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Identification of obscure yet conserved actin-associated proteins in Giardia lamblia.

ABSTRACT: Consistent with its proposed status as an early branching eukaryote, Giardia has the most divergent actin of any eukaryote and lacks core actin regulators. Although conserved actin-binding proteins are missing from Giardia, its actin is utilized similarly to that of other eukaryotes and functions in core cellular processes such as cellular organization, endocytosis, and cytokinesis. We set out to identify actin-binding proteins in Giardia using affinity purification coupled with mass spectroscopy (multidimensional protein identification technology [MudPIT]) and have identified >80 putative actin-binding proteins. Several of these have homology to conserved proteins known to complex with actin for functions in the nucleus and flagella. We validated localization and interaction for seven of these proteins, including 14-3-3, a known cytoskeletal regulator with a controversial relationship to actin. Our results indicate that although Giardia lacks canonical actin-binding proteins, there is a conserved set of actin-interacting proteins that are evolutionarily indispensable and perhaps represent some of the earliest functions of the actin cytoskeleton.

SUBMITTER: Paredez AR

PROVIDER: S-EPMC4054273 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Identification of obscure yet conserved actin-associated proteins in Giardia lamblia.

Paredez Alexander R AR Nayeri Arash A Xu Jennifer W JW Krtková Jana J Cande W Zacheus WZ

Eukaryotic cell 20140411 6

Consistent with its proposed status as an early branching eukaryote, Giardia has the most divergent actin of any eukaryote and lacks core actin regulators. Although conserved actin-binding proteins are missing from Giardia, its actin is utilized similarly to that of other eukaryotes and functions in core cellular processes such as cellular organization, endocytosis, and cytokinesis. We set out to identify actin-binding proteins in Giardia using affinity purification coupled with mass spectroscop ...[more]

PMID: 24728194

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Project description:The deep-branching protozoan parasite Giardia lamblia is the causative agent of the intestinal disease giardiasis. Consistent with its proposed evolutionary position, many pathways are minimalistic or divergent, including its actin cytoskeleton. Giardia is the only eukaryote known to lack all canonical actin-binding proteins. Previously, our lab identified a number of noncanonical Giardia lamblia actin (GlActin) interactors; however, these proteins appeared to interact only with monomeric or globular actin (G-actin) rather than with filamentous actin (F-actin). To identify F-actin interactors, we used a chemical cross-linker to preserve native interactions followed by an anti-GlActin antibody, protein A affinity chromatography, and liquid chromatography coupled to mass spectrometry. We found 46 putative actin interactors enriched under the conditions favoring F-actin. Data are available via ProteomeXchange with identifier PXD026067. None of the proteins identified contain known actin-interacting motifs, and many lacked conserved domains. Each potential interactor was then tagged with the fluorescent protein mNeonGreen and visualized in live cells. We categorized the proteins based on their primary localization; localizations included ventral disc, marginal plate, nuclei, flagella, plasma membrane, and internal membranes. One protein from each of the six categories was colocalized with GlActin using immunofluorescence microscopy. We also co-immunoprecipitated one protein from each category and confirmed three of the six potential interactions. Most of the localization patterns are consistent with previously demonstrated GlActin functions, but the ventral disc represents a new category of actin interactor localization. These results suggest a role for GlActin in ventral disc function, which has previously been controversial. IMPORTANCE Giardia lamblia is an intestinal parasite that colonizes the small intestine and causes diarrhea, which can lead to dehydration and malnutrition. Giardia actin (GlActin) has a conserved role in Giardia cells, despite being a highly divergent protein with none of the conserved regulators found in model organisms. Here, we identify and localize 46 interactors of polymerized actin. These putative interactors localize to a number of places in the cell, underlining GlActin's importance in multiple cellular processes. Surprisingly, eight of these proteins localize to the ventral disc, Giardia's host attachment organelle. Since host attachment is required for infection, proteins involved in this process are an appealing target for new drugs. While treatments for Giardia exist, drug resistance is becoming more common, resulting in a need for new treatments. Giardia and human systems are highly dissimilar, thus drugs specifically tailored to Giardia proteins would be less likely to have side effects.

Project description: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. IMPORTANCEGiardia 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.

Dataset Information

Identification of obscure yet conserved actin-associated proteins in Giardia lamblia.

Publications

Identification of obscure yet conserved actin-associated proteins in Giardia lamblia.

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