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© The Rockefeller University Press, 0021-9525/1997//365 $5.00
The Journal of Cell Biology, Volume 139, Number 2, , 1997 365-374

Article

Dynamic Distribution of Chemoattractant Receptors in Living Cells During Chemotaxis and Persistent Stimulation



Zhan Xiao*, Ning Zhang*, Douglas B. Murphy{ddagger}, and Peter N. Devreotes*

* Department of Biological Chemistry, and {ddagger} Department of Cell Biology and Anatomy, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205

While the localization of chemoattractant receptors on randomly oriented cells has been previously studied by immunohistochemistry, the instantaneous distribution of receptors on living cells undergoing directed migration has not been determined. To do this, we replaced cAR1, the primary cAMP receptor of Dictyostelium, with a cAR1-green fluorescence protein fusion construct. We found that this chimeric protein is functionally indistinguishable from wild-type cAR1. By time-lapse imaging of single cells, we observed that the receptors remained evenly distributed on the cell surface and all of its projections during chemotaxis 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 chemoattractant receptors. Some newly extended pseudopods at migration fronts showed 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, sufficient to cause a dramatic decrease in the affinity of surface binding sites and cell desensitization, also did not significantly alter the distribution profile. Hence, the induced reduction in binding activity and cellular sensitivity cannot be due to receptor relocalization. The chimeric receptors were able to "cap" rapidly during treatment with Con A, suggesting that they are mobile in the plane of the cell membrane. This capping was not influenced by pretreatment with chemoattractant.

Abbreviations used in this paper: F-actin, filamentous actin; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; LLB, loss-of-ligand binding.

Address all correspondence to Peter N. Devreotes, Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD 21205. Tel.: (410) 955-3225. Fax: (410) 955-5757.


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