The causative agent of Legionnaires' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct "Legionella-containing vacuole" (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed presence small GTPase Ran its effector RanBP1 on purified LCVs. Here we validate that localize LCVs promote intracellular growth L. pneumophila. Moreover, pneumophila protein LegG1, contains putative RCC1 guanine nucleotide...

Red fluorescent proteins (RFPs) combined with GFP are attractive probes for double-fluorescence labeling of in live cells. However, the application these is restrained by stable oligomer formation and their weak fluorescence vivo. Previous attempts to eliminate problems mutagenesis RFP from Discosoma (DsRed) resulted monomeric mRFP1 tetrameric RedStar RFP, which distinguished its enhanced Based on mutations, we have generated an mRFPmars, report spectral properties. Together green labels,...

Dictyostelium discoideum contains a full-length homologue of talin, protein implicated in linkage the actin system to sites cell-to-substrate adhesion fibroblasts and neuronal growth cones. Gene replacement eliminated talin led defects phagocytosis interaction moving cells, two processes dependent on continuous cross talk between cell surface underlying cytoskeleton. The uptake rate yeast particles was reduced, only bacteria devoid carbohydrate moiety lipopolysaccharides were adhesive enough...

Abstract The Arp2/3 complex is a ubiquitous and important regulator of the actin cytoskeleton. Here we identify this from Dictyostelium investigate its dynamics in live cells. predicted sequences subunits show strong homology to members mammalian complex, with larger generally better conserved than smaller ones. In highly motile cells , rapidly re‐distributed cytoskeleton response external stimuli. Fusions Arp3 p41‐Arc GFP reveal that phagocytosis, macropinocytosis, chemotaxis recruited...

In a motile eukaryotic cell, front protrusion and tail retraction are superimposed on each other. To single out mechanisms that result in to or transition, we separated the two processes time using cells oscillate between full state. State transitions were visualized by total internal reflection fluorescence microscopy as marker PIP3 (phosphatidylinositol [3,4,5] tris-phosphate), tumor-suppressor PTEN (phosphatase tensin homolog) degrades PIP3. Negative fluctuations layer of membrane gated...

The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting 1, Aip1p, Saccharomyces cerevisiae, related proteins Caenorhabditis, Physarum, higher eukaryotes. We show that localized dynamic regions of cell cortex are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, other pseudopodia. In cells expressing green fluorescent (GFP)-tagged DAip1, rapidly redistributes into newly formed cortical protrusions. Functions...

Actin networks are continuously reorganized in cells that rapidly change their shape. Applying total internal reflection fluorescence microscopy at acquisition rates of 10–20 Hz, we measured an average growth rate 3 μm·sec –1 for filamentous actin structures throughout the entire substrate-attached cortex Dictyostelium cells. New filaments often proceed along preexisting ones, resulting bundle formation concurrent with filament growth. In orientate a gradient chemoattractant, prominent...

In an attempt to identify unknown actin-binding proteins in cells of Dictyostelium discoideum that may be involved the control cell motility and chemotaxis, monoclonal antibodies were raised against had been enriched on F-actin affinity matrix. One antibody recognized a protein distinguished by its strong accumulation at tips filopods. These cell-surface extensions containing core bundled actin filaments are rapidly protruded retracted growth-phase stage. The 269 kD turned out resemble mouse...

Abstract Background The uptake of particles by actin-powered invagination the plasma membrane is common to protozoa and phagocytes involved in immune response higher organisms. question addressed here how a phagocyte may use geometric cues optimize force generation for particle. We survey mechanisms that enable remodel actin organization complex shape. Results Using consist two lobes separated neck, we found Dictyostelium cells transmit signals concerning curvature surface system underlying...

The environmental bacterium Legionella pneumophila causes a severe pneumonia termed Legionnaires' disease. L. employs conserved mechanism to replicate within specific vacuole in macrophages or protozoa such as the social soil amoeba Dictyostelium discoideum. Pathogen-host interactions depend on Icm/Dot type IV secretion system (T4SS), which translocates approximately 300 different effector proteins into host cells. Here we analyse effects of migration and chemotaxis amoebae,...

Porin of Dictyostelium discoideum was extracted from mitochondria with Genapol X-80 and purified by hydroxyapatite CM-cellulose chromatography. The protein displayed a single band 30 kDa in SDS-polyacrylamide gel electrophoresis. formation channels artificial lipid bilayer membranes defined its function as channel-forming component. Its average single-channel conductance 3.9 nanosiemens 1 M KCl, which suggested that the effective diameter channel is approximately 1.7 nm at small...

Ubiquitin is a highly conserved, multifunctional protein, which implicated in the heat-shock response of eukaryotes. The differential expression multiple ubiquitin genes Dictyostelium discoideum was investigated under various stress conditions. Growing D. cells express four major transcripts sizes varying from 0.6 to 1.9 kb. Upon heat shock three additional mRNAs 0.9, 1.2 and 1.4 kb accumulate within 30 min. same are expressed cold or cadmium treatment. Inhibition protein synthesis by...

Valproic acid (VPA) is used to treat epilepsy and bipolar disorder prevent migraine. It also undergoing trials for cancer therapy. However, the biochemical molecular biological actions of VPA are poorly understood. Using social amoeba Dictyostelium discoideum, we show that an acute effect inhibition chemotactic cell movement, a process partially dependent upon phospholipid signaling. Analysis this shows attenuates signal-induced translocation PH(Crac)-green fluorescent protein from cytosol...

The contractile vacuole system is an osmoregulatory organelle composed of cisternae and interconnecting ducts. Large act as bladders that periodically fuse with the plasma membrane, forming pores to expel water. To visualize entire network in vivo identify constituents vacuolar complex cell fractions, we introduced a specific marker into Dictyostelium cells, GFP-tagged dajumin. C-terminal, region this transmembrane protein responsible for sorting complex. Dajumin-GFP negligibly associates...

Abstract DdLimE regulates cell motility and cytokinesis in Dictyostelium . To specify its function, we generated knock‐out mutants analyzed mitosis by marking the mitotic apparatus with GFP‐α‐tubulin. Characteristic of DdLimE‐null cells is a late reversal caused backward movement incipient daughter cells. This process “retro‐cytokinesis” accompanied delay disassembly spindle. The length interphase microtubules increased their depolymerization at prophase impaired. These data indicate that...