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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) Z-axis two-dimensional confocal fluorescence image scanning analysis of cAR1-GFP cells. Cells adhering to a  glass coverslip were fixed in 4% paraformaldehyde/0.1% Triton  X-100 and subjected to confocal analysis. Section thickness: 0.5  μm; frame interval: 1.0 μm. (B) Three-dimensional reconstruction image of cAR1-GFP cell fluorescence. All three-dimensional  images were created from a Z-axis scanning series with the Intervision 1.6 software program. The reconstructed image was rotated around the X-axis for 150° starting from the bottom to top  image (top of cell projecting into paper plane). Images were captured every 30°. Final image shows top to bottom view. Bars, 10 μm.
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Figure 5: (A) Z-axis two-dimensional confocal fluorescence image scanning analysis of cAR1-GFP cells. Cells adhering to a glass coverslip were fixed in 4% paraformaldehyde/0.1% Triton X-100 and subjected to confocal analysis. Section thickness: 0.5 μm; frame interval: 1.0 μm. (B) Three-dimensional reconstruction image of cAR1-GFP cell fluorescence. All three-dimensional images were created from a Z-axis scanning series with the Intervision 1.6 software program. The reconstructed image was rotated around the X-axis for 150° starting from the bottom to top image (top of cell projecting into paper plane). Images were captured every 30°. Final image shows top to bottom view. Bars, 10 μm.

Mentions: To further elucidate the details of receptor distribution on various regions of the cell membrane, we carried out a Z-axis confocal analysis (Fig. 5 A). It was necessary to lightly fix the cells since they are extremely mobile. Sections of 0.5 μm in thickness were taken from the bottom of the cell close to the substratum to the upper surface at a distance of 1.0 μm per section. The cell under study was a typically flat and adhesive cell with many pseudopods and fine filopods. The bottom section (Fig. 5 A, 1) showed fluorescence in the interior of the profile, which was likely due to the upward invaginations of its basal membrane. The latter frames of Fig. 5 A displayed mostly peripheral fluorescence and little internal signal, indicating that cAR1 is highly localized on the surface. Pseudopods and filopods seemed most apparent in the lower sections. The upper surface of the cell seemed to be quite uneven: one portion of the cell (near bottom of the frame) rose up higher than the other portion. After Fig. 5 A, 3, only cross-sections of this portion were clearly present. The receptors seem to be more or less evenly distributed on the plasma membrane, although some small, randomly localized regions do contain more signals than neighboring domains.


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) Z-axis two-dimensional confocal fluorescence image scanning analysis of cAR1-GFP cells. Cells adhering to a  glass coverslip were fixed in 4% paraformaldehyde/0.1% Triton  X-100 and subjected to confocal analysis. Section thickness: 0.5  μm; frame interval: 1.0 μm. (B) Three-dimensional reconstruction image of cAR1-GFP cell fluorescence. All three-dimensional  images were created from a Z-axis scanning series with the Intervision 1.6 software program. The reconstructed image was rotated around the X-axis for 150° starting from the bottom to top  image (top of cell projecting into paper plane). Images were captured every 30°. Final image shows top to bottom view. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 5: (A) Z-axis two-dimensional confocal fluorescence image scanning analysis of cAR1-GFP cells. Cells adhering to a glass coverslip were fixed in 4% paraformaldehyde/0.1% Triton X-100 and subjected to confocal analysis. Section thickness: 0.5 μm; frame interval: 1.0 μm. (B) Three-dimensional reconstruction image of cAR1-GFP cell fluorescence. All three-dimensional images were created from a Z-axis scanning series with the Intervision 1.6 software program. The reconstructed image was rotated around the X-axis for 150° starting from the bottom to top image (top of cell projecting into paper plane). Images were captured every 30°. Final image shows top to bottom view. Bars, 10 μm.
Mentions: To further elucidate the details of receptor distribution on various regions of the cell membrane, we carried out a Z-axis confocal analysis (Fig. 5 A). It was necessary to lightly fix the cells since they are extremely mobile. Sections of 0.5 μm in thickness were taken from the bottom of the cell close to the substratum to the upper surface at a distance of 1.0 μm per section. The cell under study was a typically flat and adhesive cell with many pseudopods and fine filopods. The bottom section (Fig. 5 A, 1) showed fluorescence in the interior of the profile, which was likely due to the upward invaginations of its basal membrane. The latter frames of Fig. 5 A displayed mostly peripheral fluorescence and little internal signal, indicating that cAR1 is highly localized on the surface. Pseudopods and filopods seemed most apparent in the lower sections. The upper surface of the cell seemed to be quite uneven: one portion of the cell (near bottom of the frame) rose up higher than the other portion. After Fig. 5 A, 3, only cross-sections of this portion were clearly present. The receptors seem to be more or less evenly distributed on the plasma membrane, although some small, randomly localized regions do contain more signals than neighboring domains.

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