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 firstname.lastname@example.org email@example.com.Show MeSH
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Mentions: dSTORM recapitulated our ultrastructural studies; HEK-293T cells possessed numerous small puncta of clathrin, whereas HeLa cells displayed large, heterogeneous arrays (Figure 2, A and B). Automated segmentation allowed us to perform morphometric analysis as in Figure 1, B and C. Despite the lower resolution of dSTORM compared with EM, a distinct population of small, round CCPs was discernible in both cell types, whereas large, irregular FCLs were only common in HeLa cells (Figure 2C). We independently confirmed these observations in HeLa cells stained with a polyclonal anti–clathrin light chain antibody (Figure 2, E–G) and anti–α-adaptin (unpublished data). Frequency analysis of CCSs in HEK-293T cells by dSTORM was similar to data generated by EM (compare Figures 2D and 1Ci). In contrast, FCLs were less frequent on the ventral surface of whole HeLa cells assessed by dSTORM (10–20%; Figure 2, D and G) when compared with membrane sheets imaged by EM (35%; Figure 1Ci). However, given the likelihood of artifactually enriching for FCLs during EM sample preparation (Akisaka et al., 2003), superresolution imaging of whole cells is likely to provide a more reliable representation of the frequency of CCSs. Furthermore, dSTORM permits the systematic survey of thousands of structures across large fields of view.
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 firstname.lastname@example.org email@example.com.