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Unconventional protein secretion (UPS) plays important roles in processes for the survival of the cell and whole organisms. 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 protozoan parasite of global medical importance and reduced subcellular complexity known to release several proteins, some of them virulence factors, without canonical secretory signals, thus implicating UPS at the parasite-host interface. No dedicated machinery nor mechanism(s) for UPS in Giardia are currently known, although speculations on unique endocytic Giardia compartments 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 release of confirmed and putative virulence factors, in this study we employed affinity purification and mass spectrometry coupled to microscopy-based subcellular localization and signal correlation quantification techniques to investigate protein complexes of eleven reported unconventionally-secreted putative and confirmed virulence factors, all predicted to be cytosolic. A subset of selected putative and confirmed virulence factors, along with their interaction partners, unequivocally associate to the surface of 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 specific subset of the alpha-giardin protein family was invariably found 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 PVs/PECs, suggesting a novel link between these primarily endocytic and feeding organelles and UPS at the parasite-host interface.

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. 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. 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.

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.

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.

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.

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-β (TGFβ) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFβ receptor II (TGFβ-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 TGFβ-RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFβ-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 TGFβ-RII signalling stimulates the expression of cyclin-dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFβ-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 TGFβ-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 TGFβ as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer.

In their Spotlight article titled ‘Endocytosis in Giardia: evidence of absence’ recently published in Trends in Parasitology [1], Zamponi and colleagues comment on our recent article ‘Static clathrin assemblies at the peripheral vacuole–plasma membrane interface of the parasitic protozoan Giardia lamblia’, published in the July issue of PLoS Pathogens this year [2]. We would like to alert the readership to representations of some aspects of our data and conclusions in their opinion piece with which we disagree.

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.

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., Author Summary Organelles with endosymbiotic origin are present in virtually all extant eukaryotes and have undergone considerable remodeling during > 1 billion years of evolution. Highly diverged organelles such as mitosomes or plastids in some parasitic protozoa are the product of extensive secondary reduction. They are sufficiently unique to generate interest as targets for pharmacological intervention, in addition to providing a rich ground for evolutionary cell biologists. The so-called mitochondria-related organelles (MROs) comprise mitosomes and hydrogenosomes, with the former having lost any role in energy metabolism along with the organelle genome. The mitosomes of the intestinal pathogen Giardia lamblia are the most highly reduced MROs known and have proven difficult to investigate because of their extreme divergence and their unique biophysical properties. Here, we implemented a novel strategy aimed at systematic analysis of the organelle proteome by iterative expansion of a protein-protein interaction network. We combined serial forward and reverse co-immunoprecipitations with mass spectrometry analysis, data mining, and validation by subcellular localization and/or functional analysis to generate an interactome network centered on a giardial Tom40 homolog. This iterative ab initio proteome reconstruction provided protein-protein interaction data in addition to identifying novel organelle proteins and functions. Building on this data we generated information on organelle replication, mitosome morphogenesis and organelle dynamics in living cells.

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.

The secretory transport capacity of Giardia trophozoites is perfectly adapted to the changing environment in the small intestine of the host and is able to deploy essential protective surface coats as well as molecules which act on epithelia. These lumen-dwelling parasites take up nutrients by bulk endocytosis through peripheral vesicles or by receptor-mediated transport. The environmentally-resistant cyst form is quiescent but poised for activation following stomach passage. Its versatility and fidelity notwithstanding, the giardial trafficking systems appear to be the product of a general secondary reduction process geared towards minimization of all components and machineries identified to date. Since membrane transport is directly linked to organelle biogenesis and maintenance, less complexity also means loss of organelle structures and functions. A case in point is the Golgi apparatus which is missing as a steady-state organelle system. Only a few basic Golgi functions have been experimentally demonstrated in trophozoites undergoing encystation. Similarly, mitochondrial remnants have reached a terminally minimized state and appear to be functionally restricted to essential iron-sulfur protein maturation processes. Giardia's minimized organization combined with its genetic tractability provides unique opportunities to study basic principles of secretory transport in an uncluttered cellular environment. Not surprisingly, Giardia is gaining increasing attention as a model for the investigation of gene regulation, organelle biogenesis, and export of simple but highly protective cell wall biopolymers, a hallmark of all perorally transmitted protozoan and metazoan parasites.

The Golgi apparatus is a vital organelle in eukaryotic cells. It grabs and processes secretory materials synthesized by the endoplasmic reticulum (ER) before sorting them to their destination. The Golgi also receives materials from vacuoles/lysosomes and the plasma membrane for further recycling to other compartments within the cell (1) (Figure 1). Given the vital role of the Golgi in a cell, it is important to understand how this organelle attains and maintains its structural and functional integrity during the intense processes of membrane traffic. Despite an equally central role of the Golgi in membrane traffic in eukaryotes, the organization of this organelle has some unique features in each cell system. Therefore, the wealth of information available on the structure and activity of the Golgi in one system is not always directly transferable to others. However, certain morphological and functional aspects are common among cell systems. Therefore, studying the factors that regulate organelle biogenesis and organization of the Golgi apparatus is important in basic cell biology of eukaryotes and may also contribute to a better understanding of how different cell systems have evolved. In this study, we report on the identification of Golgi mutants in plant cells. We have developed a screen that is a promising strategy not only for the identification of genes responsible for the morphological and functional integrity of the plant Golgi but could also provide fundamental information on other multicellular systems for which the power of forward genetics cannot be exploited as easily as in Arabidopsis.

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.

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”., PLoS ONE, 8 (12), ISSN:1932-6203

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.

Undoubtedly, biotechnology has a tremendous impact on our daily lives. As a result of this and in parallel to the advancement of knowledge in this field of applied research, consumer awareness of the potential benefits and risks of this technology has steadily increased, leading to a thorough investigation of the public perception of biotechnology in the past years. Indeed, it has become clear that it is in the general interest of science and especially of applied research to inform the public of its advances. A promising next step is to strengthen biotechnology communication in scholastic institutions. In this paper, we investigate the perception of biotechnology in a specific target group, namely high-school students in the 16-20-year-old age range. We conducted a questionnaire-based survey on a total of 1410 students in six European countries to investigate students' perception, concern, scientific knowledge, and awareness. Our data revealed some unexpected patterns of acceptance and concern about biotechnology. Knowledge analysis indicated that pupils lack specific knowledge about biotechnological applications and their interest in biotechnology appeared to be linked to knowledge. Analysis of specific questions about teaching practices at schools suggests that a better targeted choice in media as vehicles for information together with selected speakers could be instrumental in increasing students' interest in science and more specifically in biotechnology.

Plant Golgi bodies possess unique morphological and functional characteristics that are key to several biological and biotechnological processes, such as transport of the cell’s building blocks to energy-rich compartments, including chloroplasts, storage vacuoles and a cellulosic cell wall. During the last decade it has become apparent that the plant Golgi apparatus has features that are remarkably different from other systems. Here we summarize the most recent findings on this organelle and we highlight pressing questions that are likely to drive the next 10 years of research on the plant Golgi apparatus.

How the endoplasmic reticulum (ER) and the Golgi apparatus maintain their morphological and functional identity while working in concert to ensure the production of biomolecules necessary for the cell's survival is a fundamental question in plant biology. Here, we isolated and characterized an Arabidopsis thaliana mutant that partially accumulates Golgi membrane markers and a soluble secretory marker in globular structures composed of a mass of convoluted ER tubules that maintain a connection with the bulk ER. We established that the aberrant phenotype was due to a missense recessive mutation in sec24A, one of the three Arabidopsis isoforms encoding the coat protomer complex II (COPII) protein Sec24, and that the mutation affects the distribution of this critical component at ER export sites. By contrast, total loss of sec24A function was lethal, suggesting that Arabidopsis sec24A is an essential gene. These results produce important insights into the functional diversification of plant COPII coat components and the role of these proteins in maintaining the dynamic identity of organelles of the early plant secretory pathway.

In this study, polyamine oxidase from maize (MPAO), which is involved in the terminal catabolism of spermidine and spermine to produce an aminoaldehyde, 1,3-diaminopropane and H(2)O(2), has been conditionally expressed at high levels in the nucleus of MCF-7 human breast cancer cells, with the aim to interfere with polyamine homeostasis and cell proliferation. Recombinant MPAO expression induced accumulation of a high amount of 1,3-diaminopropane, an increase of putrescine levels and no alteration in the cellular content of spermine and spermidine. Furthermore, recombinant MPAO expression did not interfere with cell growth of MCF-7 cells under normal conditions but it did confer higher growth sensitivity to etoposide, a DNA topoisomerase II inhibitor widely used as antineoplastic drug. These data suggest polyamine oxidases as a potential tool to improve the efficiency of antiproliferative agents despite the difficulty to interfere with cellular homeostasis of spermine and spermidine.

Maize polyamine oxidase (MPAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyses the oxidation of spermine and spermidine at the secondary amino groups. The structure of MPAO indicates a 30-A long U-shaped tunnel that forms the catalytic site, with residues Glu62 and Glu170 located close to the enzyme-bound FAD and residue Tyr298 in close proximity to Lys300, which in turn is hydrogen-bonded to the flavin N(5) atom via a water molecule (HOH309). To provide insight into the role of these residues in the catalytic mechanism of FAD reduction, we have performed steady-state and stopped-flow studies with wild-type, Glu62Gln, Glu170Gln, Tyr298Phe, and Lys300Met MPAO enzymes. We show that the steady-state enzyme activity is governed by an ionisable group with a macroscopic pK(a) of approximately 5.8. Kinetic analysis of the Glu62Gln, Glu170Gln, and Tyr298Phe MPAO enzymes have indicated (i) only small perturbations in catalytic activity as a result of mutation and (ii) steady-state pH profiles essentially unaltered when compared to the wild-type enzyme, suggesting that these residues do not play a critical role in the reaction mechanism. These kinetic observations are consistent with computational calculations that suggest that Glu62 and Glu170 are protonated over the pH range accessible to kinetic studies. Substitution of Lys300 with Met in MPAO resulted in a 1400-fold decrease in the rate of flavin reduction and a 160-fold decrease in the equilibrium dissociation constant for the Lys300Met-spermidine complex, consistent with a major role for this residue in the mechanism of substrate oxidation. A sizable solvent isotope effect (SIE = 5) accompanies FAD reduction in the wild-type enzyme and steady-state turnover (SIE = 2.3) of MPAO, consistent with the reductive half-reaction of MPAO making a major contribution to rate limitation in steady-state turnover. Studies using the enzyme-monitored turnover method indicate that oxidized FAD is the prominent form during steady-state turnover, consistent with the reductive half-reaction being rate-limiting. Our studies indicate the importance of Lys300 and probable importance of HOH309 to the mechanism of flavin reduction in MPAO. Possible roles for Lys300 and water in the mechanism of flavin reduction are discussed.