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Dynamic distribution of chemoattractant receptors in living cells during chemotaxis and persistent stimulation.

Xiao Z, Zhang N, Murphy DB, Devreotes PN - J. Cell Biol. (1997)

Bottom Line: We found that this chimeric protein is functionally indistinguishable from wild-type cAR1.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA.

ABSTRACT
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.

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cAR1-GFP rescues the developmental defect of cAR1- cells. The cAR1  cell line was transformed with 1, cAR1-GFP construct; 2, wild-type cAR1; and 3, empty vector. Transformed cells were washed with DB and developed on a 35-mm  agar plate (1 × 107 cells/plate). After 36 h, pictures were taken of  each plate for the formation of fruiting bodies.
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Figure 2: cAR1-GFP rescues the developmental defect of cAR1- cells. The cAR1 cell line was transformed with 1, cAR1-GFP construct; 2, wild-type cAR1; and 3, empty vector. Transformed cells were washed with DB and developed on a 35-mm agar plate (1 × 107 cells/plate). After 36 h, pictures were taken of each plate for the formation of fruiting bodies.

Mentions: To further test the functionality of the fusion protein, we plated the cells on non-nutrient agar. Under nutrient starvation, D. discoideum cells start a developmental program which allows them to form multicellular aggregates that differentiate into fruiting bodies. Central cells initially start to secrete cAMP, which causes neighboring cells to move chemotactically towards the center; in turn these cells secrete cAMP, attracting even more distal cells. This chain of events culminates in the formation of aggregates containing up to 105 cells. cAR1 is primarily responsible for mediating this process (Devreotes, 1994). As shown in Fig. 2, cAR1 cells cannot initiate this pathway and hence remain unaggregated (3), and wild-type cAR1 rescues the program such that normal fruiting bodies are eventually formed (2). The cAR1-GFP–expressing cell line displayed a normal developmental phenotype, reaching each stage of the program at the same time as the WT cAR1 cells (data not shown). Fig. 2, 1 shows its final fruiting body stage. These observations indicate that cAR1-GFP and WT cAR1 are functionally interchangeable.


Dynamic distribution of chemoattractant receptors in living cells during chemotaxis and persistent stimulation.

Xiao Z, Zhang N, Murphy DB, Devreotes PN - J. Cell Biol. (1997)

cAR1-GFP rescues the developmental defect of cAR1- cells. The cAR1  cell line was transformed with 1, cAR1-GFP construct; 2, wild-type cAR1; and 3, empty vector. Transformed cells were washed with DB and developed on a 35-mm  agar plate (1 × 107 cells/plate). After 36 h, pictures were taken of  each plate for the formation of fruiting bodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139806&req=5

Figure 2: cAR1-GFP rescues the developmental defect of cAR1- cells. The cAR1 cell line was transformed with 1, cAR1-GFP construct; 2, wild-type cAR1; and 3, empty vector. Transformed cells were washed with DB and developed on a 35-mm agar plate (1 × 107 cells/plate). After 36 h, pictures were taken of each plate for the formation of fruiting bodies.
Mentions: To further test the functionality of the fusion protein, we plated the cells on non-nutrient agar. Under nutrient starvation, D. discoideum cells start a developmental program which allows them to form multicellular aggregates that differentiate into fruiting bodies. Central cells initially start to secrete cAMP, which causes neighboring cells to move chemotactically towards the center; in turn these cells secrete cAMP, attracting even more distal cells. This chain of events culminates in the formation of aggregates containing up to 105 cells. cAR1 is primarily responsible for mediating this process (Devreotes, 1994). As shown in Fig. 2, cAR1 cells cannot initiate this pathway and hence remain unaggregated (3), and wild-type cAR1 rescues the program such that normal fruiting bodies are eventually formed (2). The cAR1-GFP–expressing cell line displayed a normal developmental phenotype, reaching each stage of the program at the same time as the WT cAR1 cells (data not shown). Fig. 2, 1 shows its final fruiting body stage. These observations indicate that cAR1-GFP and WT cAR1 are functionally interchangeable.

Bottom Line: We found that this chimeric protein is functionally indistinguishable from wild-type cAR1.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus