Towards a molecular understanding of human diseases using Dictyostelium discoideum

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TRENDS in Molecular Medicine

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Towards a molecular understanding of human diseases using Dictyostelium discoideum Robin S.B. Williams1, Katrina Boeckeler1, Ralph Gra¨f2, Annette Mu¨ller-Taubenberger3, Zhiru Li4, Ralph R. Isberg4,5, Deborah Wessels6, David R. Soll6, Hannah Alexander7 and Stephen Alexander7 1

Department of Biology and the Wolfson Institute for Biomedical Research, University College London, London, WC1E 6BT, UK Carl Zeiss MicroImaging GmbH, Zeppelinstrasse 4, D-85339 Hallbergmoos, Germany 3 Institut fu¨r Zellbiologie, Ludwig-Maximilians-Universita¨t Mu¨nchen, 80336 Mu¨nchen, Germany 4 Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA 5 Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, MA 02111, USA 6 W.M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA 7 Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA 2

The social amoeba Dictyostelium discoideum is increasingly being used as a simple model for the investigation of problems that are relevant to human health. This article focuses on several recent examples of Dictyostelium-based biomedical research, including the analysis of immune-cell disease and chemotaxis, centrosomal abnormalities and lissencephaly, bacterial intracellular pathogenesis, and mechanisms of neuroprotective and anti-cancer drug action. The combination of cellular, genetic and molecular biology techniques that are available in Dictyostelium often makes the analysis of these problems more amenable to study in this system than in mammalian cell culture. Findings that have been made in these areas using Dictyostelium have driven research in mammalian systems and have established Dictyostelium as a powerful model for human-disease analysis. Dictyostelium, a simple eukaryotic model Since the discovery of Dictyostelium discoideum by Raper [1] in 1935, its fascinating biology has made it a popular and productive model system for studying the molecular basis of cell and developmental biology. Dictyostelium grows by the mitotic division of single cells that feed by phagocytosis on bacteria, or by macropinocytosis on simple axenic liquid medium, making it possible to grow many cells for analysis. The term ‘social amoebae’ derives from the behaviour of cells when the food supply is exhausted or removed. Upon onset of starvation, undifferentiated single cells immediately stop division and enter what is now a well-characterized developmental program of gene expression and morphogenesis [2] (Figure 1a). This switch provides the mutually exclusive growth and differentiation stages of the life cycle. Initiation of development occurs when
105 cells aggregate to a single point by chemotaxis Corresponding authors: Williams, R.S.B. ([email protected]); Alexander, S. ([email protected]). Available online 4 August 2006. www.sciencedirect.com

to a pulsatile cAMP signal, which is relayed to cells that are more distant from the aggregation centre [3]. Following the formation of multi-cellular aggregates, cells differentiate into two basic cell types, pre-stalk and pre-spore, that ultimately become stalk and spore cells, respectively. Cell differentiation, along with specific morphogenetic cell movements, results in the formation of a mature fruiting body in which a mass of up to 80 000 spores rests on top of a multicellular stalk composed of up to 20 000 dead, vacuolated stalk cells (Figure 1b). Many features of Dictyostelium make it a very attractive model system (Box 1). Dictyostelium has been particularly useful for the study of chemotaxis and cell motility, differentiation and morphogenesis [4]. In addition, Dictyostelium has been used as a model for human-disease analysis [5], and recent studies have identified at least 33 Dictyostelium genes as orthologs of disease-related genes [6]. This review presents recent studies related to a range of humanhealth problems, including inherited conditions such as Shwachman–Bodian–Diamond syndrome (SBDS) and lissencephaly (see Glossary), bacterial pathogenesis, and neuroprotective and anti-cancer-drug mechanisms, using this simple eukaryote as a biomedical model. Human white-blood-cell disease Dictyostelium is a powerful model for investigating neutrophil chemotaxis and the molecular mechanisms that regulate this process because of similarities in the way in which both cell types move and respond to chemotactic signals [4,7]. Similar to Dictyostelium, polymorphonuclear neutrophils (PMNs) chemotax in vitro in spatial gradients [8], although neither the natural attractants nor their source have been firmly established in vivo. In nature and in vitro, Dictyostelium responds to waves of the chemoattractant cAMP. Although the spatial information at the onset of the wave results in initial cell orientation, the remaining cellular responses are dictated by the temporal cAMP gradients in the front and back of the wave, and the

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Glossary Cisplatin: a synthetic platinum-based DNA-damaging drug that is widely used for the treatment of solid tumours. fMLP: a synthetic peptide (n-formyl-Met-Leu-Phe) that mimics the activity of bacterially derived peptides. GFP–HDEL: a GFP–His-Asp-Glu-Leu fusion protein that is retained in the endoplasmic reticulum. Inositol-depletion theory: a theory for bipolar-disorder drug function, whereby the reduction of inositol recycling (caused by, for example, lithium inhibition of a family of phosphoinositide phosphatases [87]) slows the overactive production of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3], hence attenuating the elevated signalling potentially causing bipolar disorder. Legionnaires disease: a type of pneumonia caused by the bacterium Legionella pneumophila. Lissencephaly: a rare genetic disorder caused by mutations in genes that encode cytoskeleton-associated proteins, named from the Greek lissos (smooth). It occurs at a frequency of about one in 30 000 births, and it is characterized by a smooth appearance of the brain surface due to the absence of the typical folds. This results from a failure of neuronal precursors to migrate from the paraventricular area, where they originate, to the distal regions of the cerebral cortex to form the cortical layers. SHHP: the strand–helix–hairpin motif of the N-terminal module of the Dictyostelium SBDS protein comprised of a two-b-strand hairpin followed by a two-a-helix hairpin. Shwachman–Bodian–Diamond syndrome (SBDS): an autosomal recessive disorder with multi-system defects characterized by varying degrees of neutropenia (low neutrophil levels), pancreatic insufficiency, skeletal anomalies and short stature. Mutations that are associated with
70% of SBDS patients have been localized to a single SBDS gene at locus 7q11 [14]. Sphingosine-1-phosphate (S-1-P): a bioactive lipid that is synthesized from sphingosine and ATP by sphingosine kinase. S-1-P is associated with cell survival and division, and has been shown to promote cell proliferation, whereas another bioactive lipid ceramide (a precursor to S-1-P) has been shown to promote cell death. The prevailing theory on the role of S-1-P is that it is the balance between S-1-P and ceramide that controls whether a cell lives or dies. Valproic acid (VPA): 2-propyl pentanoic acid is a drug that was found to be effective in the treatment of epilepsy in 1963 [51], and has since become a commonly used treatment for bipolar disorder and migraine, schizophrenia and aggression. It is in clinical trial for cancer treatment and for reducing latent HIV levels. It also has neuroprotective effects.

absolute concentration at its peak [9]. Likewise, human PMNs respond to experimentally applied temporal gradients of n-formyl-Met–Leu–Phe (fMLP) with velocity surges and shape changes similar to those of Dictyostelium

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Box 1. The success of the social amoeba Dictyostelium as a model organism  Dictyostelium is a simple eukaryote with a cellular organization typical of higher eukaryotes. It has a plasma membrane and no cell wall.  The cells are haploid and mutant phenotypes are therefore immediately observable.  Molecular genetic tools are available, including the ability to perform homologous gene replacements rapidly [83], random insertional mutagenesis (termed restriction-enzyme-mediated integration, REMI) [84], multiple-gene deletions [85] and RNA interference (RNAi) technologies [86].  The entire 35-Mb genome has been sequenced [6], encoding
12 500 proteins. This has led to the identification of a large number of proteins closely related to their mammalian orthologs, including 33 Dictyostelium proteins that are orthologs to human diseaserelated proteins [6].  The analysis of biochemical and cell biological roles of these proteins in development and differentiation is particularly amenable in Dictyostelium because large numbers of isogenic, synchronized multicellular aggregates can be obtained at any stage of development.  A wide selection of stable cell lines containing overexpressed or ablated genes are available to facilitate analysis of signalling pathways and protein function (http://www.dictybase.org).  The commonality in protein-binding partners and signalling pathways between Dictyostelium and mammalian systems has enabled cell-signalling processes and pathways to be analysed in a genetically tractable eukaryotic model.

cells in a cAMP wave [7], suggesting that PMNs have the machinery to read all the information in a relayed wave. The remarkable similarities between the basic motile behaviour and chemotaxis of PMNs and Dictyostelium have recently been used to investigate SBDS [10,11], a human disease that affects neutrophil behaviour. Because SBDS patients have an increased incidence of infection, which is often independent of neutropenia, several early studies were performed to test whether PMNs from SBDS patients had chemotactic defects, using primarily transmembrane (Boyden chamber) assays [12]. These studies identified

Figure 1. The Dictyostelium discoideum life cycle. (a) Vegetative cells divide by mitosis until, following induction by starvation, cells enter a 24-h development cycle. During this developmental process, up to 105 cells chemotax together to form a mound and then form either a migrating slug or continue to develop directly into a mature fruiting body. (b) The mature fruiting body consists of differentiated stalk and spore cells. Scale bar is 0.1 mm. www.sciencedirect.com

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abnormalities but, because direct observation of single-cell behaviour is not possible in transmembrane assays, distinction between chemokinetic and chemotactic defects could not be made [8]. Recently, using single-cell chemotaxis assays developed to assess the responses of Dictyostelium and PMNs to spatial, temporal and concentration components of a simulated wave of chemoattractant [7], and computer-assisted methods of single-cell analysis [9], a single chemotactic defect in the PMNs of SBDS patients was identified [13]. Although SBDS PMNs migrated normally in buffer, responded normally to temporal waves of fMLP and exhibited a chemokinetic response to attractant, they could not orientate towards the fMLP source in a spatial gradient, suggesting a defect in the machinery that is involved in turning towards the chemoattractant source in a gradient. Mutations associated with
70% of SBDS patients have been localized to the single human SBDS gene [14]. Because Dictyostelium contained an SBDS ortholog and because the mechanisms regulating chemotaxis have been well studied in Dictyostelium, the function of the SBDS protein was analysed in this model system. The Dictyostelium SBDS homolog [15] encodes a protein with 38% identity and 69% similarity to the human protein and contains three conserved modules, including an RNA-recognition motif. It also contains a dyad repeat of a structural SHHP motif that might serve as a surface for interactions with ribonucleoprotein complexes [15]. Furthermore, most amino acids that are altered in the SBDS protein in patients are conserved in the Dictyostelium homolog. To investigate the function of the SDBS protein, transformants expressing SBDS–green

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fluorescent protein (GFP) were generated in Dictyostelium [15]. SBDS–GFP was distributed uniformly throughout the cytoplasm in Dictyostelium transformants migrating in buffer. However, during chemotaxis in a cAMP spatial gradient, SBDS–GFP localized to pseudopods for extended (>1 min) periods (Figure 2), starting close to the time of initial extension and remained in the pseudopod throughout its expansion (Figure 2). Pseudopodial localization was transient when chemoattractant was added globally. Although the discovery of the localization of the SBDS protein in pseudopods of chemotaxing Dictyostelium was consistent with a role for the protein in human PMN chemotaxis, it was not consistent with a role in RNA interaction, as deduced from its structure [16–18]. However, because some mRNA molecules that are involved in cytoskeletal organization and pseudopod extension localize to pseudopodia or lamellipodia in response to the appropriate stimulation [19], SBDS might have a role in mRNA localization. Other evidence points to an interaction of the SBDS protein with phosphoinositides [20] and the Rho3p GTPase [20], which are signal-transduction molecules activated in response to chemoattractant, suggesting a more-direct role for the SBDS protein in the regulation of chemotaxis. Experiments to identify SBDSprotein-associated macromolecules in Dictyostelium are currently underway, and should clarify the cellular function of this protein. Centrosome function and lissencephaly Lissencephaly is a human brain disorder caused by mutations in genes that encode cytoskeleton-associated proteins

Figure 2. Identification of a functional role of the SBDS protein in chemotaxing Dictyostelium cells has shed light on the role of this protein in neutrophil behaviour. (a) SBDS–GFP localizes to the site of new lateral pseudopod formation (80–85s, small arrow), remains localized in the pseudopod during extension and (b) exits the pseudopod upon retraction (30–40 s, small arrow). Large arrows point towards the source of cAMP. Time is in seconds (s). Abbreviations: a, anterior; u, uropod. Scale bar is 5 mm. From [15], with permission from the Company of Biologists Ltd. www.sciencedirect.com

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such as LIS1 [21]. LIS1 is an essential regulator of dynein, the microtubule minus-end-directed motor protein, and is thus indispensable for many dynein-dependent functions. Dictyostelium has been used as a model to study LIS1 function owing to its suitability for examining the role of cytoskeletal proteins in development and the high homology of human and Dictyostelium LIS1 (
60% identity). A hypomorphic Dictyostelium cell line that expresses a LIS1 protein containing a mutation frequently found in human patients was prepared [22]. Analysis of this mutant revealed that LIS1 is required for the integrity of the Golgi apparatus, proper interaction of microtubules with the cell cortex, nucleus–centrosome association and regulation of the cellular F-actin content [22] (Figure 3). Although involvement of LIS1 in Rho-GTPase-mediated actin dynamics has been previously suggested [23], research in Dictyostelium initially identified a direct interaction with a RhoGTPase, Rac1A. It is probable that alterations of actin dynamics caused by defective LIS1 also contribute to the neuronal migration defects in lissencephaly, in addition to microtubule-dependent dysfunction such as impaired microtubule interactions with the cell cortex [24], and disrupted coupling of nuclei and centrosomes [25] (Figure 3). To understand the pathogenic role of LIS1 in lissencephaly, the cellular function of LIS1 was defined by examining the binding partners of Dictyostelium LIS1 (DdLIS1). Characterization of these proteins, including dyneinassociated proteins and dynein itself [26,27], and DdCP224 (the first member of the Xenopus microtubule-associated protein 215 family, XMAP215 [28] to be identified as a LIS1 interactor [22]), in Dictyostelium has helped to understand the role of LIS1 in this disorder [22]. Interestingly, doublecortin, a marker for neuronal migratory precursors that is also frequently involved in lissencephaly [29], is absent in non-vertebrates except Dictyostelium where it is expressed during development. Taken together, among all non-vertebrate biomedical model organisms, Dictyostelium provides the only model in which all relevant proteins that are so far known to be associated with lissencephaly are present.

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Studies in Dictyostelium have also shown that LIS1 is a true component of the centrosome, which is a central component in cell-cycle progression and cytokinesis. Over a century ago, Boveri [30] proposed a role for the centrosome in spindle organization, and suggested that cancer can arise from aberrant chromosome segregation in somatic cells. Deficiencies in centrosomal proteins can lead to malfunction of centrosomes in mitosis and centrosome amplification, a hallmark of tumour cells. Dictyostelium provides a suitable model to study these effects because formation of supernumerary centrosomes has been described in various Dictyostelium mutants. For example, overexpression of two Dictyostelium centrosomal components, Nek2 [never in mitosis gene A (NIMA)-related kinase 2] [31] and DdCP224, directly affects centrosome biogenesis [31–33] (Figure 3). Nek2 seems to be required for recruitment of centrosomal material, as has been shown in Xenopus eggs [34], and might be involved in de novo centrosome formation. The XMAP215 family [28] is likely to have a role in cancer progression, as the human homologue ch-TOGp (colonic-hepatic tumour-overexpressed gene) was initially identified in a screen for genes that are overexpressed in colonic and hepatic tumours [35]. However, research in Dictyostelium showed that overexpression of these proteins is sufficient to cause the centrosome amplification that is typically observed in these tumours [32,33]. Dictyostelium has also provided the first hint that mutations in regulatory actin-associated proteins can cause centrosomal and genetic instability of malignant tumour cells. Studying mitosis in mutants that lack either actininteracting protein 1 (AIP1) or the actin-bundling protein cortexillin indicated that these mutations cause genetic instability by formation of atypical mitotic complexes and enlarged nuclei with varying DNA content [36]. These alterations were linked to moderate centrosome amplification and spindle abnormalities. Studies of supernumerary centrosome dynamics in Dictyostelium revealed important insights into cellular mechanisms to control centrosome number [33,36]. Thus, the analysis of Dictyostelium

Figure 3. LIS1 and Nek2 Dictyostelium mutants show altered cytoskeletal appearance. Comparison of wild type (a and b) and LIS1 mutants (a0 and b0 ) shows that deletion of the protein causes disarranged microtubules and a reduced F-actin content, resulting in flattening of cells and F-actin distribution in characteristic broad rims. (c) Nek2overexpressing cells show centrosome amplification, which causes the presence of more than one centrosome per nucleus in interphase cells. Maximum intensity projection of deconvolved confocal stacks for control cells (a and b), hypomorphic DdLIS1 mutants (a0 and b0 ) and cells that express GFP–DdNek2 (c). In (a), (a0 ), (b) and (b0 ), cells are stained for microtubules (anti-tubulin YL1/2, green, Alexa 488). In (a), (a0 ) and (c), centrosomes are stained with anti-DdCP224 (red); in (b) and (b0 ), actin is stained with phalloidin (Alexa 568, red), and in (c) DdNek2 is visualized by GFP fluorescence (green). Nuclei were stained with TO-PRO3 (blue). Scale bar is 2 mm. www.sciencedirect.com

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cytoskeletal and centrosomal components has enabled advances in the understanding of disorders such as lissencephaly and the role of centrosomal abnormalities in tumour genesis. Intracellular growth of pathogens Legionella pneumophila causes Legionnaires disease in humans after inhalation of contaminated aerosols. The bacteria are phagocytosed by alveolar macrophages and multiply within a membrane-bound vacuole that recruits small vesicles derived from the early secretory apparatus. These vesicles then fuse, generating an endoplasmic reticulum (ER)-like compartment [37]. The formation of the replication vacuole of L. pneumophila is controlled by the defect in organelle trafficking (Dot)–intracellular multiplication (Icm) protein translocation system, a multi-protein membrane complex that injects proteins into host cells [38]. These translocation-system substrates probably have key roles in supporting L. pneumophila intracellular growth, although the fact that mutational loss of individual effectors has little effect on intracellular growth suggests functional redundancy. Fresh-water amoebae such as Acanthamoeba castellanii are environmental hosts for L. pneumophila and are frequently found harbouring the bacteria in natural reservoirs [39]. The intracellular growth of bacteria in these amoebae is remarkably similar to L. pneumophila infection of alveolar macrophage [40], indicating a high level of evolutionary conservation. The observation that L. pneumophila grows in amoebae stimulated the use of Dictyostelium as a model for analysis of host proteins that modulate intracellular growth. Dictyostelium mimics the interaction of the bacterium with its natural host [41,42] and with growth in the macrophage, where the replication vacuole is surrounded by an ER-like compartment [42]. The association of ER markers with the L. pneumophila vacuoles, which depends on the presence of an intact Dot–Icm translocation system, further emphasizes the similarity of the replication cycle to that observed in both macrophages and amoebae hosts [43–45]. Fluorescence microscopy studies on live cells have demonstrated that L. pneumophila-containing vacuoles are rapidly transported along microtubules to a perinuclear position in Dictyostelium [45]. The vacuole then associates with ER markers such as GFP–HDEL, calnexin–GFP and GFP–calreticulin, and becomes more spacious as the compartment matures [44,45] (Figure 4). The rapid movement

Figure 4. Dictyostelium has been used to examine host–pathogen relationships in bacterial pathogenesis. Dictyostelium cells, labelled with the endoplasmic reticulum marker GFP–HDEL (green) show Legionella pneumophila (red) within vacuoles (arrow) that are (a) tight following uptake compared with (b) spacious when mature. Scale bar is 10 mm. Adapted from [44], with permission from Blackwell Publishing. www.sciencedirect.com

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of the early replication vacuole becomes more restricted during the maturation process. Several other studies have identified Dictyostelium mutants with altered ability to support intracellular replication. Some mutants that are impaired in phagocytosis have the surprising phenotype of supporting L. pneumophila replication at higher levels than wild-type Dictyostelium [46]. This might reflect the fact that competing processes that can traffic the vacuole into a degradative compartment are lacking in these mutants, enabling more-efficient replication-vacuole formation. For example, the mutation of a proton-coupled divalent metal-ion transporter (Slc11A1), which is involved in vacuolar degradation of other pathogens, shows elevated L. pneumophila replication [46]. Other mutations that seem to affect replication and vacuole biogenesis result in defective intracellular growth. In addition, mutations that eliminate the Dictyostelium Ratio A (RtoA) protein (a novel protein that contains several repeats of a serine-rich motif involved in the fusion of phospholipid vesicles) show reduced endocytosis and exocytosis rates, and this mutation causes a reduction in the integrity of the L. pneumophila replication vacuole based on lowered accumulation of an ER-associated marker [44] (Figure 4). Similar defects can be observed in mutants that lack the ER-associated proteins calnexin and calreticulin [43]. Thus, there is already a large group of Dictyostelium mutants that alters L. pneumophila intracellular growth compared with only one such mutation in the other genetic system available – the mouse baculoviral IAP repeat-containing 1e protein (BIRC1e, also known as NAIP5) – that controls a caspase-1-dependent response that restricts L. pneumophila growth [47]. Dictyostelium has also been used to study the intracellular growth of Mycobacterium marinum and Cryptococcus neoformans, both of which are commonly found in environmental reservoirs [48]. M. marinum, which causes tubercular granulomas in fish and similar cutaneous lesions in humans, shows a pattern of replication that is similar to that in macrophages. As was observed with L. pneumophila, the phagocytosis-defective Dictyostelium coronin mutant enables enhanced replication of M. marinum [42]. C. neoformans is an encapsulated fungus found in soil, and infection in immunocompromised hosts can result in meningitis [49]. C. neoformans can infect and kill Dictyostelium, and passage of the microorganism through Dictyostelium enhances its virulence in a mouse model of disease. This study supports the hypothesis that the virulence of C. neoformans for mammalian hosts is maintained by its interaction with predatory microorganisms in the environment [48]. Psychiatric-disorder signalling pathways Another area of biomedical research in which Dictyostelium has been employed is the analysis of drug-sensitive signalling pathways that are unknown or poorly characterized. The therapeutic targets and mechanisms of action of the bipolar disorder treatments lithium and valproic acid (VPA) remain unclear, despite the clinical use of these drugs for a considerable time (lithium has been used for over a century [50] and VPA since 1963 [51]). The use of Dictyostelium has helped to define how these drugs work

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[52,53] because phenotypic changes in Dictyostelium that are caused by the drugs have enabled the identification and characterization of drug-sensitive signalling pathways. To identify signalling pathways that are targeted by lithium, an insertional mutagenesis screen was employed to identify genes that confer resistance to lithium in Dictyostelium [54] (Figure 5). One identified mutant contained a disrupted gene that encodes the prolyl oligopeptidase enzyme, a protein associated with psychiatric disorders [55–57]. Prolyl oligopeptidase has a central role in regulating the levels of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] (Figure 6a) in Dictyostelium, which is a key element of various signalling pathways, including those that involve serotonin and vasopressin in mammalian systems. Analysis of Ins(1,4,5)P3 has proven to be difficult in mammalian cells because it is found at low concentrations and is highly labile. However, in Dictyostelium, the ease of handling cells and growing large

Figure 5. Schematic depiction of the identification of the genetic basis for drug resistance and the generation of isogenic, stable, multiple-gene knockouts in Dictyostelium. A mutagenesis screen for drug-resistance is possible if wild-type cells (a) show an altered phenotype when allowed to develop in the presence of a drug (b). (c) Generation of a library of mutants through the random insertion of an antibiotic-resistance cassette into genomic DNA (REMI [84]) enables the screening of this library by either: plating directly onto a bacterial lawn in the presence of a drug (d) or growing the library in high drug concentrations (e) and then plating onto a bacterial lawn, enabling single-drug-resistant cells to form plaques. (f) From each drug-resistant Dictyostelium clone, isolated genomic DNA is digested with restriction enzymes that cleave within the cassette (g) and ligation and inverse-PCR analysis enable the identification of the ablated gene that controls drug resistance (h). (i) Confirmation of the resistance phenotype caused by ablation of the identified gene using a cassette based upon the Cre–LoxP system [85] in a wild-type background, and growth of transformants in 96-well dishes to ensure single transformants enables the rapid isolation of isogenic cells that contain the ablated gene of interest (j). (k) Cells can be used for the analysis of development or grown in large numbers for further experimentation. Excision of the antibiotic-resistance cassette and further transformation with a Cre–LoxP knockout cassette based upon other genes enable the ablation of multiple genes in a single isogenic line [85]. www.sciencedirect.com

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isogenic cultures has enabled the measurement of this labile signalling molecule. The identification of Ins(1,4,5)P3 signalling in controlling resistance to lithium provided strong support for a unifying theory of bipolardisorder drug function, known as ‘the inositol-depletion theory’. Once the drug effects were discovered and characterized in Dictyostelium, the findings were extended to mammalian systems, whereby the inositol-depletiondependent increase in the size of rat dorsal root ganglia neurons, caused by three distinct bipolar disorder drugs, was reversed by the inhibition of prolyl oligopeptidase [58]. The inositol-depletion theory has also recently been supported by the identification of an indirect inhibitory effect of VPA on the de novo inositol biosynthetic enzyme inositol-1-phosphate synthase (INO1) [59]. Although the mechanism of this effect remains unclear, Ins(1,4,5)P3 depletion has been used in Dictyostelium to identify novel, potentially safer, bipolar disorder drugs. In one study [60], ten VPA derivatives were assayed for Ins(1,4,5)P3 depletion in Dictyostelium, and the identified Ins(1,4,5)P3depleting compounds were subsequently tested in mammalian neuronal growth cones. From this work, two compounds that lacked the teratogenic side effect of VPA but maintained the potential therapeutic effect of Ins(1,4,5)P3 depletion were identified. Another interesting effect of VPA is the increased activation of extracellular signal-regulated kinase 2 (ERK2), a component of the mitogen-activated protein kinase (MAPK) signalling pathway, in the hippocampus and frontal cortex of VPA-treated rats [61]. This effect that was suggested to cause the neuroprotective action of VPA occurs by an unknown mechanism. The complex regulation of ERK2 activation by both upstream kinase and poorly characterized downstream phosphatase activities has so far obstructed the identification of how VPA alters this pathway. However, a recent study [62] has shown that VPA also activates ERK2 in Dictyostelium. Analysis of this effect using Dictyostelium knockout mutants and inhibitors that modulate ERK2 activity in mammalian cells showed that reduced intracellular cAMP levels or a reduction in the activation of the cAMP target protein kinase A (PKA) phenocopied the effect of VPA [62]. The in vivo inhibition of the PKA signalling pathway by VPA was confirmed when the block in Dictyostelium development that is caused by VPA was overcome by transforming cells with a constitutively activated form of PKA. This study also showed that, in Dictyostelium, lithium inhibition of glycogen synthase kinase A [63] (the ortholog of mammalian GSK-3) caused elevated ERK2 activation, an effect also observed in the brain of lithium-treated rats [61]. These results therefore suggest that bipolar disorder drugs can have a common mechanism of action through the attenuation of the cAMP– PKA signalling pathway, thereby modulating ERK2 activation. Thus, this research confirms the existence of conserved drug-sensitive signalling pathways between Dictyostelium and mammalian model systems. Sphingolipids and cancer chemotherapy Various drugs are currently used in chemotherapy for many forms of cancer; however, in each case, therapeutic efficacy is often limited by cellular resistance [64]. In the

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Figure 6. Schematic illustration of VPA and cisplatin-sensitive signalling pathways. (a) Research using Dictyostelium identified the role of prolyl oligopeptidase (PO) in controlling basal inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] levels through the degradation of higher order inositol-phosphate compounds [54,58]. Ins(1,4,5)P3 signalling is modulated by lithium through the direct inhibition of a family of phosphoinositide phosphatases including inositol monophosphatase (IMPase) [87] and by VPA through an indirect effect on the inositol biosynthetic enzyme INO1 [59]. Lithium also directly inhibits glycogen synthase kinase (GSK-3/A) [63], and specific inhibition of this enzyme in Dictyostelium or reduction in cAMP production increased activation of extracellular signal-regulated kinase 2 (pERK2), an effect suggested to occur through modulation of the protein kinase A (PKA) and MAP kinase phosphatase (MAKP) signalling pathway; indeed, cells that contain constitutively active PKA overcomes the effect of both lithium and VPA on development [62]. Modulation of these targets regulate a potential neuroprotective role of these drugs in the mammalian brain [61]. (b) Ceramide and sphingosine-1-phosphate (S-1-P) are bioactive sphingolipids with various signalling functions. Molecular genetic experiments in Dictyostelium demonstrated that modulation of sphingosine kinase or S-1-P lyase can predictably alter the sensitivity to the anti-cancer drug cisplatin, as shown. These ideas were subsequently validated in human cells. Abbreviations: Glucose-6-P, glucose-6-phosphate; InsP1, inositol 1-phosphate; InsP2, inositol (1,4)-bisphosphate; InsP5, inositol (1,3,4,5,6)-pentakisphosphate; InsP6, inositol hexakisphosphate; Null, null mutants made by homologous recombination; OE, mutants overexpressing the protein; PIP2, phosphatidylinositol bisphosphate; PLC, Phospholipase C.

case of the drug cisplatin, some tumours are resistant at the onset of treatment (intrinsic resistance), whereas others become resistant during the course of therapy (acquired resistance). A large number of genes and proteins have been implicated in cisplatin resistance, and it is clear that resistance is complex and multifactorial [65]. Almost all studies of cisplatin resistance have used cells from cisplatin-resistant tumours or cells selected for cisplatin resistance by growth in increasing concentrations of drug in culture, which precludes any direct genetic study of resistance. Therefore, there was a clear need to analyze the mechanism of cisplatin function in a genetically tractable system to define the mechanisms of cellular drug resistance and to analyse drug function. To understand the mechanism of cisplatin resistance, an insertional mutagenesis screen was employed in Dictyostelium and identified six genes, the disruption of which conferred increased cisplatin resistance. None of the encoded proteins had been previously associated with cisplatin resistance [66]. One of these genes encoded the enzyme sphingosine-1-phosphate (S-1-P) lyase, which catalyzes the breakdown of S-1-P into phosphoethanolamine and hexadecenal [67] (Figure 6b). This was the first reported connection between the sphingolipid metabolic pathway and cisplatin resistance. It was reasoned that the increased cisplatin resistance of the S-1-P lyase mutant was due to an accumulation of S-1-P, which signalled the cells to survive and divide even in the presence of the drug. To examine this idea further, additional isogenic Dictyostelium strains were constructed with altered levels of the enzymes that synthesize (sphingosine kinase) or degrade (S-1-P lyase) S-1-P. The results of these experiments supported the model [68,69]. Surprisingly, both the S-1-P lyase overexpressors and the sphingosine kinase null mutants showed increased www.sciencedirect.com

sensitivity to carboplatin, another platinum-based chemotherapy treatment, but not DNA-damaging drugs that work through different mechanisms [68,69]. This increased sensitivity could be reversed partially with exogenously added S-1-P. Moreover, dimethyl sphingosine (DMS), a competitive inhibitor of sphingosine kinase [70], synergistically increased the sensitivity of cells to cisplatin, suggesting that it might be used therapeutically to increase the cytotoxicity of cisplatin without increasing its concentration [68,69]. The results that were obtained with Dictyostelium were validated in human cells [71]. The human S-1-P lyase was overexpressed in HEK293 cells and A549 lung cancer cells. In both cases, ectopic expression of human S-1-P lyase resulted in increased sensitivity to the platinum-based drugs cisplatin and carboplatin, and to doxorubicin but not etoposide or chlorambucil [71]. The effect was mediated via the activation of the p38 MAPK in the overexpressing cells. Thus, in agreement with results using Dictyostelium, overexpression of the S-1-P lyase in human cells did not indiscriminately increase sensitivity to all drugs. This is important because the problem of cross-resistance to chemotherapy drugs is a major obstacle in therapy. DMS synergistically increases the sensitivity of cisplatin in human cells, suggesting a role for DMS in therapy. Exogenous S-1-P reverses the effect of overexpression of the S-1-P lyase in a pertussis-toxin sensitive manner, indicating the involvement of the Gi-protein-coupled endothelial differentiation gene (EDG) receptors. In agreement, HEK293 cells that overexpressed the S-1-P lyase exhibited increased sensitivity to the stress of serum deprivation [72]. These studies demonstrated the utility of Dictyostelium in defining mechanisms of drug resistance and the development of strategies for increasing the therapeutic efficacy of chemotherapy drugs [73].

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S-1-P has been implicated in metastasis, cell invasiveness and angiogenesis but the underlying mechanisms remain unclear [74]. Analysis of the role of this protein, using the Dictyostelium S-1-P lyase null mutant, indicated that the protein was necessary to maintain a dominant anterior F-actin-filled pseudopod during aggregation and gene-ablation delayed aggregation [75]. Further analysis using computer-assisted motion analysis showed that the cells had normal chemotaxis but were unable to suppress lateral pseudopod formation and, therefore, were unable to maintain the normal velocity of cell motility [76]. This level of analysis needs to be extended to the other Dictyostelium sphingolipid mutants and human cells with altered levels of the S-1-P lyase and sphingosine kinase. Analyses of S-1-P lyase mutants in Drosophila and Caenorhabditis elegans suggest additional roles for these enzymes in multicellular development [77,78]. Concluding remarks This review presents recent studies demonstrating the use of Dictyostelium discoideum in human biomedical research. Other studies using Dictyostelium identified the target of aminobisphosphonate anti-bone resorptive drugs [79], and investigated the role of cancer-related nucleotide-excision repair enzymes [80] and of the Wiskott–Aldrich-syndromeassociated proteins [81,82]. Taken together, this body of work demonstrates the depth and breadth of human biomedical problems that can be addressed using this model organism. The use of this model is possible owing to similarities in cell structure, behaviour and intracellular signalling with mammalian cells, and this has enabled an increased understanding of the role of conserved proteins and the identification of unknown proteins involved in various biomedical problems. Limitations to the use of this model include a potential absence or reduced number of isoforms of some mammalian proteins and potential differences in the cellular role or binding partners of Dictyostelium and mammalian proteins. However, Dictyostelium often provides an advantage over mammalian and other model systems because of: (i) the ability to create isogenic strains with either tagged or disrupted genes for biochemical and cell biological analysis; (ii) the ease of manipulating both mitotically growing and developing cells; and (iii) the ability to identify mutant phenotypes (Box 1). These studies have positioned Dictyostelium as a valuable lead genetic organism for addressing problems of human health. This organism thus enables potential breakthroughs in defining conserved protein function or drug targets that have previously not been possible in more-complex systems. Box 2. Future use of Dictyostelium as a biomedical model  Analyzing the function and binding partners of conserved proteins that are involved in both heritable and induced illnesses [6].  Defining mechanisms and components of cell movement to understand processes of human cell immunity and metastasis.  Identifying proteins within cells that can be targeted to limit replication of intracellular pathogens.  Defining more novel drug targets and signalling cascades in relation to medical conditions or drugs of unknown mechanisms. www.sciencedirect.com

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Clearly, there is great scope for continued work with this adaptable model, and more areas of biomedical research that can be usefully advanced (Box 2). Acknowledgements Work in the authors’ laboratories was supported by: Wellcome Trust Career Development Award (R.S.B.W.); NIH grants GM53929 and CA95872 (H.A. and S.A.); NIH grant HD18577 and the Shwachman– Diamond Syndrome Foundation (D.R.S. and D.W.); Deutsche Forschungsgemeinschaft SFB413 (R.G. and A.M.T.) and GR1642/2–2 (R.G.); and the Howard Hughes Medical Institute (R.I.). H.A. and S.A. wish to thank Dr. J. Min for important discussions.

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