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Flat clathrin lattices: stable features of the plasma membrane.

Grove J, Metcalf DJ, Knight AE, Wavre-Shapton ST, Sun T, Protonotarios ED, Griffin LD, Lippincott-Schwartz J, Marsh M - Mol. Biol. Cell (2014)

Bottom Line: Agonist activation leads to sustained recruitment of CCR5 to FCLs.Quantitative molecular imaging indicated that FCLs partitioned receptors at the cell surface.Our observations suggest that FCLs provide stable platforms for the recruitment of endocytic cargo.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, London WC1E 6BT, United Kingdom Institute of Immunity and Transplantation, University College London, London NW3 2PF, United Kingdom j.grove@ucl.ac.uk m.marsh@ucl.ac.uk.

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CCL5 agonist triggers persistent association of CCR5 with flat clathrin lattices. CHO-CCR5 cells expressing LCb-RFP and prelabeled with mouse anti-CCR5 mAb directly conjugated to Atto 488 were imaged by TIRF microscopy for 65 min at 2 frames/min. CCL5 was added after 5 min at a final concentration of 125 nM. (A) Images from the beginning (untreated) and end (CCL5) of a representative time course, displaying the cell surface distribution of clathrin (magenta) and CCR5 (green); scale bar, 5 μm. To better illustrate the redistribution of CCR5, the images were generated by averaging four consecutive frames from T = 0 and 60 min, respectively. Images without averaging can be seen in Supplemental Video 1. (B) Stable FCLs were identified using an automated tracking algorithm (see Materials and Methods), allowing quantification of their associated CCR5 fluorescence over the time course. The data are presented as normalized fluorescence expressed relative to the signal at T = 0; n = 220 stable FCLs from three cells, surveyed across two independent experiments; error bars indicate SEM. (C) After 60 min of treatment with CCL5 ligand, CHO-CCR5 cells were imaged for 10 min at 0.33 frame/s. Kymographs display clathrin (magenta) and CCR5 (green) signals from representative CCPs and FCLs in CHO-CCR5 cells. Line plots display normalized fluorescence intensity from the lowermost kymographs (iii, vi).
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Figure 5: CCL5 agonist triggers persistent association of CCR5 with flat clathrin lattices. CHO-CCR5 cells expressing LCb-RFP and prelabeled with mouse anti-CCR5 mAb directly conjugated to Atto 488 were imaged by TIRF microscopy for 65 min at 2 frames/min. CCL5 was added after 5 min at a final concentration of 125 nM. (A) Images from the beginning (untreated) and end (CCL5) of a representative time course, displaying the cell surface distribution of clathrin (magenta) and CCR5 (green); scale bar, 5 μm. To better illustrate the redistribution of CCR5, the images were generated by averaging four consecutive frames from T = 0 and 60 min, respectively. Images without averaging can be seen in Supplemental Video 1. (B) Stable FCLs were identified using an automated tracking algorithm (see Materials and Methods), allowing quantification of their associated CCR5 fluorescence over the time course. The data are presented as normalized fluorescence expressed relative to the signal at T = 0; n = 220 stable FCLs from three cells, surveyed across two independent experiments; error bars indicate SEM. (C) After 60 min of treatment with CCL5 ligand, CHO-CCR5 cells were imaged for 10 min at 0.33 frame/s. Kymographs display clathrin (magenta) and CCR5 (green) signals from representative CCPs and FCLs in CHO-CCR5 cells. Line plots display normalized fluorescence intensity from the lowermost kymographs (iii, vi).

Mentions: To evaluate changes in receptor distribution that accompany desensitization and internalization, we monitored the codistribution of CCR5 and clathrin by live-cell TIRF microscopy. Before treatment, antibody-labeled CCR5 was evenly distributed across the cell surface with no evidence of association with clathrin (Figure 5A, top). After stimulation with CCL5 agonist, CCR5 exhibited a gradual redistribution to CCSs, which stabilized and persisted for the duration of the study (60-min acquisition at 2 frames/min; Figure 5A and Figure 5 Video 1). We identified stable FCLs in CHO-CCR5 cells using the tracking algorithm described earlier (Figure 3B) and monitored CCR5 fluorescence associated with them over the time course. CCR5 redistribution to FCLs occurs with linear kinetics until reaching a persistent plateau at ∼12 min poststimulation (Figure 5B). Preliminary FRAP analysis indicated that CCR5 association with FCLs is dynamic, with CCR5 fluorescence recovery displaying similar kinetics to clathrin turnover (unpublished data). As a further testament to the longevity of FCLs, despite subtle morphological changes, the majority of structures persevered for the entire experiment (>60 min; (Figure 5A and Figure 5 Video 1). This is also in keeping with our previous observation that agonist treatment did not affect the frequency and size of FCLs (Signoret et al., 2005).


Flat clathrin lattices: stable features of the plasma membrane.

Grove J, Metcalf DJ, Knight AE, Wavre-Shapton ST, Sun T, Protonotarios ED, Griffin LD, Lippincott-Schwartz J, Marsh M - Mol. Biol. Cell (2014)

CCL5 agonist triggers persistent association of CCR5 with flat clathrin lattices. CHO-CCR5 cells expressing LCb-RFP and prelabeled with mouse anti-CCR5 mAb directly conjugated to Atto 488 were imaged by TIRF microscopy for 65 min at 2 frames/min. CCL5 was added after 5 min at a final concentration of 125 nM. (A) Images from the beginning (untreated) and end (CCL5) of a representative time course, displaying the cell surface distribution of clathrin (magenta) and CCR5 (green); scale bar, 5 μm. To better illustrate the redistribution of CCR5, the images were generated by averaging four consecutive frames from T = 0 and 60 min, respectively. Images without averaging can be seen in Supplemental Video 1. (B) Stable FCLs were identified using an automated tracking algorithm (see Materials and Methods), allowing quantification of their associated CCR5 fluorescence over the time course. The data are presented as normalized fluorescence expressed relative to the signal at T = 0; n = 220 stable FCLs from three cells, surveyed across two independent experiments; error bars indicate SEM. (C) After 60 min of treatment with CCL5 ligand, CHO-CCR5 cells were imaged for 10 min at 0.33 frame/s. Kymographs display clathrin (magenta) and CCR5 (green) signals from representative CCPs and FCLs in CHO-CCR5 cells. Line plots display normalized fluorescence intensity from the lowermost kymographs (iii, vi).
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Figure 5: CCL5 agonist triggers persistent association of CCR5 with flat clathrin lattices. CHO-CCR5 cells expressing LCb-RFP and prelabeled with mouse anti-CCR5 mAb directly conjugated to Atto 488 were imaged by TIRF microscopy for 65 min at 2 frames/min. CCL5 was added after 5 min at a final concentration of 125 nM. (A) Images from the beginning (untreated) and end (CCL5) of a representative time course, displaying the cell surface distribution of clathrin (magenta) and CCR5 (green); scale bar, 5 μm. To better illustrate the redistribution of CCR5, the images were generated by averaging four consecutive frames from T = 0 and 60 min, respectively. Images without averaging can be seen in Supplemental Video 1. (B) Stable FCLs were identified using an automated tracking algorithm (see Materials and Methods), allowing quantification of their associated CCR5 fluorescence over the time course. The data are presented as normalized fluorescence expressed relative to the signal at T = 0; n = 220 stable FCLs from three cells, surveyed across two independent experiments; error bars indicate SEM. (C) After 60 min of treatment with CCL5 ligand, CHO-CCR5 cells were imaged for 10 min at 0.33 frame/s. Kymographs display clathrin (magenta) and CCR5 (green) signals from representative CCPs and FCLs in CHO-CCR5 cells. Line plots display normalized fluorescence intensity from the lowermost kymographs (iii, vi).
Mentions: To evaluate changes in receptor distribution that accompany desensitization and internalization, we monitored the codistribution of CCR5 and clathrin by live-cell TIRF microscopy. Before treatment, antibody-labeled CCR5 was evenly distributed across the cell surface with no evidence of association with clathrin (Figure 5A, top). After stimulation with CCL5 agonist, CCR5 exhibited a gradual redistribution to CCSs, which stabilized and persisted for the duration of the study (60-min acquisition at 2 frames/min; Figure 5A and Figure 5 Video 1). We identified stable FCLs in CHO-CCR5 cells using the tracking algorithm described earlier (Figure 3B) and monitored CCR5 fluorescence associated with them over the time course. CCR5 redistribution to FCLs occurs with linear kinetics until reaching a persistent plateau at ∼12 min poststimulation (Figure 5B). Preliminary FRAP analysis indicated that CCR5 association with FCLs is dynamic, with CCR5 fluorescence recovery displaying similar kinetics to clathrin turnover (unpublished data). As a further testament to the longevity of FCLs, despite subtle morphological changes, the majority of structures persevered for the entire experiment (>60 min; (Figure 5A and Figure 5 Video 1). This is also in keeping with our previous observation that agonist treatment did not affect the frequency and size of FCLs (Signoret et al., 2005).

Bottom Line: Agonist activation leads to sustained recruitment of CCR5 to FCLs.Quantitative molecular imaging indicated that FCLs partitioned receptors at the cell surface.Our observations suggest that FCLs provide stable platforms for the recruitment of endocytic cargo.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, London WC1E 6BT, United Kingdom Institute of Immunity and Transplantation, University College London, London NW3 2PF, United Kingdom j.grove@ucl.ac.uk m.marsh@ucl.ac.uk.

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Related in: MedlinePlus