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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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(A) Con A treatment of cAR1-GFP cells to induce receptor capping. All images are fluorescence ones. Frame 1: cell  before treatment. Frames 2–6: after application of 50 μg/ml Con  A. Con A was applied 15 s before frame 2 was taken. Time interval is 45 s. In frames 2 and 3, arrowheads indicate patching and  capping of signals. In frames 5 and 6, they show the regions devoid of signals. (B) Desensitized cells can still carry out efficient  Con A-induced receptor capping. Cells were either pretreated  with buffer (1) or 10−5 M cAMP (2) for 10 min to induce desensitization. Con A was then added to induce receptor capping. Images were taken after 5 min of Con A addition.
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Figure 7: (A) Con A treatment of cAR1-GFP cells to induce receptor capping. All images are fluorescence ones. Frame 1: cell before treatment. Frames 2–6: after application of 50 μg/ml Con A. Con A was applied 15 s before frame 2 was taken. Time interval is 45 s. In frames 2 and 3, arrowheads indicate patching and capping of signals. In frames 5 and 6, they show the regions devoid of signals. (B) Desensitized cells can still carry out efficient Con A-induced receptor capping. Cells were either pretreated with buffer (1) or 10−5 M cAMP (2) for 10 min to induce desensitization. Con A was then added to induce receptor capping. Images were taken after 5 min of Con A addition.

Mentions: The multivalent lectin Con A induces cross-linking of cell surface glycoproteins, resulting in the patching and eventual “capping” of these surface membrane proteins (Patton et al., 1990). Since cAR1-GFP maintained a uniform distribution on cell surface under all physiological conditions, we tested whether the Con A treatment would shift its location along with other surface proteins. As shown in Fig. 7 A, the distribution does change drastically. Fig. 7 A, 1 and 2, shows an elongated cell involved in random movement, with a number of pseudopods. Con A (final volume 20–50 μg/ml) was added 10 s before Fig. 7 A, 2 was taken. A detectable enrichment of signal on the bottom right corner of the cell quickly occurred (Fig. 7 A, 2, arrowhead). Massive patching and/or capping of the receptor then took place at three regions (Fig. 7 A, 3, arrowheads). After this, the cell gradually rounded up, and one major capping event proceeded to completion at the bottom edge of the cell. The patch on the opposite end persisted but did not culminate into an equally impressive cap. During this stage, certain regions of the membrane lost receptors, as was evident from the depletion of fluorescence signals (Fig. 7 A, 5 and 6, arrowheads). The rounded cells were not able to respond to micropipettes containing cAMP. Higher Con A concentration (>0.5 mg/ml) resulted in very inefficient patching and no subsequent capping (data not shown), as might be expected for a multivalent ligand.


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)

(A) Con A treatment of cAR1-GFP cells to induce receptor capping. All images are fluorescence ones. Frame 1: cell  before treatment. Frames 2–6: after application of 50 μg/ml Con  A. Con A was applied 15 s before frame 2 was taken. Time interval is 45 s. In frames 2 and 3, arrowheads indicate patching and  capping of signals. In frames 5 and 6, they show the regions devoid of signals. (B) Desensitized cells can still carry out efficient  Con A-induced receptor capping. Cells were either pretreated  with buffer (1) or 10−5 M cAMP (2) for 10 min to induce desensitization. Con A was then added to induce receptor capping. Images were taken after 5 min of Con A addition.
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Related In: Results  -  Collection

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Figure 7: (A) Con A treatment of cAR1-GFP cells to induce receptor capping. All images are fluorescence ones. Frame 1: cell before treatment. Frames 2–6: after application of 50 μg/ml Con A. Con A was applied 15 s before frame 2 was taken. Time interval is 45 s. In frames 2 and 3, arrowheads indicate patching and capping of signals. In frames 5 and 6, they show the regions devoid of signals. (B) Desensitized cells can still carry out efficient Con A-induced receptor capping. Cells were either pretreated with buffer (1) or 10−5 M cAMP (2) for 10 min to induce desensitization. Con A was then added to induce receptor capping. Images were taken after 5 min of Con A addition.
Mentions: The multivalent lectin Con A induces cross-linking of cell surface glycoproteins, resulting in the patching and eventual “capping” of these surface membrane proteins (Patton et al., 1990). Since cAR1-GFP maintained a uniform distribution on cell surface under all physiological conditions, we tested whether the Con A treatment would shift its location along with other surface proteins. As shown in Fig. 7 A, the distribution does change drastically. Fig. 7 A, 1 and 2, shows an elongated cell involved in random movement, with a number of pseudopods. Con A (final volume 20–50 μg/ml) was added 10 s before Fig. 7 A, 2 was taken. A detectable enrichment of signal on the bottom right corner of the cell quickly occurred (Fig. 7 A, 2, arrowhead). Massive patching and/or capping of the receptor then took place at three regions (Fig. 7 A, 3, arrowheads). After this, the cell gradually rounded up, and one major capping event proceeded to completion at the bottom edge of the cell. The patch on the opposite end persisted but did not culminate into an equally impressive cap. During this stage, certain regions of the membrane lost receptors, as was evident from the depletion of fluorescence signals (Fig. 7 A, 5 and 6, arrowheads). The rounded cells were not able to respond to micropipettes containing cAMP. Higher Con A concentration (>0.5 mg/ml) resulted in very inefficient patching and no subsequent capping (data not shown), as might be expected for a multivalent ligand.

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