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.