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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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An electron microscopy survey of clathrin heterogeneity. Plasma membrane sheets from the ventral and dorsal cell surfaces were prepared for electron microscopy. (A) Representative images of the ventral plasma membrane of HEK-293T (i, ii) and HeLa cells (iii); Scale bar, 500 nm. In HEK-293T cells, small, homogeneous CCPs (arrowheads) predominate. HeLa cells show similar CCPs but had a propensity to also form large FCLs (L), largely composed of hexagonal clathrin basketwork (arrows). FCLs often displayed budding structures at their periphery (asterisks), which appeared morphologically similar to classical CCPs. (B) Morphometric analysis of clathrin heterogeneity was performed by manual image segmentation (see Materials and Methods). Scatter plots display the circularity and two-dimensional surface area of individual CCSs from the ventral surface of i) HEK-293T (n = 2256 structures) and ii) HeLa cells (n = 1329 structures). Shading indicates CCSs defined as small, <30,000 nm2 (white); medium, 30,000–100,000 nm2 (light gray); or large, >100,000 nm2 (dark gray). (C) Stacked histograms expressing the relative frequency of small, medium, and large structures on the ventral (i) and dorsal (ii) surface of HEK-293T and HeLa cells; n = 24 and 19 cells, respectively, surveyed across three independent experiments. Error bars indicate SEM.
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Figure 1: An electron microscopy survey of clathrin heterogeneity. Plasma membrane sheets from the ventral and dorsal cell surfaces were prepared for electron microscopy. (A) Representative images of the ventral plasma membrane of HEK-293T (i, ii) and HeLa cells (iii); Scale bar, 500 nm. In HEK-293T cells, small, homogeneous CCPs (arrowheads) predominate. HeLa cells show similar CCPs but had a propensity to also form large FCLs (L), largely composed of hexagonal clathrin basketwork (arrows). FCLs often displayed budding structures at their periphery (asterisks), which appeared morphologically similar to classical CCPs. (B) Morphometric analysis of clathrin heterogeneity was performed by manual image segmentation (see Materials and Methods). Scatter plots display the circularity and two-dimensional surface area of individual CCSs from the ventral surface of i) HEK-293T (n = 2256 structures) and ii) HeLa cells (n = 1329 structures). Shading indicates CCSs defined as small, <30,000 nm2 (white); medium, 30,000–100,000 nm2 (light gray); or large, >100,000 nm2 (dark gray). (C) Stacked histograms expressing the relative frequency of small, medium, and large structures on the ventral (i) and dorsal (ii) surface of HEK-293T and HeLa cells; n = 24 and 19 cells, respectively, surveyed across three independent experiments. Error bars indicate SEM.

Mentions: The distinctive polygonal basketwork of CCSs make them easy to identify and particularly amenable to ultrastructural analysis (Heuser, 1980). We first evaluated clathrin heterogeneity in a variety of cell lines by preparing ventral (bottom) and dorsal (top) plasma membrane sheets for EM. Concordant with the standard model of CME, we observed abundant small, round structures characteristic of CCPs. In some cell types (e.g., HEK-293T and BSC-1 cells), these structures predominated (Figure 1A, i and ii, and unpublished data). However, other cell types (e.g., HeLa and CHO cells) also formed large, irregular FCLs (Figure 1Aiii and Supplemental Figure S4; examples of ventral membrane sheets from HEK-293T and HeLa cells are provided in Supplemental Figures S1 and S2).


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)

An electron microscopy survey of clathrin heterogeneity. Plasma membrane sheets from the ventral and dorsal cell surfaces were prepared for electron microscopy. (A) Representative images of the ventral plasma membrane of HEK-293T (i, ii) and HeLa cells (iii); Scale bar, 500 nm. In HEK-293T cells, small, homogeneous CCPs (arrowheads) predominate. HeLa cells show similar CCPs but had a propensity to also form large FCLs (L), largely composed of hexagonal clathrin basketwork (arrows). FCLs often displayed budding structures at their periphery (asterisks), which appeared morphologically similar to classical CCPs. (B) Morphometric analysis of clathrin heterogeneity was performed by manual image segmentation (see Materials and Methods). Scatter plots display the circularity and two-dimensional surface area of individual CCSs from the ventral surface of i) HEK-293T (n = 2256 structures) and ii) HeLa cells (n = 1329 structures). Shading indicates CCSs defined as small, <30,000 nm2 (white); medium, 30,000–100,000 nm2 (light gray); or large, >100,000 nm2 (dark gray). (C) Stacked histograms expressing the relative frequency of small, medium, and large structures on the ventral (i) and dorsal (ii) surface of HEK-293T and HeLa cells; n = 24 and 19 cells, respectively, surveyed across three independent experiments. Error bars indicate SEM.
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Related In: Results  -  Collection

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Figure 1: An electron microscopy survey of clathrin heterogeneity. Plasma membrane sheets from the ventral and dorsal cell surfaces were prepared for electron microscopy. (A) Representative images of the ventral plasma membrane of HEK-293T (i, ii) and HeLa cells (iii); Scale bar, 500 nm. In HEK-293T cells, small, homogeneous CCPs (arrowheads) predominate. HeLa cells show similar CCPs but had a propensity to also form large FCLs (L), largely composed of hexagonal clathrin basketwork (arrows). FCLs often displayed budding structures at their periphery (asterisks), which appeared morphologically similar to classical CCPs. (B) Morphometric analysis of clathrin heterogeneity was performed by manual image segmentation (see Materials and Methods). Scatter plots display the circularity and two-dimensional surface area of individual CCSs from the ventral surface of i) HEK-293T (n = 2256 structures) and ii) HeLa cells (n = 1329 structures). Shading indicates CCSs defined as small, <30,000 nm2 (white); medium, 30,000–100,000 nm2 (light gray); or large, >100,000 nm2 (dark gray). (C) Stacked histograms expressing the relative frequency of small, medium, and large structures on the ventral (i) and dorsal (ii) surface of HEK-293T and HeLa cells; n = 24 and 19 cells, respectively, surveyed across three independent experiments. Error bars indicate SEM.
Mentions: The distinctive polygonal basketwork of CCSs make them easy to identify and particularly amenable to ultrastructural analysis (Heuser, 1980). We first evaluated clathrin heterogeneity in a variety of cell lines by preparing ventral (bottom) and dorsal (top) plasma membrane sheets for EM. Concordant with the standard model of CME, we observed abundant small, round structures characteristic of CCPs. In some cell types (e.g., HEK-293T and BSC-1 cells), these structures predominated (Figure 1A, i and ii, and unpublished data). However, other cell types (e.g., HeLa and CHO cells) also formed large, irregular FCLs (Figure 1Aiii and Supplemental Figure S4; examples of ventral membrane sheets from HEK-293T and HeLa cells are provided in Supplemental Figures S1 and S2).

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.

Show MeSH
Related in: MedlinePlus