Role of SpdA in Cell Spreading and Phagocytosis in Dictyostelium.

Dictyostelium discoideum is a widely used model to study molecular mechanisms controlling cell adhesion, cell spreading on a surface, and phagocytosis. In this study we isolated and characterize a new mutant created by insertion of a mutagenic vector in the heretofore uncharacterized spdA gene. SpdA-ins mutant cells produce an altered, slightly shortened version of the SpdA protein. They spread more efficiently than WT cells when allowed to adhere to a glass substrate, and phagocytose particles more efficiently. On the contrary, a functional spdA knockout mutant where a large segment of the gene was deleted phagocytosed less efficiently and spread less efficiently on a substrate. These phenotypes were highly dependent on the cellular density, and were most visible at high cell densities, where secreted quorum-sensing factors inhibiting cell motility, spreading and phagocytosis are most active. These results identify the involvement of SpdA in the control of cell spreading and phagocytosis. The underlying molecular mechanisms, as well as the exact link between SpdA and cell spreading, remain to be established

Synergistic control of cellular adhesion by transmembrane 9 proteins

The transmembrane 9 (TM9) family of proteins contains numerous members in eukaryotes. Although their function remains essentially unknown in higher eukaryotes, the Dictyostelium discoideum Phg1a TM9 protein was recently reported to be essential for cellular adhesion and phagocytosis. Herein, the function of Phg1a and of a new divergent member of the TM9 family called Phg1b was further investigated in D. discoideum. The phenotypes of PHG1a, PHG1b, and PHG1a/PHG1b double knockout cells revealed that Phg1a and Phg1b proteins play a synergistic but not redundant role in cellular adhesion, phagocytosis, growth, and development. Complementation analysis supports a synergistic regulatory function rather than a receptor role for Phg1a and Phg1b proteins. Together, these results suggest that Phg1 proteins act as regulators of cellular adhesion, possibly by controlling the intracellular transport in the endocytic pathway and the composition of the cell surface

S<i>pdA-ins</i> mutant cells phagocytose particles faster than WT cells.

<p>Cells were incubated for 20 min in HL5 medium containing fluorescent dextran or fluorescent latex beads. Cells were then washed, and internalized fluorescence was measured by flow cytometry. (A) Uptake of fluorescent dextran was expressed as a percentage of the value obtained for the WT cells. (B) Phagocytosis of fluorescent beads was expressed as the average number of beads ingested per cell. The average and SEM of 6 independent samples are presented. *: p<0.01 (Student t-test). (C) Cells were incubated for 0, 5, 10, 15, 20, 30, 60, 90, 120, or 150 min in HL5 medium containing fluorescent latex beads. The average and SEM of 4 independent experiments are presented. <i>SpdA-ins</i> mutant cells ingested particles faster than WT cells.</p

The actin organization is not significantly altered in <i>spdA-ins</i> cells.

<p>Cells were allowed to adhere to a glass coverslip for 10 min in HL5. After fixation filamentous actin was labeled with fluorescent phalloidin. The contact area between cells and their substrate was visualized by confocal microscopy, and did not reveal gross alterations of actin organization in <i>spdA-ins</i> cells. When cells were incubated in phosphate buffer (PB), formation of filopodia was induced in both WT and <i>spdA-ins</i> cells.</p

Characterization of <i>spdA-ins</i> mutant cells.

<p>(A) <i>SpdA-ins</i> 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 <i>Dictyostelium</i> on bacteria, we applied 10'000, 1'000, 100 or 10 <i>Dictyostelium</i> cells on a lawn of <i>K</i>. <i>pneumoniae</i> or <i>M</i>. <i>luteus</i> bacteria (black). WT cells created a phagocytic plaque (white). <i>SpdA</i> mutant cells grew as efficiently as WT cells on a lawn of <i>K</i>. <i>pneumoniae</i> but less efficiently in the presence of <i>M</i>. <i>luteus</i>. (C) Growth of <i>Dictyostelium</i> mutant strains in the presence of different bacterial species.</p

Role of SpdA in the regulation of phagocytosis by cell density.

<p>(A) Cells were grown to the indicated densities, and allowed to phagocytose fluorescent latex beads for 20 minutes. Phagocytosis was measured by flow cytometry. The results of three independent experiments were pooled in this figure. (B) In the experiment described in A, phagocytosis in mutant cells was directly compared to phagocytosis by WT cells grown at the same density. While both mutant cells phagocytosed like WT cells at low cell density, marked differences appeared when cellular density increased.</p

<i>SpdA-ins</i> cells adhere more efficiently than WT cells to their substrate.

<p>(A) Side view of a cell attached to its substrate and exposed to a flow of medium. The adhesion of the cell to its substrate can be assessed by determining the speed of a flow of medium that is necessary to detach the cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160376#pone.0160376.ref019" target="_blank">19</a>]. The strength applied by the flow of medium on the cell is σh², and its mechanical moment (σh³) is balanced by the adhesive force (F). Inspired from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160376#pone.0160376.ref018" target="_blank">18</a>]. (B) Percentage of detached cells as a function of the applied shear stress. At a low flow (between 0 and 0.5 Pa), <i>spdA-ins</i> cells detached less readily than WT cells from the substrate. At higher flow (>0.5 Pa) no significant difference can be seen between WT cells and <i>spdA-ins</i> cells. Data from three independent experiments is represented in this graph. A decrease in cell detachment can be the result of an increase in the adhesion force (F) or of a decrease in h (i.e. of a more efficient cell spreading).</p

WT and <i>spdA-ins</i> cells have similar sizes.

<p>(A) Cell size was analyzed by electric current exclusion using a CASY 1 cell counter. (B) The packed cell volume of a known number of cells was determined in graded tubes. (C) The amount of protein per cell was determined using a Lowry assay. For each experiment, the average and SEM of three independent experiments is indicated. No significant differences were seen between WT and <i>spdA-ins</i> cells.</p

The cellular amounts of SibA, Phg1 and Talin are similar in WT cells and in <i>spdA-ins</i> cells.

<p>To determine the cellular amount of SibA, Pgh1A or Talin, cellular proteins were separated by electrophoresis and specific proteins revealed with antibodies against SibA (A), Talin (B) or Phg1A (C). The intensity of the signal was quantified and expressed in arbitrary units (a.u.). The average and SEM of four independent experiments are represented. The amounts of SibA, Phg1a and Talin were similar in WT cells and in <i>spdA-ins</i> cells.</p

The phenotype of <i>spdA-ins</i> mutant cells is cell autonomous.

<p>(A) <i>SpdA</i> mutant cells expressing GFP were mixed with WT cells and cultured for three days. We then incubated the cells with rhodamine-labeled latex beads and assessed phagocytosis by flow cytometry. Expression of GFP allowed to distinguish WT cells from <i>spdA</i> mutant cells, and revealed that <i>spdA</i> mutant cells co-cultured with WT cells phagocytosed more efficiently than WT cells. (B) The phagocytosis of WT and <i>spdA-ins</i> cells cultured separately or co-cultured is indicated (mean±SEM; n = 6). *: p<0.01 (Student t-test).</p