The Golgi apparatus in parasitic protists

This review summarizes the current reports on the Golgi apparatus of parasitic protists. Numerous recent publications have demonstrated that studies on intracellular traffic in parasites essentially advanced our knowledge on the Golgi structure and function, which has been traditionally based on research on yeast and mammalian cultured cells. It has been reported that the parasitic lifestyle determines the functional and structural peculiarities of the secretory systems in unrelated groups of unicellular parasites that make them different from those in mammalian and yeast cells. This review covers the best-studied protists, predominantly those of high medical importance, belonging to the following taxa: Parabasalia (Trichomonas), Diplomonada (Giardia), Entamoebidae (Entamoeba), parasitic Alveolata of the phyllum Apicomplexa (Toxoplasma, Plasmodium), and Kinetoplastida (Trypanosoma, Leishmania). The morphology of the Golgi organelle in eukaryotes from various taxonomic groups has been compared. Within three of the six highest taxa of Eukaryota (Adl et al., 2005) a minimum of eight groups are represented by species lacking Golgi dictiosomes. However, biochemical and/or molecular (genomic) evidence indicate that an organelle with the functions of the Golgi was present in every lineage of eukaryotes studied thus far. Loss of the Golgi organelle is a secondary event as proven by identification of Golgi genes in the genomes of Golgi-lacking lineages. The loss might have occurred independently several times in evolution. Neither the number of stacks, nor the size of the organelle correlates with the intensity of secretion or the position of the species on the evolutionary tree (in terms of presumably early/lately diverged lineages).     

Biogenesis of iron-sulphur clusters in amitochondriate and apicomplexan protists

During the last 4 years there has been an enormous interest in the question how iron-sulphur ([Fe-S]) clusters, which are essential building blocks for life, are synthesised and assembled into apo-proteins, both in prokaryotes and in eukaryotes. The emerging picture is that the basic mechanism of this pathway has been well conserved during evolution. In yeast and probably all other eukaryotes the mitochondrion is the place where [Fe-S] clusters are synthesised, even for extramitochondrial [Fe-S] cluster-containing proteins, and a number of proteins have been functionally characterised to a certain extent within this pathway. However, almost nothing is known about this aspect in parasitic protists, although recent studies of amitochondriate protists and on the plastid-like organelle of apicomplexan parasites, the apicoplast, have started to change this. In this article I will summarise the current view of [Fe-S] cluster biogenesis in eukaryotes and discuss its implications for amitochondriate protists and for the plastid-like organelle of apicomplexan parasites.     

The secretory pathway of protists: spatial and functional organization and evolution.

All cells secrete a diversity of macromolecules to modify their environment or to protect themselves. Eukaryotic cells have evolved a complex secretory pathway consisting of several membrane-bound compartments which contain specific sets of proteins. Experimental work on the secretory pathway has focused mainly on mammalian cell lines or on yeasts. Now, some general principles of the secretory pathway have become clear, and most components of the secretory pathway are conserved between yeast cells and mammalian cells. However, the structure and function of the secretory system in protists have been less extensively studied. In this review, we summarize the current knowledge about the secretory pathway of five different groups of protists: Giardia lamblia, one of the earliest lines of eukaryotic evolution, kinetoplastids, the slime mold Dictyostelium discoideum, and two lineages within the "crown" of eukaryotic cell evolution, the alveolates (ciliates and Plasmodium species) and the green algae. Comparison of these systems with the mammalian and yeast system shows that most elements of the secretory pathway were presumably present in the earliest eukaryotic organisms. However, one element of the secretory pathway shows considerable variation: the presence of a Golgi stack and the number of cisternae within a stack. We suggest that the functional separation of the plasma membrane from the nucleus-endoplasmic reticulum system during evolution required a sorting compartment, which became the Golgi apparatus. Once a Golgi apparatus was established, it was adapted to the various needs of the different organisms.     

Evidence for Golgi bodies in proposed 'Golgi-lacking' lineages

Golgi bodies are nearly ubiquitous in eukaryotic cells. The apparent lack of such structures in certain eukaryotic lineages might be taken to mean that these protists evolved prior to the acquisition of the Golgi, and it raises questions of how these organisms function in the absence of this crucial organelle. Here, we report gene sequences from five proposed 'Golgi-lacking' organisms (Giardia intestinalis, Spironucleus barkhanus, Entamoeba histolytica, Naegleria gruberi and Mastigamoeba balamuthi). BLAST and phylogenetic analyses show these genes to be homologous to those encoding components of the retromer, coatomer and adaptin complexes, all of which have Golgi-related functions in mammals and yeast. This is, to our knowledge, the first molecular evidence for Golgi bodies in two major eukaryotic lineages (the pelobionts and heteroloboseids). This substantiates the suggestion that there are no extant primitively 'Golgi-lacking' lineages, and that this apparatus was present in the last common eukaryotic ancestor, but has been altered beyond recognition several times.     

Interorganellar communication and membrane contact sites in protozoan parasites

A key characteristic of eukaryotic cells is the presence of organelles with discrete boundaries and functions. Such subcellular compartmentalization into organelles necessitates platforms for communication and material exchange between each other which often involves vesicular trafficking and associated processes. Another way is via the close apposition between organellar membranes, called membrane contact sites (MCSs). Apart from lipid transfer, MCSs have been implicated to mediate in various cellular processes including ion transport, apoptosis, and organelle dynamics. In mammalian and yeast cells, contact sites have been reported between the membranes of the following: the endoplasmic reticulum (ER) and the plasma membrane (PM), ER and the Golgi apparatus, ER and endosomes (i.e., vacuoles, lysosomes), ER and lipid droplets (LD), the mitochondria and vacuoles, the nucleus and vacuoles, and the mitochondria and lipid droplets, whereas knowledge of MCSs in non-model organisms such as protozoan parasites is extremely limited. Growing evidence suggests that MCSs play more general and conserved roles in cell physiology. In this mini review, we summarize and discuss representative MCSs in divergent parasitic protozoa, and highlight the universality, diversity, and the contribution of MCSs to parasitism.     

Structural-functional organization of Golgi apparatus

This review is dedicated to the structure and function of Golgi apparatus (GA). It summarizes contemporary data published in numerous experimental papers and in several reviews. Possible ways of intra-Golgi transport of proteins, existent models of structural and functional organization of Golgi organelle, as well as the issues of its biogenesis, posttranslational modification and sorting of proteins and lipids, and mechanisms of their traffic-king are discussed. Special attention is paid to the role of coatomer proteins (COPI, COPII and clathrin), fusion proteins (SNAREs), and small GTPases (ARF, SARI) in the secretory pathway. In addition, the phenomena of ultrastructural alterations of GA due to various functional conditions and physiological stimuli are specifically accented. We included in this review our original data on a probable involvement of GA in water transport, and on the organization of atypical GA in microsporidia--intracellular parasitic protists.     

Structural-functional organization of Golgi apparatus

This review is dedicated to the structure and function of Golgi apparatus (GA). It summarizes contemporary data published in numerous experimental papers and in several reviews. Possible ways of intra-Golgi transport of proteins, existent models of structural and functional organization of Golgi organelle, as well as the issues of its biogenesis, posttranslational modification and sorting of proteins and lipids, and mechanisms of their trafficking are discussed. Special attention is paid to the role of coatomer proteins (COPI, COPII and clathrin), fusion proteins (SNAREs), and small GTPases (ARF, SARI) in the secretory pathway. In addition, the phenomena of ultrastructural alterations of GA due to various functional conditions and physiological stimuli are specifically accented. We included in this review our original data on a probable involvement of GA in water transport, and on the organization of atypical GA in microsporidia--intracellular parasitic protists.     

RNAi pathways in parasitic protists and worms

Tropical diseases caused by parasitic worms and protists are of major public health concern affecting millions of people worldwide. New therapeutic and diagnostic tools would be of great help in dealing with the public health and economic impact of these diseases. RNA interference (RNAi) pathways utilize small non-coding RNAs to regulate gene expression in a sequence-specific manner. In recent years, a wealth of data about the mechanisms and biological functions of RNAi pathways in distinct groups of eukaryotes has been described. Often, RNAi pathways have unique features that are restricted to groups of eukaryotes. The focus of this review will be on RNAi pathways in specific groups of parasitic eukaryotes that include Trypanosoma cruzi, Plasmodium and Schistosoma mansoni. These parasites are the causative agents of Chagas disease, Malaria, and Schistosomiasis, respectively, all of which are tropical diseases that would greatly benefit from the development of new diagnostic and therapeutic tools. In this context, we will describe specific features of RNAi pathways in each of these parasitic eukaryotic groups and discuss how they could be exploited for the treatment of tropical diseases.     

Evolution and diversity of the Golgi

The Golgi is an ancient and fundamental eukaryotic organelle. Evolutionary cell biological studies have begun establishing the repertoire, processes, and level of complexity of membrane-trafficking machinery present in early eukaryotic cells. This article serves as a review of the literature on the topic of Golgi evolution and diversity and reports a novel comparative genomic survey addressing Golgi machinery in the widest taxonomic diversity of eukaryotes sampled to date. Finally, the article is meant to serve as a primer on the rationale and design of evolutionary cell biological studies, hopefully encouraging readers to consider this approach as an addition to their cell biological toolbox. It is clear that the major machinery involved in vesicle trafficking to and from the Golgi was already in place by the time of the divergence of the major eukaryotic lineages, nearly 2 billion years ago. Much of this complexity was likely generated by an evolutionary process involving gene duplication and coevolution of specificity encoding membrane-trafficking proteins. There have also been clear cases of loss of Golgi machinery in some lineages as well as innovation of novel machinery. The Golgi is a wonderfully complex and diverse organelle and its continued exploration promises insight into the evolutionary history of the eukaryotic cell.     

Genetic diversity of microbial eukaryotes in anoxic sediment around fumaroles on a submarine caldera floor based on the small-subunit rDNA phylogeny

Recent culture-independent molecular analyses have shown the diversity and ecological importance of microbial eukaryotes (protists) in various marine environments. In the present study we directly extracted DNA from anoxic sediment near active fumaroles on a submarine caldera floor at a depth of 200 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit (SSU) rDNA. By sequencing cloned SSU rDNA of the libraries and their phylogenetic analyses, it was shown that most sequences have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, stramenopiles and Opisthokonta). In particular, some sequences were closely related to those of representatives of eukaryotic parasites, such as Phagomyxa and Cryothecomonas of Cercozoa, Pirsonia of stramenopiles and Ichthyosporea of Opisthokonta, although it is not clear whether the organisms occur in free-living or parasitic forms. In addition, other sequences did not seem to be related to any described eukaryotic lineages suggesting the existence of novel eukaryotes at a high-taxonomic level in the sediment. The community composition of microbial eukaryotes in the sediment we surveyed was different overall from those of other anoxic marine environments previously investigated.     

Biochemistry and Evolution of Anaerobic Energy Metabolism in Eukaryotes

Summary: Major insights into the phylogenetic distribution, biochemistry, and evolutionary significance of organelles involved in ATP synthesis (energy metabolism) in eukaryotes that thrive in anaerobic environments for all or part of their life cycles have accrued in recent years. All known eukaryotic groups possess an organelle of mitochondrial origin, mapping the origin of mitochondria to the eukaryotic common ancestor, and genome sequence data are rapidly accumulating for eukaryotes that possess anaerobic mitochondria, hydrogenosomes, or mitosomes. Here we review the available biochemical data on the enzymes and pathways that eukaryotes use in anaerobic energy metabolism and summarize the metabolic end products that they generate in their anaerobic habitats, focusing on the biochemical roles that their mitochondria play in anaerobic ATP synthesis. We present metabolic maps of compartmentalized energy metabolism for 16 well-studied species. There are currently no enzymes of core anaerobic energy metabolism that are specific to any of the six eukaryotic supergroup lineages; genes present in one supergroup are also found in at least one other supergroup. The gene distribution across lineages thus reflects the presence of anaerobic energy metabolism in the eukaryote common ancestor and differential loss during the specialization of some lineages to oxic niches, just as oxphos capabilities have been differentially lost in specialization to anoxic niches and the parasitic life-style. Some facultative anaerobes have retained both aerobic and anaerobic pathways. Diversified eukaryotic lineages have retained the same enzymes of anaerobic ATP synthesis, in line with geochemical data indicating low environmental oxygen levels while eukaryotes arose and diversified.     

Rewiring and regulation of cross-compartmentalized metabolism in protists

Plastid acquisition, endosymbiotic associations, lateral gene transfer, organelle degeneracy or even organelle loss influence metabolic capabilities in many different protists. Thus, metabolic diversity is sculpted through the gain of new metabolic functions and moderation or loss of pathways that are often essential in the majority of eukaryotes. What is perhaps less apparent to the casual observer is that the sub-compartmentalization of ubiquitous pathways has been repeatedly remodelled during eukaryotic evolution, and the textbook pictures of intermediary metabolism established for animals, yeast and plants are not conserved in many protists. Moreover, metabolic remodelling can strongly influence the regulatory mechanisms that control carbon flux through the major metabolic pathways. Here, we provide an overview of how core metabolism has been reorganized in various unicellular eukaryotes, focusing in particular on one near universal catabolic pathway (glycolysis) and one ancient anabolic pathway (isoprenoid biosynthesis). For the example of isoprenoid biosynthesis, the compartmentalization of this process in protists often appears to have been influenced by plastid acquisition and loss, whereas for glycolysis several unexpected modes of compartmentalization have emerged. Significantly, the example of trypanosomatid glycolysis illustrates nicely how mathematical modelling and systems biology can be used to uncover or understand novel modes of pathway regulation.     

Deciphering the Golgi apparatus: from imaging to genes

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.     

Eukaryotes devoid of the most important cellular organelles (flagella, Golgi apparatus, mitochondria) and the main task of organellology]

Comparative evidence on the lack of three important organelles (flagella, Golgi-complex, mitochondria) in cells and organisms at the cellular level of organization has been summarized for all the four eukaryotic kingdoms--Protista, Fungi, Plantae and Animalia (Metazoa). It is established that in the course of evolution these organelles may undergo the total reduction. There is no cellular organelle to be regarded as universal, indispensable. There are only three main obligatory cell components--the plasmalemma, nucleus and cytoplasm (with applied cytoskeleton, cytomembranes and ribosomes). The reduction of flagella (cilia) is occurring in different taxa independent of the transition of protists from the flagellate type of locomotion to the amoeboid, gliding of metabolizing ones, and in the number of metazoan cells. The members of Protista and Fungi, which line in microaerobic or anaerobic conditions, nearly inevitably lose their mitochondria. The tendency to lose Golgi-complex is demonstrated in protists with parasitic mode of life, especially in combination with anaerobiosis. There is so far no satisfied morphological criterium that could say with certainty whether the lacking of flagella, Golgi complex or mitochondria in the low eukaryotes may be primary or secondary (as the result of reduction). Data on the composition, structure and RNA nucleotide sequences cannot be either the straight evidence. A comparative analysis of these data shows that the ribosomes of the primary eukaryotes were, presumably, of a prokaryotic type. Their eukaryotization was carried out for a long time during the evolution of the low eukaryotes (Protista and Fungi), probably, independently in different phylogenetic lines. It is unknown at what steps and in what main phylogenetic lines the three above mentioned organelles may have appeared. It is proposed to single out a special division of cytology--organellology (organoidology)--as an individual science whose main purpose may be investigation of the origination, evolution and disappearance of organelles.     

Genetic Diversity of Microbial Eukaryotes in Anoxic Sediment of the Saline Meromictic Lake Namako-ike (Japan): On the Detection of Anaerobic or Anoxic-tolerant Lineages of Eukaryotes

Available sequence data on eukaryotic small-subunit ribosomal DNA (SSU rDNA) directly retrieved from various environments have increased recently, and the diversity of microbial eukaryotes (protists) has been shown to be much greater than previously expected. However, the molecular information accumulated to date does still not thoroughly reveal ecological distribution patterns of microbial eukaryotes. In the ongoing challenge to detect anaerobic or anoxic-tolerant lineages of eukaryotes, we directly extracted DNA from the anoxic sediment of a saline meromictic lake, constructed genetic libraries of PCR-amplified SSU rDNA, and performed phylogenetic analyses with the cloned SSU rDNA sequences. Although a few sequences could not be confidently assigned to any major eukaryotic groups in the analyses and are debatable regarding their taxonomic positions, most sequences obtained have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, Stramenopiles, and Opisthokonta). Among these sequences, some branched with lineages predominantly composed of uncultured environmental clones retrieved from other anoxic environments, while others were closely related to those of eukaryotic parasites (e.g. Phytomyxea of Cercozoa, Gregarinea of Alveolata, and Ichthyosporea of Opisthokonta).     

Intermediate organelles of the plant secretory pathway: identity and function

The secretory pathway of eukaryotic cells comprises a network of organelles that connects three large membranes, the plasma membrane, the vacuole and the endoplasmic reticulum. The Golgi apparatus and the various post-Golgi organelles that control vacuolar sorting, secretion and endocytosis can be regarded as intermediate organelles of the endocytic and biosynthetic routes. Many processes in the secretory pathway have evolved differently in plants and cannot be studied using yeast or mammalian cells as models. The best characterized organelles are the Golgi apparatus and the prevacuolar compartment, but recent work has shed light on the role of the trans Golgi network, which has to be regarded as a separate organelle in plants. In this study, we wish to highlight recent findings regarding the late secretory pathway and its crosstalk with the early secretory pathway as well as the endocytic route in plants. Recently published findings and suggested models are discussed within the context of known features of the equivalent pathway in other eukaryotes.     

Cellular responses to endoplasmic reticulum stress and apoptosis

The endoplasmic reticulum (ER) is the cell organelle where secretory and membrane proteins are synthesized and folded. Correctly folded proteins exit the ER and are transported to the Golgi and other destinations within the cell, but proteins that fail to fold properly—misfolded proteins—are retained in the ER and their accumulation may constitute a form of stress to the cell—ER stress. Several signaling pathways, collectively known as unfolded protein response (UPR), have evolved to detect the accumulation of misfolded proteins in the ER and activate a cellular response that attempts to maintain homeostasis and a normal flux of proteins in the ER. In certain severe situations of ER stress, however, the protective mechanisms activated by the UPR are not sufficient to restore normal ER function and cells die by apoptosis. Most research on the UPR used yeast or mammalian model systems and only recently Drosophila has emerged as a system to study the molecular and cellular mechanisms of the UPR. Here, we review recent advances in Drosophila UPR research, in the broad context of mammalian and yeast literature.     

Export of cyst wall material and Golgi organelle neogenesis in Giardia lamblia depend on endoplasmic reticulum exit sites

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 ofnewly 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.     

GPI-anchor synthesis

The glycosyl phosphatidylinositol (GPI) anchor of membrane proteins is widely distributed in eukaryotes and parasitic protozoa. The structure and biosynthetic pathway of its core have been elucidated and appear to be conserved in various species. Some of the genes involved in mammalian GPI-anchor biosynthesis have recently been isolated using GPI-anchor-deficient mutant cell lines and expression cloning methods. One of these genes proved to be responsible for a GPI-anchor deficiency known as paroxysmal nocturnal hemoglobinuria. Since the core of the GPI anchor is variously modified in different species and since there may be other differences between its biosynthetic pathway in parasites and their hosts, this pathway could be a target for chemotherapy. In this review, Taroh Kinoshita and Junji Takeda focus on the GPI-anchor biosynthetic pathway and the genes involved in it.