JCS03692 Supplementary Material

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• Supplemental Table S1 -

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• Supplemental Table S2 -

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• Supplemental Figure S1 -

  Fig. S1. Receptor mobility controls. (A) Cumulative probability distribution of squared displacements of concanavalin A (labelled by Alexa 647) in the anterior (gray circles) and posterior (black circles) top membrane of cAR1-eYFP/car1⁻ cells. The distributions were equivalent in terms of a two-sample Kolmogorov-Smirnoff test with an acceptance level set to 93.5%. Fluorescent labelled lipids to assay membrane viscosity were not successful because of their fast uptake by D. discoideum cells. (B) Cumulative probability distributions of squared displacements of cm1234-eYFP from the top membrane of polarized wt cells from the anterior (n=398) and from the posterior (n=349), respectively. The data were taken in the natural assay. Fitting the distributions to the two-population model revealed 72±7% and 42±4% of the receptors were mobile, respectively. (C) Cumulative probability distribution of squared displacements of cAR1-eYFP in the top membrane of control (16 hours in LoFlo and subsequently 2 hours in PB) cAR1-eYFP/car1⁻ cells. In relation to the data presented in Fig. 2A, these data were taken in a similar but independent experiment prior to the addition of the actin-depolymerization drug, Latrunculin B. The distributions of cAR1-eYFP in cells treated with 5 µM and 10 µM Latrunculin B were significantly different from cells that were not treated with Latrunculin B as tested by a 2-sample Kolmogorov-Smirnoff test with an acceptance level set to 93.5%, respectively. 58±6% and 59±6% of the receptors were found to be mobile in the 5 an 10 µM Latrunculin B-treated cells, respectively, by fitting the distributions to the two-population model. In cells not treated with Latrunculin B 38 ±4% of the receptors were found to be mobile. (D) Cumulative probability distribution of squared displacements of cAR1-eYFP from the top membrane of the anterior (n=141) and posterior (n=207) of polarized pi3k⁻/⁻ cells in a chemotaxis needle assay. The distributions of the anterior and posterior were identical to the anterior and posterior distributions of cAR1-eYFP in car1⁻ cells, respectively, whereas they were different from each other. The significance was tested in terms of a 2-sample Kolmogorov-Smirnoff test with an acceptance level set to 93.5%. 46±5% and 30±3% of the receptors were found to be mobile, respectively. (E) Cumulative probability distribution of squared displacements of cAR1-eYFP from the top membrane of cells globally stimulated with 1 µM cAMP for less than 5 minutes (open grey circles, n=385) in comparison to those at the anterior (closed grey circles, n=225), and posterior (closed black circles, n=367) of polarized car1⁻ cells in a chemotaxis needle assay (also see, for the latter two Fig. 2D). The global stimulation distribution of squared displacements was identical to the distribution at the anterior of chemotaxing cells in terms of a two-sample Kolmogorov-Smirnoff test with an acceptance level set to 93.5%; it was different to the distribution at the posterior of chemotaxing cells. 54±5% of the receptors were found to be mobile on global stimulation.

• Supplemental Figure S2 -

  Fig. S2. Details of the model. Description of the components in the finite element model implemented in VCell. Extracellular cAMP binds to the cAR1 receptor with an on-rate of 9.3 µM⁻¹seconds⁻¹ leading to an activated receptor cAR1*. cAR1* inactivation is set by the off-rate of 0.39 seconds⁻¹. The activated receptor catalyses the dissociation of the G-protein Gα2βγ hetero-trimer into a membrane bound Gβγ and a cytosolic Gα2 subunit. This reaction was assumed to be diffusion limited with a reaction rate of 0.66 µm²/molecule/second. The diffusion-limited back-reaction of Gα2 re-coupling to Gβγ is characterized by a rate constant of 100 µM⁻¹second⁻¹. This reaction thereby deactivates G-protein signalling.