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Biomedical subjects

Markus Maniak

Publications and source records attributed to Markus Maniak.

8 recordsLinked to original sources
PubMed2006

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.

Amino Acid Sequence↗
PubMed2006

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.

Acid Phosphatase↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2003

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.

Actin Depolymerizing Factors↗
PubMed2003

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.

Animals↗
PubMed2003

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.

Amino Acid Sequence↗
PubMed2002

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. Parallels to phagocytes of the mammalian immune system are emphasized. The major distinctive feature between mammalian phagocytes and free-living cells is the need for osmoregulation. Therefore Dictyostelium cells possess a contractile vacuole that has occasionally obscured analysis of endocytosis but is now found to be entirely separate from endocytic organelles. In conclusion, the potential of Dictyostelium amoebas to provide a model system of mammalian phagocytes is ever increasing.

Animals↗