Supplementary table and figures from Collective regulation of cell motility using an accurate density-sensing system

The capacity of living cells to sense their population density and to migrate accordingly is essential for the regulation of many physiological processes. However, the mechanisms used to achieve such functions are poorly known. Here, based on the analysis of multiple trajectories of vegetative <i>Dictyostelium discoideum</i> cells, we investigate such a system extensively. We show that the cells secrete a high-molecular-weight quorum-sensing factor (QSF) in their medium. This extracellular signal induces, in turn, a reduction of the cell movements, in particular, through the downregulation of a mode of motility with high persistence time. This response appears independent of cAMP and involves a G-protein-dependent pathway. Using a mathematical analysis of the cells' response function, we evidence a negative feedback on the QSF secretion, which unveils a powerful generic mechanism for the cells to detect when they exceed a density threshold. Altogether, our results provide a comprehensive and dynamical view of this system enabling cells in a scattered population to adapt their motion to their neighbours without physical contact

Intraendosomal membranes association with pycnosomes.

<p><i>D</i>. <i>discoideum</i> cells were treated for 2h with U18666A to induce the formation of intraluminal membranes in the endocytic compartments, then fixed and processed for electron microscopy. Pycnosomes (stars) often appeared continuous with internal membranes. Arrowheads point to regions where the continuity between pycnosomal material and endosomal membranes was most apparent. Bar: 500 nm.</p

Pycnosomes present in <i>D</i>. <i>discoideum</i> endocytic compartments are secreted in the extracellular medium.

<p><i>D</i>. <i>discoideum</i> cells grown in axenic medium were fixed and processed for electron microscopy. (A) Most endocytic compartments appeared empty or contained occasionally a few vesicles (arrowhead). (B) Dense bodies (pycnosomes) appeared as amorphous structures in the endosomal lumen (star). (C) Secreted pycnosomes were recovered from the extracellular medium by differential centrifugation. Bars: 500 nm.</p

The Sct family of proteins.

<p>The SctA protein was identified by mass spectrometry as the product of the DDB_G0278725 gene. The <i>D</i>. <i>discoideum</i> genome encodes three proteins exhibiting sequence homology to SctA: SctB, SctC and SctD. All Sct proteins harbor a signal peptide (not shown) and a conserved pair of cysteines residues (*). The regions of homology between all Sct proteins were aligned using the Multalin software and treated with Boxshade. The consensus sequence is indicated. The SctC protein contains a long extension rich in glycines and serines, just downstream of the predicted signal peptide (not shown on the figure).</p

SctA-positive material appears continuous with intraendosomal membranes.

<p><i>D</i>. <i>discoideum</i> cells were treated for 2h with U18666A, then fixed and processed for immuno-electron microscopy. (A) In sections labeled with the H161 anti-p80 antibody, the p80 protein was seen associated with the limiting endosomal membrane as well as with internal membranes, but was not detected in pycnosomes (stars). (B-C) SctA-positive structures with the dense morphology of pycnosomes were also detected, and often appeared continuous with intraluminal membranes (arrowhead). Bar: 500 nm.</p

Electron microscopy reveals SctA-enriched endosomal pycnosomes.

<p><i>D</i>. <i>discoideum</i> cells grown in axenic medium were processed for immuno-electron microscopy. (A-B) Sections were labeled with the H161 anti-p80 antibody. The p80 protein was abundantly present in endosomal membranes, and only small amounts of p80 were found associated with pycnosomes (stars) in the lumen. (C-D) The B4.2 antibody revealed a high concentration of SctA in endosomal pycnosomes. In some pictures, pycnosomes appeared associated with some membranous elements (arrowheads). Bar: 500 nm.</p

SctA is a major constituent of secreted dense bodies.

<p>(A) <i>D</i>. <i>discoideum</i> cells were pelleted by centrifugation at 600 x g (pellet P6) and the supernatant (S6) was sequentially subjected to centrifugations at 15’000 and 100’000 x g. The corresponding supernatants (S15, S100) and pellets (P15, P100) resuspended in an equivalent volume were diluted in reducing sample buffer and separated on a 15% acrylamide gel. Equal volume of samples were loaded except for P6 that was diluted 1/10. The main proteins were revealed by silver staining. (B) The same amount of the 15 000 x g pellet was resuspended in reducing (+DTT) or non reducing (-DTT) sample buffer and analyzed by SDS-PAGE and Coomassie staining. Molecular weights (in kDa) are indicated, as well as the SctA protein (arrowheads).</p

SctA is massively associated with secreted pycnosomes.

<p>(A) The SctA and B proteins were expressed in bacteria as GST-fusion proteins, purified, and analyzed by Western blot with anti-GST and B4.2 antibodies. While an anti-GST antibody labeled both proteins, the B4.2 antibody only recognized GST-SctA and not SctB. (B) Supernatants (S6, S15, S100) and corresponding pellets (P15, P100) obtained by differential centrifugation of <i>D</i>. <i>discoideum</i> cell culture medium (600, 15’000, 100’000 x <i>g</i>, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154875#pone.0154875.g002" target="_blank">Fig 2</a>) were analyzed by Western blot using the B4.2 antibody. Equal volumes were loaded for all samples. (C) <i>D</i>. <i>discoideum</i> cells were cultured for 4 days. Cells and a fraction enriched in secreted pycnosomes were recovered by successive centrifugation of cell suspension at 600 x <i>g</i> and 100’000 x <i>g</i> respectively. Serial two-fold dilutions from the two fractions were analyzed by Western blot using the B4.2 antibody, the H161 anti-p80 and an antibody against mitochondrial porin. The number of cells loaded on each lane is indicated above each band. The fraction of each protein associated to secreted pycnosomes is indicated on the right.</p