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First published online May 8, 2008
doi: 10.1242/10.1242/jcs.030692

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A spatially restricted increase in receptor mobility is involved in directional sensing during Dictyostelium discoideum chemotaxis

¹ Physics of Life Processes, Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands
² Cell Biology, Leiden Institute of Biology, Leiden University, PO Box 9504, 2300 RA Leiden, The Netherlands

View larger version (37K): [in this window] [in a new window]   Fig. 1. Generation of cell lines expressing cAMP-receptor/eYFP fusion proteins at endogenous levels. (A) Detection of cAR1-eYFP fusion protein by western blot using an anti-GFP antibody. Free YFP showed the expected band at 30 kDa (lane3). As cAR1 has a size of 40 kDa, the correct size for the fusion protein is 70 kDa, which was observed (lane2). Transformed car1– cells with the cAR1-eYFP fusion protein exhibited a protein-band at the correct size (lane2), whereas car1– cells did not (lane1). Free eYFP was not detected in cAR1-eYFP/car1– cells. (B) cAR1-eYFP was localized at the plasma membrane of car1– cells, as detected by confocal microscopy. (C) The first image shows the aggregation-deficient phenotype of car1– mutant 24 hours after starvation. These cells were not able to initiate the developmental cycle. The following images display the different developmental stages of car1– cells transformed with the cAR1-eYFP construct. The developmental defect of car1– cells was rescued by the cAR1-eYFP transformation. (D) The left picture shows a fluorescence image of the top membrane of a typical unstimulated car1– cell transformed with cAR1-eYFP. After a brief photobleaching pulse (2.5-5.0 seconds) individual receptors were detected (peaks of fluorescence in the right image). (E) Fluorescence signal of an individual cAR1-eYFP molecule as a function of time, showing a single-step photobleaching event characteristic for individual molecules. (F) Two examples of trajectories of individual cAR1-eYFP molecules diffusing in the top plasma membrane.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Diffusion of cAR1-eYFP for resting cells and polarized cells. (A) Cumulative probability distribution of squared displacements, P(r2), of the trajectories (n=2060) of cAR1-eYFP from the top membrane of resting cells (control, black circles), recorded with tlag=44mseconds lag time between subsequent images. Data were fitted to a two-component model (equation 1) (grey curve), resulting in a fraction of immobile receptors, a fraction of mobile receptors and the mean squared displacement (MSD) of mobile receptors. The diffusion constant, D, was determined from: MSD=4Dtlag + 42=0.034±0.003 µm2 with a lateral accuracy =40nm given by our experimental conditions. 38±4% (mean±s.e.m.) of the receptors were mobile, characterized by a diffusion constant D=0.17±0.02 µm2/s. Fit to a one-component model (broken line, grey) did clearly fail to describe the data. (B) Cumulative probability distribution of squared displacements of cAR1-eYFP from the top membrane of the anterior (grey circles, n=526), and posterior (black circles, n=282) of polarized car1– cells in the natural assay. By fitting both P(r2) with the two-population model, 44±4% and 39±4% of the anterior and posterior receptors, respectively, were found to be mobile, which was significantly different according to a two-sample Kolmogorov-Smirnov test (KS-test) with an acceptance level of 93.5%. (C) Cumulative probability distribution of squared displacements of cAR1-eYFP from the top membrane of the anterior (n=193) and posterior (n=368) sides of gradient-sensing competent car1– cells in relation to the position of the needle before the cAMP gradient was initiated. The mobile fraction of the receptors was not different between anterior and posterior, and was equal to the control, as tested with a KS-test with an acceptance level of 93.5%. (D) Cumulative probability distribution of squared displacements of cAR1-eYFP from the top membrane of the anterior (n=225) and posterior (n=367) of gradient-sensing car1– cells. After the needle filled with 10 µM cAMP was placed, 54±5% and 31±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 the anterior (n=404) and posterior (n=509) of gradient-sensing g2– cells before the needle with cAMP was placed. 57±6% and 59±6% of the receptors were found to be mobile, respectively. (F) Cumulative probability distribution of squared displacements of cAR1-eYFP from the top membrane of the anterior (n=531) and posterior (n=687) of gradient-sensing g2– cells after the needle filled with 10 µM cAMP was placed. 51±5% and 49±5% of the anterior and posterior receptors, respectively, were found to be mobile.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Mobile fraction. Fraction of the mobile receptors (grey) characterized by a diffusion constant D=0.17±0.02 µm2/s and that of immobile receptors (black). The fractions were compared in resting cells (control), and in anterior and posterior of gradient-sensing polarized cells. The polarized cells were measured either in the chemotaxis needle assay.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Model describing accelerated signalling at the leading edge. (A) In resting cells, we found two receptor populations: immobile and mobile receptors. A fraction of the immobile receptors is coupled to G2GDP/Gβ ,which, in turn, is coupled to protein-protein networks and/or the cytoskeleton which inhibits diffusion (1). This fraction of immobile receptors becomes mobile by uncoupling of G2GTP upon cAMP activation. Free G2GTP and free Gβ subunits activate intracellular signalling (2). The mobile receptors have the ability to further activate other G2GDP/Gβ complexes in a diffusion-limited process (3). In a final step, re-association of the receptor with G2GDP/Gβ and corresponding loss of cAMP immobilizes the receptor again (4). (B) An ellipsoidal cell is exposed to a gradient of 0.4nM/µm cAMP. The concentration at the leading edge is 66nM and that at the trailing edge 58nM. The density of active cAR1 receptor (cAR1*) is plotted versus the position along the cell membrane. At the leading edge the density of active cAR1 is higher by a factor of 1.05 when compared with the density at the trailing edge (6.1 molecules/µm2 versus 5.8 molecules/µm2) following the cAMP gradient. The density of activated Gβ at the leading edge was 73.0 molecules/µm2, whereas that at the trailing edge was 71.4 molecules/µm2. Hence, diffusion leads to a linear amplification of the gradient by a factor of 5.

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