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First published online 12 April 2005
doi: 10.1242/jcs.02317

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RasGEF-containing proteins GbpC and GbpD have differential effects on cell polarity and chemotaxis in Dictyostelium

¹ Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
² MicroSpectroscopy Centre, Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
³ W. M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA

View larger version (25K): [in a new window]   Fig. 6. cAMP and cGMP binding to the lysates of mutant cells. (A) Binding of 50 nM [3H]cGMP or [3H]cAMP to the cytosolic fraction of a cell lysate was measured. The results are expressed as the amount of cyclic nucleotide bound per mg protein. The black bar represent lysates of gbpC–/gbpD– cells, the grey bar, gbpC–/gbpD–/N-GbpD-GFPOE cells and the white bar, lysates of gbpC–/gbpD–/GbpDOE cells. (B) Binding of 5 nM [3H]cGMP to the cytosolic fraction of lysates of gbpC– cells (black bar) and gbpC–/N-GbpC-GFPOE cells (grey bar) was measured. The means±s.d. of at least two independent experiments with quadruple determinations are shown.

View larger version (36K): [in a new window]   Fig. 1. Chemotaxis of gbpC and gbpD mutants. (A) Cell tracks of representative wild-type (WT) and mutant Dictyostelium cells in a spatial gradient of cAMP. Cells were starved until the ripple stage was reached. Subsequently, they were placed in a chemotaxis chamber filled with phosphate buffer at one end and phosphate buffer with cAMP at the other end. Images were taken every 10 seconds for a period of 5 minutes and the cell outlines of subsequent images were placed on top of each other to obtain the final picture. (B) Cell tracks and separate images of a wild-type and a gbpD– cell that were placed in a chemotaxis chamber. The tracks are composed of stacked images that were taken every 4 seconds. Every third frame is also shown as a separate image.

View larger version (15K): [in a new window]   Fig. 2. Adhesion strength of vegetative gbpD– and GbpDOE cells. The percentage of cells that became detached upon shaking of the culture dish at 150 rpm is plotted against the time of shaking. Symbols indicate the wild type (x), gbpD– (x) and gbpD– /GbpD OE cells (x). Error bars represent the s.d. of three independent experiments.

View larger version (54K): [in a new window]   Fig. 3. Morphology of Dictyostelium cells overexpressing GbpD protein. Confocal images of starved gbpD– and gbpD–/GbpDOE cells expressing the F-actin binding domain of LimE fused to GFP. The DIC image and the uppermost fluorescent image were taken just above the glass surface. The second fluorescent image was taken 2 µm above the glass surface. The thickness of the images corresponds to 1 µm. The cross section image was obtained by making use of the projection option of the Zeiss LSM Image browser 5 program. White arrowheads indicate the height of the upper two fluorescent images. Bar, 10 µm.

View larger version (17K): [in a new window]   Fig. 4. cAMP-induced F-actin formation as measured with a phalloidin binding assay. Aggregation competent wild-type (WT) and mutant cells were stimulated with cAMP and the F-actin content was determined at the indicated time points. The presented values are the average of two independent experiments with error bars indicating standard deviations.

View larger version (43K): [in a new window]   Fig. 5. cAMP-induced myosin II translocation. Wild-type cells and mutant cells expressing myosin II-GFP were starved and stimulated with cAMP. (A) Confocal fluorescent images were taken every 5 seconds and are shown at the indicated time points after cAMP stimulation. The gbpC– cells contained unusual amounts of vacuoles in this particular experiment. (B) Quantification of the data using the QuimP software developed by T. Bretschneider (Dormann et al., 2002). Fluorescence at the membrane and in the cytosol was calculated and the ratio of the fluorescence at the membrane divided by the average cytosolic fluorescence was plotted against time. In gbpD– (x) and wild-type cells (x), myosin translocates to the cortex after an initial depletion. In gbpC– cells (x), this myosin translocation to the cortex is absent and depletion of the fluorescent signal from the cortex is prolonged and enhanced. The displayed data are averaged values from 13 wild-type cells, nine gbpC– cells and seven gbpD– cells from two or three experiments.

View larger version (17K): [in a new window]   Fig. 7. The effect of GbpD overexpression on the adhesion strength of mutants with undetectable cyclic nucleotide levels. The adhesion strength of several mutants was studied by measuring the percentage of cells that became detached from the substrate after shaking for 60 minutes at 150 rpm. The requirement of cGMP for GbpD function was studied by overexpressing GbpD in a guanylyl cyclase double knockout, sgc–/gca–; cGMP levels of this cell line were below the detection limit (0.019 pmol/107 cells or 3 nM). To study the requirement of cAMP for GbpD function, the phosphodiesterase GbpB was overexpressed. This led to cAMP levels that were below the detection limit of the assay (0.024 pmol/107 cells or 4 nM). Finally, GbpD was overexpressed in a cell line that was devoid of cAMP and cGMP by overexpressing GbpB protein in the sgc–/gca– cell line (both cGMP and cAMP levels were below the detection limit). Error bars represent the s.d. of at least two independent experiments.