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      Static Clathrin Assemblies at the Peripheral Vacuole—Plasma Membrane Interface of the Parasitic Protozoan Giardia lamblia

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          Abstract

          Giardia lamblia is a parasitic protozoan that infects a wide range of vertebrate hosts including humans. Trophozoites are non-invasive but associate tightly with the enterocyte surface of the small intestine. This narrow ecological specialization entailed extensive morphological and functional adaptations during host-parasite co-evolution, including a distinctly polarized array of endocytic organelles termed peripheral vacuoles (PVs), which are confined to the dorsal cortical region exposed to the gut lumen and are in close proximity to the plasma membrane (PM). Here, we investigated the molecular consequences of these adaptations on the Giardia endocytic machinery and membrane coat complexes. Despite the absence of canonical clathrin coated vesicles in electron microscopy, Giardia possesses conserved PV-associated clathrin heavy chain ( GlCHC), dynamin-related protein ( GlDRP), and assembly polypeptide complex 2 (AP2) subunits, suggesting a novel function for GlCHC and its adaptors. We found that, in contrast to GFP-tagged AP2 subunits and DRP, CHC::GFP reporters have no detectable turnover in living cells, indicating fundamental differences in recruitment to the membrane and disassembly compared to previously characterized clathrin coats. Histochemical localization in electron tomography showed that these long-lived GlCHC assemblies localized at distinctive approximations between the plasma and PV membrane. A detailed protein interactome of GlCHC revealed all of the conserved factors in addition to novel or highly diverged proteins, including a putative clathrin light chain and lipid-binding proteins. Taken together, our data provide strong evidence for giardial CHC as a component of highly stable assemblies at PV-PM junctions that likely have a central role in organizing continuities between the PM and PV membranes for controlled sampling of the fluid environment. This suggests a novel function for CHC in Giardia and the extent of molecular remodeling of endocytosis in this species.

          Author Summary

          In canonical clathrin mediated endocytosis (CME) models, the concerted action of ca. 50 proteins mediates the uptake of extracellular components. The key player in this process is clathrin which coats transport intermediates called clathrin coated vesicles (CCV). The intestinal parasite Giardia lamblia has undergone extensive remodeling during colonization of the mammalian duodenum. Here, we report on unique features of this parasite’s endocytic system, consisting of fixed peripheral vacuoles (PV) in close proximity to the exposed plasma membrane (PM), with no discernible CCVs. Using state-of-the-art imaging strategies, we show that the surface of Giardia trophozoites is pock-marked with PM invaginations reaching to the underlying PV membrane. Co-immunoprecipitation and analysis of protein dynamics reveal that, in line with the absence of CCVs, giardial clathrin assemblies have no dynamic behavior. CHC still remains associated to AP2 and dynamin, both conserved dynamic CME components, and to a newly identified putative clathrin light chain. The emerging model calls for giardial clathrin organized into static cores surrounded by dynamic interaction partners, and most likely involved in the regulation of fusion between the PM and the PVs in a “kiss-and-flush”-like mechanism. This suggests that Giardia harbors a conceptually novel function for clathrin in endocytosis, which might be a consequence of host-parasite co-evolution.

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          Most cited references 88

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          Directed evolution of APEX2 for electron microscopy and proteomics

          APEX is an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. Because the limited sensitivity of APEX precludes applications requiring low APEX expression, we used yeast display evolution to improve its catalytic efficiency. Our evolved APEX2 is far more active in cells, enabling the superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins and the use of electron microscopy to resolve the sub-mitochondrial localization of calcium uptake regulatory protein MICU1.
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            Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy

            Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments 1 or require light and are difficult to use 2 . Here we report the development of a simple and robust EM genetic tag, called “APEX,” that is active in all cellular compartments and does not require light. APEX is a monomeric 28 kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins. We also fused APEX to the N- or C-terminus of the mitochondrial calcium uniporter (MCU), a newly identified channel whose topology is disputed 3,4 . MCU-APEX and APEX-MCU give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N-and C-termini of MCU face the matrix.
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              Tickets to ride: selecting cargo for clathrin-regulated internalization.

               Michael Traub (2009)
              Clathrin-mediated endocytosis oversees the constitutive packaging of selected membrane cargoes into transport vesicles that fuse with early endosomes. The process is responsive to activation of signalling receptors and ion channels, promptly clearing post-translationally tagged forms of cargo off the plasma membrane. To accommodate the diverse array of transmembrane proteins that are variably gathered into forming vesicles, a dedicated sorting machinery cooperates to ensure that non-competitive uptake from the cell surface occurs within minutes. Recent structural and functional data reveal remarkable plasticity in how disparate sorting signals are recognized by cargo-selective clathrin adaptors, such as AP-2. Cargo loading also seems to govern whether coats ultimately bud or dismantle abortively at the cell surface.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                20 July 2016
                July 2016
                : 12
                : 7
                Affiliations
                [1 ]Institute of Parasitology, University of Zurich, Zurich, Switzerland
                [2 ]Center for Microscopy and Image Analysis, University of Zurich, Zurich, Switzerland
                University of California Los Angeles, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JPZ LC SR AK CF ABH. Performed the experiments: JPZ LC SR AK. Analyzed the data: JPZ LC CF ABH. Contributed reagents/materials/analysis tools: AK. Wrote the paper: JPZ CF ABH.

                [¤]

                Current address: Department of Chemistry and Biochemistry, University of Berne, Berne, Switzerland

                Article
                PPATHOGENS-D-16-00456
                10.1371/journal.ppat.1005756
                4954726
                27438602
                © 2016 Zumthor et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 11, Tables: 0, Pages: 33
                Product
                Funding
                Funded by: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (CH)
                Award ID: 31-140803/1
                Award Recipient :
                Funded by: Forschungskredit der Universität Zürich
                Award ID: 55080507
                Award Recipient :
                Funded by: Forschungskredit der Universität Zürich
                Award ID: K-52201-05-01
                Award Recipient :
                Funded by: Forschungskredit der Universität Zürich
                Award ID: K-52201-06-01
                Award Recipient :
                This work was partially supported by grant nr. 31-140803/1, awarded to ABH by the Swiss National Science Fund ( www.snf.ch). JPZ received grant nrs. 55080507 followed by K-52201-05-01, and CF received grant nr. K-52201-06-01, all as fellowships from the Forschungskredit der Universität Zürich ( www.researchers.uzh.ch). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cell Membranes
                Biology and Life Sciences
                Parasitology
                Parasite Groups
                Apicomplexa
                Trophozoites
                Research and Analysis Methods
                Microscopy
                Light Microscopy
                Fluorescence Recovery after Photobleaching
                Biology and Life Sciences
                Organisms
                Protozoans
                Parasitic Protozoans
                Giardia
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cell Membranes
                Membrane Proteins
                Biology and Life Sciences
                Organisms
                Protozoans
                Parasitic Protozoans
                Giardia
                Giardia Lamblia
                Research and Analysis Methods
                Microscopy
                Electron Microscopy
                Transmission Electron Microscopy
                Custom metadata
                Data are freely retrievable using project accession number PXD003718 and project DOI 10.6019/PXD003718.

                Infectious disease & Microbiology

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