DYNAMIC DISTRIBUTION OF CHEMOATTRACTANT RECEPTORS IN LIVING CELLS DURING CHEMOTAXIS AND PERSISTENT STIMULATION

While the localization of chemoattractant receptors on randomly orient ed cells has been previously studied by immunohistochemistry, the inst antaneous distribution of receptors on living cells undergoing directe d migration has not been determined. To do this, we replaced cAR1, the primary cAMP receptor of Dictyostelium, with a cAR1-green fluorescenc e protein fusion construct. We found that this chimeric protein is fun ctionally indistinguishable from wild-type cAR1. By time-lapse imaging of single cells, we observed that the receptors remained evenly distr ibuted on the cell surface and all of its projections during chemotaxi s involving turns and reversals of polarity directed by repositioning of a chemoattractant-filled micropipet. Thus, cell polarization cannot result from a gradient-induced asymmetric distribution of chemoattrac tant receptors. Some newly extended pseudopods at migration fronts sho wed a transient drop in fluorescence signals, suggesting that the flow of receptors into these zones may slightly lag behind the protrusion process. Challenge with a uniform increase in chemoattractant, suffici ent to cause a dramatic decrease in the affinity of surface binding si tes and cell desensitization, also did not significantly alter the dis tribution profile. Hence, the induced reduction in binding activity an d cellular sensitivity cannot bi-due to receptor relocalization. The c himeric receptors were able to ''cap'' rapidly during treatment with C on A, suggesting that they are mobile in the plane of the cell membran e. This capping was not influenced by pretreatment with chemoattractan t.