ER-dependent membrane repair of mycobacteria-induced vacuole damage.

IMPORTANCE: Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis, the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the Dictyostelium discoideum/Mycobacterium marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as M. tuberculosis.

Phospholipids containing ether-bound hydrocarbon-chains are essential for efficient phagocytosis and neutral lipids of the ester-type perturb development in Dictyostelium.

Lipids are the building blocks for cellular membranes; they provide signalling molecules for membrane dynamics and serve as energy stores. One path of their synthesis is initiated by glycerol-3-phosphate acyltransferase (GPAT), which in Dictyostelium resides on the endoplasmic reticulum. When an excess of fatty acids is present, it redistributes to storage organelles, the lipid droplets. Mutants, where the GPAT was eliminated by homologous recombination, produce fewer lipid droplets and are almost devoid of triacylglycerols (TAG), rendering them more resistant to cell death and cell loss in the developmental stages preceding fruiting body formation. The enzyme most closely related to GPAT is called FARAT, because it combines a fatty acyl-reductase (FAR) and an acyltransferase (AT) domain in its sequence. The protein is confined to the lumen of the peroxisome, where it transfers a fatty acid to dihydroxyacetone-phosphate initiating the synthesis of ether lipids, later completed at the endoplasmic reticulum. A mutant lacking FARAT produces lipid droplets that are devoid of the storage lipid monoalkyl-diacyl-glycerol (MDG), but the efficiency of spore formation in the developmental cycle is largely unaltered. Instead, these mutants are strongly impaired in phagocytosis of yeast particles, which is attributed to reduced synthesis of membrane phospholipids containing ether-linked chains.

Fat-containing cells are eliminated during Dictyostelium development.

Triacylglycerol is a universal storage molecule for metabolic energy in living organisms. However, Dictyostelium amoebae, that have accumulated storage fat from added fatty acids do not progress through the starvation period preceding the development of the durable spore. Mutants deficient in genes of fat metabolism, such as fcsA, encoding a fatty acid activating enzyme, or dgat1 and dgat2, specifying proteins that synthesize triacylglycerol, strongly increase their chances to contribute to the spore fraction of the developing fruiting body, but lose the ability to produce storage fat efficiently. Dictyostelium seipin, an orthologue of a human protein that in patients causes the complete loss of adipose tissue when mutated, does not quantitatively affect fat storage in the amoeba. Dictyostelium seiP knockout mutants have lipid droplets that are enlarged in size but reduced in number. These mutants are as vulnerable as the wild type when exposed to fatty acids during their vegetative growth phase, and do not efficiently enter the spore head in Dictyostelium development.

The C Isoform of Dictyostelium Tetraspanins Localizes to the Contractile Vacuole and Contributes to Resistance against Osmotic Stress.

Tetraspanins (Tsps) are membrane proteins that are widely expressed in eukaryotic organisms. Only recently, Tsps have started to acquire relevance as potential new drug targets as they contribute, via protein-protein interactions, to numerous pathophysiological processes including infectious diseases and cancer. However, due to a high number of isoforms and functional redundancy, knowledge on specific functions of most Tsps is still scarce. We set out to characterize five previously annotated Tsps, TspA-E, from Dictyostelium discoideum, a model for studying proteins that have human orthologues. Using reverse transcriptase PCRs, we found mRNAs for TspA-E in the multicellular slug stage, whereas vegetative cells expressed only TspA, TspC and, to a lesser extent, TspD. We raised antibodies against TspA, TspC and TspD and detected endogenous TspA, as well as heterologously expressed TspA and TspC by Western blot. N-deglycosylation assays and mutational analyses showed glycosylation of TspA and TspC in vivo. GFP-tagged Tsps co-localized with the proton pump on the contractile vacuole network. Deletion strains of TspC and TspD exibited unaltered growth, adhesion, random motility and development. Yet, tspC- cells showed a defect in coping with hypo-osmotic stress, due to accumulation of contractile vacuoles, but heterologous expression of TspC rescued their phenotype. In conclusion, our data fill a gap in Dictyostelium research and open up the possibility that Tsps in contractile vacuoles of e.g. Trypanosoma may one day constitute a valuable drug target for treating sleeping sickness, one of the most threatening tropical diseases.

Lipid droplet dynamics at early stages of Mycobacterium marinum infection in Dictyostelium.

Lipid droplets exist in virtually every cell type, ranging not only from mammals to plants, but also to eukaryotic and prokaryotic unicellular organisms such as Dictyostelium and bacteria. They serve among other roles as energy reservoir that cells consume in times of starvation. Mycobacteria and some other intracellular pathogens hijack these organelles as a nutrient source and to build up their own lipid inclusions. The mechanisms by which host lipid droplets are captured by the pathogenic bacteria are extremely poorly understood. Using the powerful Dictyostelium discoideum/Mycobacterium marinum infection model, we observed that, immediately after their uptake, lipid droplets translocate to the vicinity of the vacuole containing live but not dead mycobacteria. Induction of lipid droplets in Dictyostelium prior to infection resulted in a vast accumulation of neutral lipids and sterols inside the bacterium-containing compartment. Subsequently, under these conditions, mycobacteria accumulated much larger lipid inclusions. Strikingly, the Dictyostelium homologue of perilipin and the murine perilipin 2 surrounded bacteria that had escaped to the cytosol of Dictyostelium or microglial BV-2 cells respectively. Moreover, bacterial growth was inhibited in Dictyostelium plnA knockout cells. In summary, our results provide evidence that mycobacteria actively manipulate the lipid metabolism of the host from very early infection stages.

Dictyostelium discoideum Dgat2 can substitute for the essential function of Dgat1 in triglyceride production but not in ether lipid synthesis.

Triacylglycerol (TAG), the common energy storage molecule, is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in Dictyostelium discoideum are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the endoplasmic reticulum (ER)-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite from what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The Dictyostelium Dgat1, however, produces TAG, wax esters, and, most interestingly, also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG through either Dgat1 or Dgat2 activity is essential for Dictyostelium to grow on bacteria, its natural food substrate.

Dictyostelium lipid droplets host novel proteins.

Across all kingdoms of life, cells store energy in a specialized organelle, the lipid droplet. In general, it consists of a hydrophobic core of triglycerides and steryl esters surrounded by only one leaflet derived from the endoplasmic reticulum membrane to which a specific set of proteins is bound. We have chosen the unicellular organism Dictyostelium discoideum to establish kinetics of lipid droplet formation and degradation and to further identify the lipid constituents and proteins of lipid droplets. Here, we show that the lipid composition is similar to what is found in mammalian lipid droplets. In addition, phospholipids preferentially consist of mainly saturated fatty acids, whereas neutral lipids are enriched in unsaturated fatty acids. Among the novel protein components are LdpA, a protein specific to Dictyostelium, and Net4, which has strong homologies to mammalian DUF829/Tmem53/NET4 that was previously only known as a constituent of the mammalian nuclear envelope. The proteins analyzed so far appear to move from the endoplasmic reticulum to the lipid droplets, supporting the concept that lipid droplets are formed on this membrane.

The isoform B of the Dictyostelium long-chain fatty-acyl-coenzyme A synthetase is initially inserted into the ER and subsequently provides peroxisomes with an activity important for efficient phagocytosis.

Long-chain fatty-acyl-coenzyme A synthetases activate fatty acids for anabolic or catabolic metabolism. They often localize to more than one organelle within eukaryotic cells. Dictyostelium contains two of these proteins, FcsA and FcsB with the latter being targeted to the membrane of the endoplasmic reticulum by virtue of an N-terminal signal sequence and from there appears to move on to peroxisomes. Deletion of this signal favors the peripheral association of the protein with the mitochondrial surface instead. A strain lacking the activity of the FcsB enzyme was constructed by homologous recombination. It has a severe deficiency in the phagocytic uptake of particles, which can be partially alleviated by a peroxisomally targeted, soluble FcsA enzyme. It is, however, not rescued by expressing FcsA in the cytoplasm or targeting it to the ER, indicating that peroxisomal ß-oxidation is important for phagocytosis. In a fcsA(-)/B(-) double mutant phagocytosis efficiency is similar to fcsB(-) cells. However, unlike the single mutants, the fcsA(-)/B(-) strain is delayed in morphogenesis, but forms viable spores, albeit within a small fruiting body. This developmental defect is also seen in other mutants affecting peroxisomal enzymes involved in ß-oxidation and the glyoxylate cycle.

Dictyostelium as a model for human lysosomal and trafficking diseases.

Dictyostelium cells are genetically haploid and therefore easily analyzed for mutant phenotypes. In the past, many tools and molecular markers have been developed for a quantitative and qualitative analysis of the endocytic pathway in these amoebae. This review outlines parallels and discrepancies between mutants in Dictyostelium, the corresponding mammalian cells and the symptoms of human patients affected by lysosomal and trafficking defects. Situations where knowledge from Dictyostelium may potentially help understand human disease and vice versa are also addressed.

Targeting the actin-binding protein VASP to late endosomes induces the formation of giant actin aggregates.

In vitro, the vasodilator-stimulated phosphoprotein (VASP) acts as a regulator of actin filament assembly in many ways. In cells it localizes to sites where actin is rapidly polymerized such as filopodia, lamellipodia, and focal adhesions. We have mistargeted VASP to the surface of the late endosome in Dictyostelium cells thereby inducing the formation of a dense actin aggregate which sequesters various actin-binding proteins and endosomal components. Depletion of these proteins from the cytoplasm leads to phenotypes mimicking the corresponding knockout cells. Some properties of the actin aggregate are reminiscent of Hirano bodies that are often observed in nerve tissue from patients suffering from neurodegenerative diseases, opening the possibility that protein sequestration contributes to neuronal malfunction.

Competition between targeting signals in hybrid proteins provides information on their relative in vivo affinities for subcellular compartments.

After their translation and folding in the cytoplasm, proteins may be imported into an organelle, associate with a membrane, or rather become part of large, highly localised cytoplasmic structures such as the cytoskeleton. The localisation of a protein is governed by the strength of binding to its immediate target, such as an import receptor for an organelle or a major component of the cytoskeleton, e.g. actin. We have experimentally provided a set of actin-binding proteins with competing targeting information and expressed them at various concentrations to analyse the strength of the signal that governs their subcellular localisation. Our microscopic observations indicate that organellar sorting signals override the targeting preference of most cytoskeletal proteins. Among these signals, the nuclear localisation signal of SV40 is strongest, followed by the oligomerised PHB domain that targets vacuolin to the endosomal surface, and finally the tripeptide SKL mediating transport into the peroxisome. The actin-associated protein coronin, however, can only be misled by the nuclear localisation signal. Interestingly, the targeting behaviour of this model set of hybrid proteins in living Dictyostelium amoebae correlates surprisingly well with the affinities of their constituent signals derived from in vitro experiments conducted in various other organisms. Accordingly, this approach allows estimating the in vivo affinity of a protein to its target even if the latter is not known, as in the case of vacuolin.

The BEACH protein LvsB is localized on lysosomes and postlysosomes and limits their fusion with early endosomes.

The Chediak-Higashi syndrome (CHS) is a genetic disorder caused by the loss of the BEACH protein Lyst. Impaired lysosomal function in CHS patients results in many physiological problems, including immunodeficiency, albinism and neurological problems. Dictyostelium LvsB is the ortholog of mammalian Lyst and is also important for lysosomal function. A knock-in approach was used to tag LvsB with green fluorescent protein (GFP) and express it from its single chromosomal locus. GFP-LvsB was observed on late lysosomes and postlysosomes. Loss of LvsB resulted in enlarged postlysosomes, in the abnormal localization of proton pumps on postlysosomes and their abnormal acidification. The abnormal postlysosomes in LvsB-null cells were produced by the inappropriate fusion of early endosomal compartments with postlysosomal compartments. The intermixing of compartments resulted in a delayed transit of fluid-phase marker through the endolysosomal system. These results support the model that LvsB and Lyst proteins act as negative regulators of fusion by limiting the heterotypic fusion of early endosomes with postlysosomal compartments.

Attenuation of phospholipid signaling provides a novel mechanism for the action of valproic acid.

Valproic acid (VPA) is used to treat epilepsy and bipolar disorder and to prevent migraine. It is also undergoing trials for cancer therapy. However, the biochemical and molecular biological actions of VPA are poorly understood. Using the social amoeba Dictyostelium discoideum, we show that an acute effect of VPA is the inhibition of chemotactic cell movement, a process partially dependent upon phospholipid signaling. Analysis of this process shows that VPA attenuates the signal-induced translocation of PH(Crac)-green fluorescent protein from cytosol to membrane, suggesting the inhibition of phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) production. Direct labeling of lipids in vivo also shows a reduction in PIP and PIP(2) phosphorylation following VPA treatment. We further show that VPA acutely reduces endocytosis and exocytosis-processes previously shown to be dependent upon PIP(3) production. These results suggest that in Dictyostelium, VPA rapidly attenuates phospholipid signaling to reduce endocytic trafficking. To examine this effect in a mammalian model, we also tested depolarization-dependent neurotransmitter release in rat nerve terminals, and we show that this process is also suppressed upon application of VPA and an inhibitor of phosphatidylinositol 3-kinase. Although a more comprehensive analysis of the effect of VPA on lipid signaling will be necessary in mammalian systems, these results suggest that VPA may function to reduce phospholipid signaling processes and thus may provide a novel therapeutic effect for this drug.

Quantitative and microscopic methods for studying the endocytic pathway.

Endocytosis is a process that is essential to the life of all eukaryotic cells. Laboratory strains of Dictyostelium are extremely efficient in the uptake of both particles and fluid. Many different cellular processes feed into the endocytic pathway, and many organelle-associated and cytoplasmic proteins, including the ones from the cytoskeleton, contribute to the efficiency of transit. Therefore mutants, especially in genes of unknown function, must be characterized regarding their endocytic performance. We describe the most common tools and protocols to visualize and quantify all of the individual steps in endocytic transit.

Vacuolin, a flotillin/reggie-related protein from Dictyostelium oligomerizes for endosome association.

We have analysed the domain structure of vacuolin, a Dictyostelium protein binding to the cytoplasmic surface of late endosomes. Localisation studies using GFP fusions together with a yeast two-hybrid analysis and co-immunoprecipitation experiments reveal that a region close to the C-terminus mediates oligomer formation of the protein through a coiled-coil mechanism which in turn is a prerequisite for the efficient binding to endosomal membranes via a prohibitin (PHB) domain in the middle of the molecule. Overexpression of the coiled-coil domain strongly competes with endogenous vacuolin in the oligomers and reduces the efficiency of membrane targeting. The domain arrangement of vacuolin is most similar to flotillin/reggie, a protein found on late endosomes of mammalian cells.

A Dictyostelium mutant with reduced lysozyme levels compensates by increased phagocytic activity.

Lysozymes are bacteria-degrading enzymes and play a major role in the immune defense of animals. In free-living protozoa, lysozyme-like proteins are involved in the digestion of phagocytosed bacteria. Here, we purified a protein with lysozyme activity from Dictyostelium amoebae, which constitutes the founding member, a novel class of lysozymes. By tagging the protein with green fluorescent protein or the Myc epitope, a new type of lysozyme-containing vesicle was identified that was devoid of other known lysosomal enzymes. The most highly expressed isoform, encoded by the alyA gene, was knocked out by homologous recombination. The mutant cells had greatly reduced enzymatic activity and grew inefficiently when bacteria were the sole food source. Over time the mutant gained the ability to internalize bacteria more efficiently, so that the defect in digestion was compensated by increased uptake of food particles.

A Dictyostelium long chain fatty acyl coenzyme A-synthetase mediates fatty acid retrieval from endosomes.

We have identified a subset of Dictyostelium endosomes that carry a long chain fatty acyl coenzyme A-synthetase (LC-FACS 1) on their cytosolic surface. Immunofluorescence studies and observations using GFP-fusion proteins collectively suggest that LC-FACS 1 associates with endosomes a few minutes after their formation, remains bound through the acidic phase of endocytic maturation and dissociates early in the phase where the endosomal content is neutralised prior to exocytosis. Mutants in the fcsA gene, encoding the LC-FACS 1 protein, were constructed by homologous recombination. These cells show a strong defect in the intracellular accumulation of fatty acids, either taken up together with the liquid medium or bound to the surface of particles. Because the mutant cells are otherwise fully competent for macropinocytosis and phagocytosis, we conclude that the LC-FACS 1 protein mediates the retrieval of fatty acids from the lumen of endosomes into the cytoplasm.

A coat of filamentous actin prevents clustering of late-endosomal vacuoles in vivo.

The endocytic pathway depends on the actin cytoskeleton. Actin contributes to internalization at the plasma membrane and to subsequent trafficking steps like propulsion through the cytoplasm, fusion of phagosomes with early endosomes, and transport from early to late endosomes. In vitro studies with mammalian endosomes and yeast vacuoles implicate actin in membrane fusion. Here, we investigate the function of the actin coat that surrounds late endosomes in Dictyostelium. Latrunculin treatment leads to aggregation of these endosomes into grape-like clusters and completely blocks progression of endocytic marker. In addition, the cells round up and stop moving. Because this drug treatment perturbs all actin assemblies in the cell simultaneously, we used a novel targeting approach to specifically study the function of the cytoskeleton in one subcellular location. To this end, we constructed a hybrid protein targeting cofilin, an actin depolymerizing protein, to late endosomes. As a consequence, the endosomal compartments lost their actin coats and aggregated, but these cells remained morphologically normal, and the kinetics of endocytic marker trafficking were unaltered. Therefore, the actin coat prevents the clustering of endosomes, which could be one safeguard mechanism precluding their docking and fusion.

Fusion and fission events in the endocytic pathway of Dictyostelium.

The endocytic pathway in Dictyostelium appears as a short circuit between endocytosis and exocytosis. Within the hour that elapses between internalization of nutrients and release of remnants, digestion by lysosomal enzymes occurs. Meanwhile, the maturing endosome undergoes a complex series of fusion and fission events, which change its character profoundly and which are far from being fully understood. This review attempts to order the dynamic events into a sequence of stages that is most consistent with present knowledge.