34 research outputs found
Supplementary Information from Inference of the drivers of collective movement in two cell types: <i>Dictyostelium</i> and melanoma
Supplements, supplementary figures and supplementary table
Two distinct behaviours are spatially separated in the migrating population.
<p>Cells migrating in the 10 μM folate under-agarose assay. Those circled in green are travelling away from the well (x component of velocity >1 μm/min), those in red are travelling toward it. Larger circles indicate faster movement in the appropriate direction. Cells marked in blue show no substantial movement either way. doi: <a href="http://dx.doi.org/10.5525/gla.researchdata.252" target="_blank">10.5525/gla.researchdata.252</a>.</p
Limitations of guidance by point sources.
<p>(A) Chemotactic regions for a localised source of attractant. Assuming chemotaxis requires a 1% relative occupancy difference, we can indicate the regions where a directional signal can be resolved (however poorly). Each panel shows a snapshot of a simulation of attractant diffusion from a point source at the right-hand side. Dotted lines bound the region in which chemotaxis can occur. Graphs above each simulation show receptor occupancy difference, with regions of ≥1% occupancy difference filled in green, and regions of <1% filled in red. The leftmost column shows time points for a source of low concentration. The middle column shows a source of high concentration. The right-hand side shows a self-generated gradient, in which the area in which chemotaxis can occur follows the density wave as it travels. (B) The fraction of the region in which chemotaxis can occur for short- and long-range point source, point-sink systems in the absence of degradation, displayed as a function of source concentration (on the <i>y</i>-axis) and time given to diffuse (on the <i>x</i>-axis). Hotter colours indicate better coverage of the region, with red indicating guidance over the entire domain and blue indicating no guidance. Note that this colour does not reflect on the strength of the guiding cues, only on their extent in space. The graph assumes a diffusion coefficient of 300 μm<sup>2</sup>/s. doi: <a href="http://dx.doi.org/10.5525/gla.researchdata.252" target="_blank">10.5525/gla.researchdata.252</a>.</p
Mass spectrometry confirms model prediction of migratory wave as an attractant sink.
<p>(A) Distribution of population (red), average x component of velocity (the component directed away from the well) (green), and folate concentration, as determined by mass spectrometry. As predicted by the model, the folate concentration profile descends to a sink in the population wave. Overhead shows a microscope image of the assay from a single technical repeat aligned with the graph. (B) Diagram of the situation in A. Cells are distributed with a density peak toward the front. Attractant (blue) is metabolised by this density peak, leaving none (or almost none) behind it. Only the cells in the density peak are given strong chemotactic signals and migrate forward. (C) A model of mutual reinforcement by the attractant and density waves. The density wave shapes attractant by degradation, and the resulting environmental profile causes a central tendency in chemotaxing cells, maintaining the higher density region. doi: <a href="http://dx.doi.org/10.5525/gla.researchdata.252" target="_blank">10.5525/gla.researchdata.252</a>.</p
The population wave is diagnostic of a self-generated gradient.
<p>(A) Migratory dynamics of a typical 10 μM simulation. (B) Migratory dynamics for contact inhibition with and without attractant degradation. Graphs below show that CIL makes the additional prediction that cells that start near the well end near the well. (C) Inclusion of a diffusible degrading enzyme. Even where all degrader is free to diffuse, the migratory wave forms and decays. (D) Inclusion of receptor internalisation. (E) Migratory dynamics and population distribution at 7 h with secreted repellent. doi: <a href="http://dx.doi.org/10.5525/gla.researchdata.252" target="_blank">10.5525/gla.researchdata.252</a>.</p
Degradation is important to chamber assays.
<p>(A) Diagram of the experiment. Cells are evenly distributed in a chamber with an outer well containing attractant and an inner well containing attractant-free medium. In the absence of external influences, this will create by diffusion a linear gradient of the attractant leading towards the outer well. We observe the migratory behaviour of cells on a bridge between these two wells and graph the profile of velocity toward the outer well. Cartoons of this behaviour are shown above (B) and (C), with cell movement shown by green arrows. (B, D, and F) Velocity in the direction of the imposed gradient for <i>Dictyostelium</i> cells in an Insall chamber for (B) 10 μM cAMP, (D) 200 μM Sp-cAMPS and (F) 600 nM Sp-cAMPS. (C, E, and G) Velocity of cells in a simulated Insall chamber-like condition, with (C) high cAMP and no degradation, (E) low cAMP and no degradation, and (G) high cAMP with degradation. (H) Gradient predicted by the simulations with (blue) and without (black, dashed) degradation. This shows how much degradation can cause the profile to deviate from the originally imposed linear gradient. doi: <a href="http://dx.doi.org/10.5525/gla.researchdata.252" target="_blank">10.5525/gla.researchdata.252</a>.</p
Leading edge dynamics are caused by singular exposure to chemoattractant.
<p>(A, B) The x component of cell velocity across the population for 10 μM (A) and 100 μM (B) simulations. The population is sorted by proximity to the well and binned into 2% sections. The population percentile is shown on the <i>y</i>-axis, with cells nearest the well shown at the top and cells furthest from the well shown at the bottom. Time progresses along the <i>x</i>-axis. Colours indicate velocity directed away from the well, as is indicated in the legend. White dots in (A) are placed at the divide between chemotaxing and nonchemotaxing cells for three different time points and correspond to the positions of the dotted lines in the illustrative stills of a 10 μM simulation at these time points. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002404#pbio.1002404.s004" target="_blank">S3 Movie</a> demonstrates the construction of (A). (C, D) Receptor occupancy differences for A and B. (E, F) Directed velocity of cells in 10 μM (E) and 100 μM (F) folate experiments. The movement of cells in the region labelled ‘b’ showed clear directionality, where those in the region labelled ‘a’ were indistinguishable from 0. (G, H) Comparison of starting positions of cells with their positions after 5 h for simulated (G) and real (H) cells. Bars are arranged according to the position of cells at the end of the experiment, with cells finishing in the trailing quintile represented in the leftmost bar and those further forward shown to the right, up to the leading quintile in the rightmost bar. Colours indicate the starting quintile of the cells, as shown in the left-hand still of the simulation, with red indicating those cells that began at the rear through to blue indicating those that began at the front. doi: <a href="http://dx.doi.org/10.5525/gla.researchdata.252" target="_blank">10.5525/gla.researchdata.252</a>.</p
A self-generated gradient model can explain directed motility in a spreading assay.
<p>(A) <i>Dictyostelium</i> cells migrating in the under-agarose assay. Agarose initially contains 20 μM folate. (B) A simulation of cells migrating in response to a self-generated gradient. (Inset) Cells move in a 2-D persistent random walk, biased by local attractant gradient and degrade local attractant over time. The data for this figure can be accessed at doi: <a href="http://dx.doi.org/10.5525/gla.researchdata.252" target="_blank">10.5525/gla.researchdata.252</a>.</p
fAR1/G protein machinery mediates LPS-induced cell migration and particle engulfment.
<p>(A) EZ-TAXIScan chemotaxis toward a linear LPS gradient of vegetative <i>WT</i>, <i>gβ</i><sup><i>−</i></sup>, <i>far1</i><sup><i>−</i></sup>, and fAR1-Y/<i>far1</i><sup><i>−</i></sup> cells. Migration paths toward LPS are shown. (B) Ten cells of each strain from (A) were used for tracing. The mean and SD resulting from quantification of chemotaxis parameters are shown. A Student <i>t</i> test indicated a statistically significant difference between <i>gβ</i><sup><i>−</i></sup>, <i>far1</i><sup><i>−</i></sup>, and <i>WT</i> cells (* indicates <i>P</i> < 0.01). (C) LPS on particle surface triggers localized PIP3 signaling and engulfment. Engulfment of 1 μm LPS-coated beads (red) by <i>WT</i> but not <i>far1</i><sup><i>−</i></sup> or <i>gβ</i><sup><i>−</i></sup> cells expressing PH<sub>CRAC</sub>-GFP (green). Scale bar: 2 μm. (D) LPS on particle surface triggers localized actin polymerization to form phagocytic cup. Engulfment of 1 μm LPS-coated beads (red) by <i>WT</i> but not <i>far1</i><sup><i>−</i></sup> or <i>gβ</i><sup><i>−</i></sup> cells expressing LimEΔcoil-GFP (green). Scale bar: 2 μm. (E) LPS triggers engulfment through fAR1 and Gβ. Quantitation of engulfment movies from C and D to compare engulfment ability between <i>WT</i>, <i>far1</i><sup><i>−</i></sup>, and <i>gβ</i><sup><i>−</i></sup> cells. A Student <i>t</i> test indicated a statistically significant difference in percentage of cell-engulfing LPS-beads between <i>far1</i><sup><i>−</i></sup>, <i>gβ</i><sup><i>−</i></sup>, and <i>WT</i> cells (<i>P</i> < 0.01). Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005754#pbio.2005754.s007" target="_blank">S1 Data</a>. fAR1, folic acid receptor 1; LimEΔcoil, partial sequences of LimE protein; LPS, lipopolysaccharide; PH<sub>CRAC</sub>, PH domain of cytosolic regulator of adenylyl cyclase; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; WT, wild-type; LimEΔcoil, partial sequences of LimE protein</p
fAR1/G protein machinery mediates live <i>K</i>. <i>aerogenes</i> engulfment.
<p>(A) <i>WT</i> and mutants were mixed with pHrodo-labeled <i>K</i>. <i>aerogenes</i> at a 1:50 ratio. After 20 min, cells were mounted on a slide in basic pH buffer and analyzed by confocal microscopy. The representative data are shown. The engulfed pHrodo-labeled <i>K</i>. <i>aerogenes</i> are shown as red; Scale bars, 5 μm. (B) The engulfed bacterial number in each cell from (A) was measured and plotted for <i>WT</i> and mutant cells. A Student <i>t</i> test indicated a statistically significant difference in number of engulfed bacteria per cell between <i>far1</i><sup><i>−</i></sup>, <i>gβ</i><sup><i>−</i></sup>, and <i>WT</i> cells (* indicates <i>P</i> < 0.01). (C) <i>WT</i> and mutant cells were mixed with pHrodo-labeled live <i>K</i>. <i>aerogenes</i> at a 1:100 ratio for the indicated time. Cells were suspended in basic pH buffer and analyzed for the percentage of pHrodo positive cells by flow cytometry, which represents the cells that engulfed <i>K</i>. <i>aerogenes</i>. Quantification of engulfed <i>K</i>. <i>aerogenes</i> is compared between different <i>Dictyostelium</i> strains. (D) The mean and SD resulting from quantification of 3 independent repetitions of the experiments exemplified in (C) are plotted. (E) fAR1 recognizes not only diffusible chemoattractant but also immobilized ligand on bacterial surface to mediate both migration and engulfment. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005754#pbio.2005754.s007" target="_blank">S1 Data</a>. ERK2, extracellular signal-regulated kinase 2; fAR1, folic acid receptor 1; LPS, lipopolysaccharide; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase abbreviation SSC, side scatter; WT, wild type.</p