LPIN2 is the phosphatase dominating the penultimate step of neutral lipid biosynthesis in .

amoebae store surplus fatty acids from the diet in form of lipid droplets. Some of the enzymes governing neutral lipid synthesis are already known. For the phosphatidic acid-specific phosphatases, six genes were found, one of which was automatically annotated as LPIN2.

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

Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. , 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.

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

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 resides on the endoplasmic reticulum. When an excess of fatty acids is present, it redistributes to storage organelles, the lipid droplets.

Fat-containing cells are eliminated during development.

Triacylglycerol is a universal storage molecule for metabolic energy in living organisms. However, 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 , encoding a fatty acid activating enzyme, or and , 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Conserved features of endocytosis in Dictyostelium.

Endocytosis in protozoa is often regarded as largely different from the pathways operating in mammalian cells. Experiments in the amoeba Dictyostelium, one of the genetically tractable single-celled organisms, have allowed us to manipulate the flow through endocytic compartments and to study the dynamic distribution of molecules by means of green fluorescent protein fusions. This review attempts to compile the molecular data available from Dictyostelium and assign them to specific steps of internalization by phagocytosis or macropinocytosis and to subsequent stages of the endocytic pathway.