Flat clathrin lattices: stable features of the plasma membrane.
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.
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 email@example.com firstname.lastname@example.org.Show MeSH
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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association with FCLs is dynamic, with CCR5 fluorescence recovery displaying similar kinetics to clathrin turnover (unpublished data).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).
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 email@example.com firstname.lastname@example.org.