Fig 1.
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.
Fig 2.
SpdA-ins mutant cells phagocytose particles faster than WT cells.
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. SpdA-ins mutant cells ingested particles faster than WT cells.
Fig 3.
The phenotype of spdA-ins mutant cells is cell autonomous.
(A) SpdA 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 spdA mutant cells, and revealed that spdA mutant cells co-cultured with WT cells phagocytosed more efficiently than WT cells. (B) The phagocytosis of WT and spdA-ins cells cultured separately or co-cultured is indicated (mean±SEM; n = 6). *: p<0.01 (Student t-test).
Fig 4.
SpdA-ins cells adhere more efficiently than WT cells to their substrate.
(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 [19]. 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 [18]. (B) Percentage of detached cells as a function of the applied shear stress. At a low flow (between 0 and 0.5 Pa), spdA-ins 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 spdA-ins 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).
Fig 5.
SpdA-ins cells spread more efficiently than WT cells.
(A) WT and spdA-ins cells attached to a glass substrate were examined by scanning electron microscopy. Three representative pictures of each cell line are shown. SpdA cells spread more efficiently on their substrate than WT cells. Scalebar: 10μm. (B) To quantify cell spreading, cells were examined by phase contrast and RICM (Reflexion Interference Contrast Microscopy). The contact area between cells and their substrate appears black and it was quantified for WT and spdA-ins cells (mean±SEM; n = 4 independent experiments. In each experiment 20 cells were analysed for each sample). *: p<0.01(Student t-test). (C) The contact area of individual cells is represented for the whole population of cells analyzed. (D) Kinetics of cell spreading was determined. SpdA-ins cells spread more and faster than WT cells. Thin lines: average spreading kinetics of cells (11 and 6 cells, respectively, representative of 3 independent experiments). Solid lines: fit with eq. 7 in ref 13, A(t) = Amax tanh (αt).
Fig 6.
The cellular amounts of SibA, Phg1 and Talin are similar in WT cells and in spdA-ins cells.
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 spdA-ins cells.
Fig 7.
The actin organization is not significantly altered in spdA-ins cells.
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 spdA-ins cells. When cells were incubated in phosphate buffer (PB), formation of filopodia was induced in both WT and spdA-ins cells.
Fig 8.
WT and spdA-ins cells have similar sizes.
(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 spdA-ins cells.
Fig 9.
Role of SpdA in the regulation of phagocytosis by cell density.
(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.