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  • Isolation of spdA-ins mutant cells: All Dictyostelium strains used in this study were derived from the subclone DH1-10 [5] of the DH1 strain, referred to as wild-type (WT) for simplicity. Cells were grown at 21°C in HL5 medium (14.3 g/L peptone (Oxoid, Hampshire, England), 7.15 g/L yeast extract, 18 g/L maltose monohydrate, 0.641 g/L Na2HPO4.2H2O, 0.490 g/L KH2PO4) and subcultured twice a week to maintain a maximal density of 106 cells/ml. Unless otherwise specified, all experiments presented in this study were done with cells grown to a density of approximately 500’000 cells per mL. Random mutants were generated by restriction enzyme-mediated integration (REMI) mutagenesis [10], subcloned individually, then tested for their ability to grow on a lawn of bacteria as described previously [11]. In this study, one mutant growing inefficiently on a laboratory strain of Micrococcus luteus (Ml) bacteria was selected for further analysis. The genomic DNA from these spdA-ins mutant cells was recovered, digested with ClaI and religated, and the mutagenic plasmid was recovered together with the flanking regions of its genomic insertion site (Fig 1). This plasmid was sequenced to identify the insertion site. It was also used after ClaI digestion to transfect WT cells and generate targeted spdA-ins mutant cells. Three independent spdA-ins mutant clones were generated, and used in parallel in this study, with indistinguishable results (Fig 1). Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0160376.g001 Characterization of spdA-ins mutant cells. (A) SpdA-ins mutant cells were originally created by the random insertion of a REMI mutagenic vector (pSC) in the coding sequence of gene DDB_G0287845 (position 2635). (B) To quantify growth of Dictyostelium on bacteria, we applied 10'000, 1'000, 100 or 10 Dictyostelium cells on a lawn of K. pneumoniae or M. luteus bacteria (black). WT cells created a phagocytic plaque (white). SpdA mutant cells grew as efficiently as WT cells on a lawn of K. pneumoniae but less efficiently in the presence of M. luteus. (C) Growth of Dictyostelium mutant strains in the presence of different bacterial species. Phagocytosis and Fluid Phase Uptake: Phagocytosis and fluid phase uptake were determined as described previously [5] by incubating cells for 20 min in suspension in HL5 medium containing either 1-μm-diameter Fluoresbrite YG carboxylate microspheres (Polysciences, Warrington, PA) or Alexa647-dextran (Life Technologies, Eugene, OR). Cells were then washed twice with HL5 containing 0.2% NaN3, and the internalized fluorescence was measured by flow cytometry [5]. Kinetics of phagocytosis were determined similarly after 0, 5, 10, 15, 20, 30, 40, 60, 90, 120 and 150 min of incubation. Since these experiments required a large number of cells, the cells were grown to a higher concentration than for all other experiments described in this study (1.5x106 cells per mL vs 500’000 cells per mL). This accounts for the fact that phagocytosis was less efficient for all strains in this set of experiments. To determine if the elevated phagocytosis observed in spdA-ins mutant cells was a cell-autonomous phenotype, we mixed WT cells and spdA-ins mutant cells expressing GFP (ratio 1:1). After three days of co-culture in HL5, phagocytosis was analyzed as described above, but using latex beads fluorescent in the rhodamine channel (Polysciences, Inc). During flow cytometry, GFP fluorescence allowed to distinguish WT from spdA-ins mutant/GFP cells. In order to visualize cell spreading on a substrate, 1.5x105 cells were allowed to adhere for 20 min on a glass surface in a FluoroDish (World Precision Instr., Sarasota, FL). To monitor the presence and spreading of D. discoideum cells we used an inverted microscope (Olympus IX71 or Zeiss Axiovert 100M) and imaged by phase contrast and Reflection Interference Contrast Microscopy (RICM) as previously described [12]. Images and movies (15 frames per second) were acquired with an Olympus DP30 CCD camera or a High resolution black/white CCD camera (Hamamatsu CCD cooled camera). RICM images were sub-sampled at 1 image per 1.2 s, the background was subtracted and flattened and the noise filtered. Dark cell-surface contact zones were defined by segmentation and quantified as described [13]. To measure cell motility, cells were observed for 60 min (picture every 30 sec) by phase contrast with a Plan-Neofluar 10x magnification. Pictures were taken with a Hamamatsu CCD cooled camera. We used Particle tracking from the Metamorph software to track individual cell trajectories. In these experiments, borosilicate glass plates were first cleaned with detergent in alkaline conditions, then briefly detached with a 14 M NaOH solution, thoroughly rinsed and dried. Radial flow experiments were performed essentially as described previously [14, 15]. Briefly, cells were resuspended in HL5 (106 cells/mL) and allowed to attach on the glass surface during 10 min, then a radial hydrodynamic flow was applied for 10 min and the density of the remaining attached cells was determined as a function of the distance to the center of the flow. The results were expressed as the percentage of detached cells as a function of the applied shear stress. To visualize filamentous actin, cells were allowed to adhere on a glass coverslip for 10 min in HL5 and were fixed in PB containing 4% paraformaldehyde for 30 min. This fixation was sufficient to permeabilize the cells. The actin cytoskeleton was labeled by incubating the cells for 1h in phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin and 1μg/ml tetramethylrhodamine B isothiocyanate (TRITC)-labeled phalloidin. The coverslips were then washed twice, mounted and observed by laser scanning confocal microscopy (Zeiss LSM 510). The pictures presented represent optical sections at the site of contact between the cells and the substrate. For scanning electron microscopy, cells were incubated on glass coverslips overnight in HL5. Cells were then fixed with 2% glutaraldehyde in HL5 for 30 min followed by 2% glutaraldehyde in 100 mM PB (pH 7.14) for 30 min. Cells were rinsed and postfixed in 1% osmium tetroxide in 100 mM PB (pH 7.14) for 1 h. The fixative was removed, and cells were progressively dehydrated through a 25–100% ethanol series. After air-drying, cells were sputter-coated in gold and viewed on a JEOL-JSM-7001 FA Field Emission Scanning Electron Microscope. To measure cell size based on electric current exclusion (CASY technology), cells were grown to a density of 50x104 cells/mL, diluted to 1x104 cells/mL, and 10 mL were analyzed using a CASY 1 cell counter (Roche; CASY Model TTC). To determine packed cell volume, cells are grown to a density of 3x106 cells/mL. Cells were counted under a Nikon eclipse TS100 microscope with a cell counting Neubauer chamber. 3x106 cells in 1 mL were transferred in the packed cell volume (PCV) tube with calibrated capillary and volume graduation (5μL) (TPP Techno Plastic Products AG; Product no 87005). Cells were centrifuged 2 min at 1500 rpm, and the pellet volume measured in the calibrated capillary. The ratio μL of pellet/number of cells was calculated. To measure the amount of protein per cell, 106 cells were collected by centrifugation, washed once in 1ml PBS, resuspended in 50 μL PBS containing triton X-100, 0.05% and transferred to a 96 well plate. To quantify protein content using a Lowry assay (DC Protein Assay, Bio-RAD) we added to each well 25 μL of reagent A and 200μL reagent B. After 15 min the absorbance at 750 nm was measured in a microplate reader, and compared to a set of calibrated serial dilutions. To determine the cellular amounts of SibA, Phg1 and Talin, we resuspended cell pellets in sample buffer and separated the proteins on a polyacrylamide gel (7% for SibA and Talin and 10% for Phg1), after which they were transferred to a nitrocellulose membrane (Invitrogen, Carlsbad, CA). The membranes were incubated with a polyclonal anti-SibA antibody (SibA) [12], the YC1 rabbit antipeptide antiserum to a Phg1 peptide [5], or the anti-talin 169.477.5 [16], a kind gift from Prof. G. Gerisch (Martinsried, Germany). Binding of antibodies was revealed by ECL using a secondary HRP-coupled antibody (Amersham Biosciences). The signal intensity was evaluated using Quantity One software (Bio-Rad).
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