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Arrestins function in cAR1 GPCR-mediated signaling and cAR1 internalization in the development of Dictyostelium discoideum.

Cao X, Yan J, Shu S, Brzostowski JA, Jin T - Mol. Biol. Cell (2014)

Bottom Line: At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6-min intervals, directing cell migration.The adcB(-)C(-) cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation.In addition, ligand-induced cAR1 internalization is compromised in adcB(-)C(-) cells, suggesting that arrestins are involved in elimination of high-affinity cAR1 receptors from cell surface after the aggregation stage of multicellular development.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

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Arrestins play a role in controlling oscillations of cAMP signaling. (A) cAMP oscillations were observed in the wild-type, adcB−C−, and adcB−C− cells expressing AdcC-YFP during early development by capturing dark-field images every 1 min with a stereo microscope. Time plots were generated by measuring intensity changes in the frame-subtracted image sequence. Stills showing the differences in the dark-field wave patterns between the strains appear below the respective time plots. Scale bar, ∼0.5 mm. (B) The period of cAMP oscillation was analyzed with three mounds per movie and graphed. Means (n = 3) and SDs are shown. Statistical significance was assessed by t test, **p < 0.01. Independent experiments were performed at least twice.
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Figure 5: Arrestins play a role in controlling oscillations of cAMP signaling. (A) cAMP oscillations were observed in the wild-type, adcB−C−, and adcB−C− cells expressing AdcC-YFP during early development by capturing dark-field images every 1 min with a stereo microscope. Time plots were generated by measuring intensity changes in the frame-subtracted image sequence. Stills showing the differences in the dark-field wave patterns between the strains appear below the respective time plots. Scale bar, ∼0.5 mm. (B) The period of cAMP oscillation was analyzed with three mounds per movie and graphed. Means (n = 3) and SDs are shown. Statistical significance was assessed by t test, **p < 0.01. Independent experiments were performed at least twice.

Mentions: A previous study showed that ERK2 activity oscillates in phase with extracellular cAMP waves during specific stages of development of D. discoideum (Maeda et al., 2004). To test whether arrestins play a role in cAR1-controlled oscillations of cAMP signaling, we assessed the frequency of cAMP oscillations in wild-type, adcB−C−, and adcB−C− cells expressing AdcC-YFP. Cells were plated on Petri dishes containing starvation buffer to initiate development, and the aggregation stage was recorded for cAMP wave analysis. cAMP oscillations (waves) can be measured by proxy by recording optical density oscillations of cells imaged by dark-field microscopy (Siegert and Weijer, 1989; see Figure 5A). Changes in grayscale values from subregions in frame-subtracted images were measured using ImageJ. We found that wild-type cells produced waves with ∼6-min period as expected (Figure 5, A and B) and displayed a robust dark-field wave pattern with broad spirals arms propagating from signaling centers (Figure 5A shows a still from the image sequence). In contrast, adcB−C− cells produced cAMP waves with a faster, ∼3-min period (Figure 5, A and B). The shorter distance between propagating wavefronts from multiple signaling centers can be observed in the still shown in Figure 5A. Of importance, this disrupted wave pattern was rescued by expressing AdcC-YFP in the adcB−C− cells (Figure 5, A and B). Furthermore, adcB− and adcC− cells did not display cAMP waves of higher frequencies (Supplemental Figure S9), suggesting that AdcB and AdcC can compensate for the loss of either one, and only in cells lacking both AdcC and AdcB is a defect in the formation of cAMP waves obvious. Our data indicate that AdcB and AdcC are involved in cAR1-mediated oscillations of cAMP signaling during the aggregation stage of D. discoideum development.


Arrestins function in cAR1 GPCR-mediated signaling and cAR1 internalization in the development of Dictyostelium discoideum.

Cao X, Yan J, Shu S, Brzostowski JA, Jin T - Mol. Biol. Cell (2014)

Arrestins play a role in controlling oscillations of cAMP signaling. (A) cAMP oscillations were observed in the wild-type, adcB−C−, and adcB−C− cells expressing AdcC-YFP during early development by capturing dark-field images every 1 min with a stereo microscope. Time plots were generated by measuring intensity changes in the frame-subtracted image sequence. Stills showing the differences in the dark-field wave patterns between the strains appear below the respective time plots. Scale bar, ∼0.5 mm. (B) The period of cAMP oscillation was analyzed with three mounds per movie and graphed. Means (n = 3) and SDs are shown. Statistical significance was assessed by t test, **p < 0.01. Independent experiments were performed at least twice.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Arrestins play a role in controlling oscillations of cAMP signaling. (A) cAMP oscillations were observed in the wild-type, adcB−C−, and adcB−C− cells expressing AdcC-YFP during early development by capturing dark-field images every 1 min with a stereo microscope. Time plots were generated by measuring intensity changes in the frame-subtracted image sequence. Stills showing the differences in the dark-field wave patterns between the strains appear below the respective time plots. Scale bar, ∼0.5 mm. (B) The period of cAMP oscillation was analyzed with three mounds per movie and graphed. Means (n = 3) and SDs are shown. Statistical significance was assessed by t test, **p < 0.01. Independent experiments were performed at least twice.
Mentions: A previous study showed that ERK2 activity oscillates in phase with extracellular cAMP waves during specific stages of development of D. discoideum (Maeda et al., 2004). To test whether arrestins play a role in cAR1-controlled oscillations of cAMP signaling, we assessed the frequency of cAMP oscillations in wild-type, adcB−C−, and adcB−C− cells expressing AdcC-YFP. Cells were plated on Petri dishes containing starvation buffer to initiate development, and the aggregation stage was recorded for cAMP wave analysis. cAMP oscillations (waves) can be measured by proxy by recording optical density oscillations of cells imaged by dark-field microscopy (Siegert and Weijer, 1989; see Figure 5A). Changes in grayscale values from subregions in frame-subtracted images were measured using ImageJ. We found that wild-type cells produced waves with ∼6-min period as expected (Figure 5, A and B) and displayed a robust dark-field wave pattern with broad spirals arms propagating from signaling centers (Figure 5A shows a still from the image sequence). In contrast, adcB−C− cells produced cAMP waves with a faster, ∼3-min period (Figure 5, A and B). The shorter distance between propagating wavefronts from multiple signaling centers can be observed in the still shown in Figure 5A. Of importance, this disrupted wave pattern was rescued by expressing AdcC-YFP in the adcB−C− cells (Figure 5, A and B). Furthermore, adcB− and adcC− cells did not display cAMP waves of higher frequencies (Supplemental Figure S9), suggesting that AdcB and AdcC can compensate for the loss of either one, and only in cells lacking both AdcC and AdcB is a defect in the formation of cAMP waves obvious. Our data indicate that AdcB and AdcC are involved in cAR1-mediated oscillations of cAMP signaling during the aggregation stage of D. discoideum development.

Bottom Line: At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6-min intervals, directing cell migration.The adcB(-)C(-) cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation.In addition, ligand-induced cAR1 internalization is compromised in adcB(-)C(-) cells, suggesting that arrestins are involved in elimination of high-affinity cAR1 receptors from cell surface after the aggregation stage of multicellular development.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

Show MeSH
Related in: MedlinePlus