcid.org/0000-0003-3719-2483
                http://orcid.org/0000-0002-9298-3264
                Article
                mSphere00248-17
                10.1128/mSphere.00248-17
                5597967
                28932813
                66a3ef79-9146-424c-ad95-3b399e2bd14f
                Copyright © 2017 Krtková et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                History
                : 26 May 2017
                : 21 August 2017
                Page count
                supplementary-material: 6, Figures: 8, Tables: 0, Equations: 0, References: 56, Pages: 16, Words: 10089
                Funding
                Funded by: Czech Ministry of Education, Youth, and Sports
                Award ID: NPUI (LO1417)
                Award Recipient : Jana Krtková
                Funded by: HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) https://doi.org/10.13039/100000060
                Award ID: 1R01AI110708-01A1
                Award Recipient : Alexander R. Paredez
                Categories
                Research Article
                Molecular Biology and Physiology
                Custom metadata
                September/October 2017

                14-3-3,evolutionary cell biology,rho gtpase,actin
                14-3-3, evolutionary cell biology, rho gtpase, actin

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