ABSTRACT
Understanding cellular ultrastructure is tightly bound to microscopic resolution and the ability to identify individual components at that resolution. Expansion microscopy has revolutionised this topic. Here we present and compare two protocols of ultrastructure expansion microscopy that allow for 4.5-fold mostly isotropic expansion and the use of antibodies, metabolic labelling, and DNA stains to demarcate individual regions such as the endoplasmic reticulum, the nuclei, the peripheral endocytic compartments as well as the ventral disc and the cytoskeleton in Giardia lamblia. We present an optimised, shortened, and modular protocol that can be swiftly adjusted to the investigators needs in this important protozoan model organism.
ABSTRACT
Functional gene and protein characterizations in parasitic protists are often limited by their genetic tractability. Despite the development of CRISPR-Cas9-derived or inspired approaches for a handful of protist parasites, the overall genetic tractability of these organisms remains limited. The intestinal parasite Giardia lamblia is one such species, with the added challenge of a paucity of reliable selection markers. To address this limitation, we tested the feasibility of using Nourseothricin as an effective selection agent in Giardia. Here, we report that axenically-grown WB Giardia cells are sensitive to Nourseothricin and that engineering expression of the streptothricin acetyltransferase (SAT-1) gene from Streptomyces rochei in transgenic parasites confers resistance to this antibiotic. Furthermore, we determine that SAT-1-expressing parasites are cross-resistant neither to Neomycin nor Puromycin, which are widely used to select for transgenic parasites. Consequently, we show that Nourseothricin can be used in sequential combination with both Neomycin and Puromycin to select for dual transfection events. This work increases the number of reliable selection agents and markers for Giardia genetic manipulation, expanding the limited molecular toolbox for this species of global medical importance.
Subject(s)
Giardia lamblia , Streptothricins , Giardia lamblia/genetics , Giardia lamblia/drug effects , Streptothricins/pharmacology , Acetyltransferases/genetics , Drug Resistance/genetics , Streptomyces/genetics , Streptomyces/drug effects , Antiprotozoal Agents/pharmacology , Organisms, Genetically Modified , CRISPR-Cas SystemsABSTRACT
Unconventional protein secretion (UPS) plays important roles in cell physiology. In contrast to canonical secretory routes, UPS does not generally require secretory signal sequences and often bypasses secretory compartments such as the ER and the Golgi apparatus. Giardia lamblia is a protist parasite with reduced subcellular complexity which releases several proteins, some of them virulence factors, without canonical secretory signals. This implicates UPS at the parasite-host interface. No dedicated machinery nor mechanism(s) for UPS in Giardia are currently known, although speculations on the involvement of endocytic organelles called PV/PECs, have been put forth. To begin to address the question of whether PV/PECs are implicated in virulence-associated UPS and to define the composition of molecular machinery involved in protein release, we employed affinity purification and mass spectrometry, coupled to microscopy-based subcellular localization and signal correlation quantification to investigate the interactomes of 11 reported unconventionally secreted proteins, all predicted to be cytosolic. A subset of these are associated with PV/PECs. Extended and validated interactomes point to a core PV/PECs-associated UPS machinery, which includes uncharacterized and Giardia-specific coiled-coil proteins and NEK kinases. Finally, a subset of the alpha-giardin protein family was enriched in all PV/PECs-associated protein interactomes, highlighting a previously unappreciated role for these proteins at PV/PECs and in UPS. Taken together, our results provide the first characterization of a virulence-associated UPS protein complex in Giardia lamblia at PV/PECs, suggesting a novel link between these primarily endocytic and feeding organelles and UPS at the parasite-host interface.
Subject(s)
Giardia lamblia , Giardiasis , Parasites , Animals , Giardia lamblia/genetics , Giardiasis/parasitology , Protein Transport , Proteins , Protozoan Proteins/genetics , Protozoan Proteins/metabolismABSTRACT
BACKGROUND: Giardia lamblia, a parasitic protist of the Metamonada supergroup, has evolved one of the most diverged endocytic compartment systems investigated so far. Peripheral endocytic compartments, currently known as peripheral vesicles or vacuoles (PVs), perform bulk uptake of fluid phase material which is then digested and sorted either to the cell cytosol or back to the extracellular space. RESULTS: Here, we present a quantitative morphological characterization of these organelles using volumetric electron microscopy and super-resolution microscopy (SRM). We defined a morphological classification for the heterogenous population of PVs and performed a comparative analysis of PVs and endosome-like organelles in representatives of phylogenetically related taxa, Spironucleus spp. and Tritrichomonas foetus. To investigate the as-yet insufficiently understood connection between PVs and clathrin assemblies in G. lamblia, we further performed an in-depth search for two key elements of the endocytic machinery, clathrin heavy chain (CHC) and clathrin light chain (CLC), across different lineages in Metamonada. Our data point to the loss of a bona fide CLC in the last Fornicata common ancestor (LFCA) with the emergence of a protein analogous to CLC (GlACLC) in the Giardia genus. Finally, the location of clathrin in the various compartments was quantified. CONCLUSIONS: Taken together, this provides the first comprehensive nanometric view of Giardia's endocytic system architecture and sheds light on the evolution of GlACLC analogues in the Fornicata supergroup and, specific to Giardia, as a possible adaptation to the formation and maintenance of stable clathrin assemblies at PVs.
Subject(s)
Giardia lamblia , Clathrin/metabolism , Clathrin Heavy Chains/genetics , Clathrin Heavy Chains/metabolism , Clathrin Light Chains/metabolism , Endocytosis , Giardia lamblia/genetics , Giardia lamblia/metabolism , PhylogenyABSTRACT
BACKGROUND: Comparing a parasitic lineage to its free-living relatives is a powerful way to understand how that evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardia's pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis, and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species. RESULTS: We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36 and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the endoplasmic reticulum and, for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. CONCLUSIONS: Our findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, associates to multiple cellular locations, and presents changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.
Subject(s)
Endosomal Sorting Complexes Required for Transport , Giardia lamblia , Biological Evolution , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Endosomes/metabolism , Giardia lamblia/genetics , Giardia lamblia/metabolism , Protein TransportABSTRACT
Protein secretion in eukaryotic cells is a well-studied process, which has been known for decades and is dealt with by any standard cell biology textbook. However, over the past 20 years, several studies led to the realization that protein secretion as a process might not be as uniform among different cargos as once thought. While in classic canonical secretion proteins carry a signal sequence, the secretory or surface proteome of several organisms demonstrated a lack of such signals in several secreted proteins. Other proteins were found to indeed carry a leader sequence, but simply circumvent the Golgi apparatus, which in canonical secretion is generally responsible for the modification and sorting of secretory proteins after their passage through the endoplasmic reticulum (ER). These alternative mechanisms of protein translocation to, or across, the plasma membrane were collectively termed "unconventional protein secretion" (UPS). To date, many research groups have studied UPS in their respective model organism of choice, with surprising reports on the proportion of unconventionally secreted proteins and their crucial roles for the cell and survival of the organism. Involved in processes such as immune responses and cell proliferation, and including far more different cargo proteins in different organisms than anyone had expected, unconventional secretion does not seem so unconventional after all. Alongside mammalian cells, much work on this topic has been done on protist parasites, including genera Leishmania, Trypanosoma, Plasmodium, Trichomonas, Giardia, and Entamoeba. Studies on protein secretion have mainly focused on parasite-derived virulence factors as a main source of pathogenicity for hosts. Given their need to secrete a variety of substrates, which may not be compatible with canonical secretion pathways, the study of mechanisms for alternative secretion pathways is particularly interesting in protist parasites. In this review, we provide an overview on the current status of knowledge on UPS in parasitic protists preceded by a brief overview of UPS in the mammalian cell model with a focus on IL-1ß and FGF-2 as paradigmatic UPS substrates.
ABSTRACT
Phosphorylated derivatives of phosphatidylinositol (PIPs) are key membrane lipid residues involved in clathrin-mediated endocytosis (CME). CME relies on PIP species PI(4,5)P2 to mark endocytic sites at the plasma membrane (PM) associated to clathrin-coated vesicle (CCV) formation. The highly diverged parasitic protist Giardia lamblia presents disordered and static clathrin assemblies at PM invaginations, contacting specialized endocytic organelles called peripheral vacuoles (PVs). The role for clathrin assemblies in fluid phase uptake and their link to internal membranes via PIP-binding adaptors is unknown. Here we provide evidence for a robust link between clathrin assemblies and fluid-phase uptake in G. lamblia mediated by proteins carrying predicted PX, FYVE and NECAP1 PIP-binding modules. We show that chemical and genetic perturbation of PIP-residue binding and turnover elicits novel uptake and organelle-morphology phenotypes. A combination of co-immunoprecipitation and in silico analysis techniques expands the initial PIP-binding network with addition of new members. Our data indicate that, despite the partial conservation of lipid markers and protein cohorts known to play important roles in dynamic endocytic events in well-characterized model systems, the Giardia lineage presents a strikingly divergent clathrin-centered network. This includes several PIP-binding modules, often associated to domains of currently unknown function that shape and modulate fluid-phase uptake at PVs.
Subject(s)
Giardia lamblia/genetics , Giardia lamblia/metabolism , Phosphatidylinositols/metabolism , Biological Transport , Carrier Proteins/metabolism , Cell Membrane/metabolism , Clathrin/metabolism , Clathrin-Coated Vesicles , Endocytosis/physiology , Giardia lamblia/parasitology , Giardiasis/metabolism , Vacuoles/metabolismABSTRACT
Phosphoinositides (or phosphatidylinositol phosphates, PIPs) are low-abundance membrane phospholipids that act, in conjunction with their binding partners, as important constitutive signals defining biochemical organelle identity as well as membrane trafficking and signal transduction at eukaryotic cellular membranes. In this review, we present roles for PIP residues and PIP-binding proteins in endocytosis and autophagy in protist parasites such as Trypanosoma brucei, Toxoplasma gondii, Plasmodium falciparum, Entamoeba histolytica, and Giardia lamblia. Molecular parasitologists with an interest in comparative cell and molecular biology of membrane trafficking in protist lineages beyond the phylum Apicomplexa, along with cell and molecular biologists generally interested in the diversification of membrane trafficking in eukaryotes, will hopefully find this review to be a useful resource.
Subject(s)
Apicomplexa/metabolism , Carrier Proteins/metabolism , Phosphatidylinositols/metabolism , Autophagy , Endocytosis , Protein Transport , Signal TransductionABSTRACT
Over the past years, the subcellular organization of the Excavata member Giardia lamblia (syn. duodenalis, intestinalis) has been investigated in considerable detail. There are several reasons for this endeavour which go beyond this parasite's medical importance and are mostly concerned with its reduced subcellular complexity and debated evolutionary status. One may say that simplification has emerged as a paradigm for the evolution of Giardia's subcellular architecture. However, a complete appreciation of the evolutionary and ecological significance of this phenomenon is far from complete. In this chapter, we present and discuss the most recent data on the main trafficking pathways in G. lamblia which include endo- and exo-cytosis, organellar import and function. We provide perspectives on open questions concerning organelle replication and inheritance and include a technical outlook on methods and approaches to genetic manipulations in G. lamblia. A better understanding of G. lamblia subcellular organization at the morphological and molecular level complements any effort aimed at elucidating this parasitic species' evolutionary status and could provide us with the basis for novel strategies to interfere with parasite transmission and/or pathogenesis.
Subject(s)
Giardia lamblia/metabolism , Giardiasis/parasitology , Protozoan Proteins/metabolism , Giardiasis/transmission , Protein TransportSubject(s)
Giardia lamblia/pathogenicity , Animals , Diarrhea/etiology , Diarrhea/parasitology , Diarrhea/prevention & control , Giardia lamblia/physiology , Giardia lamblia/ultrastructure , Giardiasis/etiology , Giardiasis/parasitology , Giardiasis/prevention & control , Humans , Protozoan Vaccines/isolation & purification , Protozoan Vaccines/pharmacology , Vacuoles/physiology , Vacuoles/ultrastructureABSTRACT
The genome of the protozoan parasite Giardia lamblia is organized in two diploid nuclei, which has so far precluded complete analysis of gene function. Here we use a previously developed Cre/loxP-based knock-out and selection marker salvage strategy in the human-derived isolate WB-C6 to eliminate all four copies of the Cyst-Wall-Protein-1 locus (CWP1). Because these loci are silenced in proliferating trophozoites and highly expressed only in encysting cells, CWP1 ablation allows functional characterization of a conditional phenotype in parasites induced to encyst. We show that encysting Δcwp1 cells are unable to establish the stage-regulated trafficking machinery with Golgi-like encystation-specific vesicles required for cyst-wall formation but show morphological hallmarks of cyst development and karyokinesis. This 'pseudocyst' phenotype is rescued by transfection of Δcwp1 cells with an episomally maintained CWP1 expression vector. Genome editing in genera Giardia and Trypanosoma are the only reported examples addressing questions on pathogen transmission within the Excavata supergroup.
Subject(s)
Biosynthetic Pathways , Giardia lamblia/metabolism , Golgi Apparatus/metabolism , Organelle Biogenesis , Protozoan Proteins/metabolism , Cell Differentiation , Cell Membrane/metabolism , Cell Survival , Cell Wall/metabolism , Cell Wall/ultrastructure , Cytoplasmic Vesicles/metabolism , Genes, Reporter , Genetic Complementation Test , Giardia lamblia/cytology , Humans , Mutation/genetics , SolubilityABSTRACT
Protozoan parasites of the genus Giardia are highly prevalent globally, and infect a wide range of vertebrate hosts including humans, with proliferation and pathology restricted to the small intestine. This narrow ecological specialization entailed extensive structural and functional adaptations during host-parasite co-evolution. An example is the streamlined mitosomal proteome with iron-sulphur protein maturation as the only biochemical pathway clearly associated with this organelle. Here, we applied techniques in microscopy and protein biochemistry to investigate the mitosomal membrane proteome in association to mitosome homeostasis. Live cell imaging revealed a highly immobilized array of 30-40 physically distinct mitosome organelles in trophozoites. We provide direct evidence for the single giardial dynamin-related protein as a contributor to mitosomal morphogenesis and homeostasis. To overcome inherent limitations that have hitherto severely hampered the characterization of these unique organelles we applied a novel interaction-based proteome discovery strategy using forward and reverse protein co-immunoprecipitation. This allowed generation of organelle proteome data strictly in a protein-protein interaction context. We built an initial Tom40-centered outer membrane interactome by co-immunoprecipitation experiments, identifying small GTPases, factors with dual mitosome and endoplasmic reticulum (ER) distribution, as well as novel matrix proteins. Through iterative expansion of this protein-protein interaction network, we were able to i) significantly extend this interaction-based mitosomal proteome to include other membrane-associated proteins with possible roles in mitosome morphogenesis and connection to other subcellular compartments, and ii) identify novel matrix proteins which may shed light on mitosome-associated metabolic functions other than Fe-S cluster biogenesis. Functional analysis also revealed conceptual conservation of protein translocation despite the massive divergence and reduction of protein import machinery in Giardia mitosomes.
Subject(s)
Giardia lamblia/physiology , Giardia lamblia/ultrastructure , Homeostasis/physiology , Protozoan Proteins/metabolism , Fluorescent Antibody Technique , Immunoblotting , Immunoprecipitation , Mass Spectrometry , Microscopy, Electron, Transmission , Organelles , Organisms, Genetically Modified , Polymerase Chain Reaction , Trophozoites/physiology , Trophozoites/ultrastructureABSTRACT
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.
Subject(s)
Cell Membrane/metabolism , Clathrin/metabolism , Endocytosis/physiology , Giardia lamblia/metabolism , Vacuoles/metabolism , Cell Membrane/ultrastructure , Fluorescent Antibody Technique , Giardia lamblia/ultrastructure , Immunoblotting , Immunoprecipitation , Mass Spectrometry , Microscopy, Confocal , Microscopy, Electron , Vacuoles/ultrastructureABSTRACT
Determining signalling pathways that regulate pancreatic regeneration following pancreatitis is critical for implementing therapeutic interventions. In this study we elucidated the molecular mechanisms underlying the effects of transforming growth factor-ß (TGFß) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFß receptor II (TGFß-RII) using a Cre-LoxP system under the control of pancreas transcription factor 1a (PTF1a) promoter, specific for the pancreatic epithelium, and evaluated the molecular and cellular changes in a mouse model of cerulein-induced pancreatitis. We show that TGFß-RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFß-RII signalling not only restricts acinar cell replication during the regenerative phase of the disease but also limits ADM formation in vivo and in vitro in a cell-autonomous manner. Analyses of molecular mechanisms underlying the observed phenotype revealed that TGFß-RII signalling stimulates the expression of cyclin-dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFß-RII ablation in epithelial cells resulted in increased infiltration of inflammatory cells in the early phases of pancreatitis and increased activation of pancreatic stellate cells in the later stages of pancreatitis, thus highlighting a TGFß-based crosstalk between epithelial and stromal cells regulating the development of pancreatic inflammation and fibrosis. Collectively, our data not only contribute to clarifying the cellular processes governing pancreatic tissue regeneration, but also emphasize the conserved role of TGFß as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer.
Subject(s)
Acinar Cells/pathology , Pancreas/pathology , Pancreatitis/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Amylases/metabolism , Animals , Carcinoma, Pancreatic Ductal/pathology , Cell Cycle Checkpoints/physiology , Cell Proliferation/physiology , Cell Transformation, Neoplastic/pathology , Cells, Cultured , Ceruletide/toxicity , Epithelial Cells/pathology , Fibrosis/pathology , Irritants/toxicity , Lipase/metabolism , Male , Metaplasia/pathology , Mice, Knockout , Mice, Transgenic , Pancreas/enzymology , Pancreatic Neoplasms/pathology , Pancreatitis/enzymology , Receptor, Transforming Growth Factor-beta Type IIABSTRACT
The bacteriophage-derived Cre/loxP system is a valuable tool that has revolutionised genetic and cell biological research in many organisms. We implemented this system in the intestinal parasite Giardia lamblia, an evolutionarily diverged protozoan whose binucleate and tetraploid genome organisation severely limits the application of reverse genetic approaches. We show that Cre-recombinase is functionally expressed in G. lamblia and demonstrate "recycling" of selectable markers. Providing the means for more complex and versatile genetic modifications, this technique massively increases the scope of functional investigations in G. lamblia and other protozoa with similar limitations with respect to genetic manipulation.
Subject(s)
Genetics, Microbial/methods , Giardia lamblia/genetics , Molecular Biology/methods , Recombination, Genetic , Bacteriophages/enzymology , Bacteriophages/genetics , Gene Expression , Integrases/genetics , Integrases/metabolism , TetraploidyABSTRACT
Giardia lamblia is an intestinal protozoan parasite required to survive in the environment in order to be transmitted to a new host. To ensure parasite survival, flagellated trophozoites colonizing the small intestine differentiate into non-motile environmentally-resistant cysts which are then shed in the environment. This cell differentiation process called encystation is characterized by significant morphological remodeling which includes secretion of large amounts of cyst wall material. Although much is known about the transcriptional regulation of encystation and the synthesis and trafficking of cyst wall material, the investigation of global changes in protein content and abundance during G. lamblia encystation is still unaddressed. In this study, we report on the quantitative analysis of the G. lamblia proteome during encystation using tandem mass spectrometry. Quantification of more than 1000 proteins revealed major changes in protein abundance in early, mid and late encystation, notably in constitutive secretory protein trafficking. Early stages of encystation were marked by a striking decrease of endoplasmic reticulum-targeted variant-specific surface proteins and significant increases in cytoskeleton regulatory components, NEK protein kinases and proteins involved in protein folding and glycolysis. This was in stark contrast to cells in the later stages of encystation which presented a surprisingly similar proteome composition to non-encysting trophozoites. Altogether these data constitute the first quantitative atlas of the Giardia proteome covering the whole process of encystation and point towards an important role for post-transcriptional control of gene expression in Giardia differentiation. Furthermore, our data provide a valuable resource for the community-based annotation effort of the G. lamblia genome, where almost 70% of all predicted gene models remains "hypothetical".
Subject(s)
Giardia lamblia/metabolism , Giardia lamblia/pathogenicity , Protozoan Proteins/metabolism , Animals , Gene Expression Regulation, Developmental , Giardia lamblia/growth & development , Giardiasis/parasitology , Humans , Proteome , Protozoan Proteins/genetics , Tandem Mass Spectrometry , Trophozoites/growth & development , Trophozoites/metabolismABSTRACT
Giardia lamblia parasitism accounts for the majority of cases of parasitic diarrheal disease, making this flagellated eukaryote the most successful intestinal parasite worldwide. This organism has undergone secondary reduction/elimination of entire organelle systems such as mitochondria and Golgi. However, trophozoite to cyst differentiation (encystation) requires neogenesis of Golgi-like secretory organelles named encystation-specific vesicles (ESVs), which traffic, modify and partition cyst wall proteins produced exclusively during encystation. In this work we ask whether neogenesis of Golgi-related ESVs during G. lamblia differentiation, similarly to Golgi biogenesis in more complex eukaryotes, requires the maintenance of distinct COPII-associated endoplasmic reticulum (ER) subdomains in the form of ER exit sites (ERES) and whether ERES are also present in non-differentiating trophozoites. To address this question, we identified conserved COPII components in G. lamblia cells and determined their localization, quantity and dynamics at distinct ERES domains in vegetative and differentiating trophozoites. Analogous to ERES and Golgi biogenesis, these domains were closely associated to early stages of newly generated ESV. Ectopic expression of non-functional Sar1 GTPase variants caused ERES collapse and, consequently, ESV ablation, leading to impaired parasite differentiation. Thus, our data show how ERES domains remain conserved in G. lamblia despite elimination of steady-state Golgi. Furthermore, the fundamental eukaryotic principle of ERES to Golgi/Golgi-like compartment correspondence holds true in differentiating Giardia presenting streamlined machinery for secretory organelle biogenesis and protein trafficking. However, in the Golgi-less trophozoites ERES exist as stable ER subdomains, likely as the sole sorting centres for secretory traffic.
Subject(s)
Cell Differentiation , Endoplasmic Reticulum/metabolism , Giardia lamblia/physiology , Spores, Protozoan/physiologyABSTRACT
Eukaryotic cells use COPII-coated carriers for endoplasmic reticulum (ER)-to-Golgi protein transport. Selective cargo capture into ER-derived carriers is largely driven by the SEC24 component of the COPII coat. The Arabidopsis genome encodes three AtSEC24 genes with overlapping expression profiles but it is yet to be established whether the AtSEC24 proteins have overlapping roles in plant growth and development. Taking advantage of Arabidopsis thaliana as a model plant system for studying gene function in vivo, through reciprocal crosses, pollen characterization, and complementation tests, evidence is provided for a role for AtSEC24A in the male gametophyte. It is established that an AtSEC24A loss-of-function mutation is tolerated in the female gametophyte but that it causes defects in pollen leading to failure of male transmission of the AtSEC24A mutation. These data provide a characterization of plant SEC24 family in planta showing incompletely overlapping functions of the AtSEC24 isoforms. The results also attribute a novel role to SEC24 proteins in a multicellular model system, specifically in male fertility.