Limits...
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.

Show MeSH

Related in: MedlinePlus

A superresolution microscopy survey of clathrin heterogeneity. Whole fixed cells were permeabilized and labeled with a mouse anti–clathrin heavy chain mAb, followed by secondary anti-mouse Alexa Fluor 647. We imaged CCSs on the ventral cell surface by dSTORM using TIRF illumination. (A) dSTORM superresolution images of CCSs in HEK-293T and HeLa cells. Images were reconstructed using a 25-nm pixel size; scale bar, 4 μm. Insets display standard diffraction-limited TIRF images of the same area. (B) Enlarged areas from dSTORM images in A, displaying small, round CCPs in HEK-293T and large, pleomorphic FCLs in HeLa cells; scale bar, 1 μm. Heat map intensity scale indicates density of localizations/μm2. (C) Morphometric analysis of clathrin heterogeneity was performed by automated image segmentation (see Materials and Methods). Scatter plots display the circularity and two-dimensional surface area of individual CCSs from HEK-293T and HeLa cells; each plot displays 2000 representative structures. Color coding indicates CCSs defined as small, <30,000 nm2 (white); medium, 30,000–100,000 nm2 (light gray); or large, >100,000 nm2 (dark gray). (D) Stacked histograms displaying the relative frequency of small, medium, and large structures in HEK-293T and HeLa cells from n = 8 and 13 cells, respectively, surveyed across three independent experiments; error bars indicate SEM. (E) HeLa cells were stained with rabbit anti–clathrin light chain polyclonal serum, followed by secondary anti-rabbit Alexa Fluor 647. Representative dSTORM image (i). The image was reconstructed with a 25-nm pixel size; scale bar, 4 μm. An enlarged area is shown in ii; scale bar, 1 μm. (F, G) Morphometric analysis of CCSs stained with clathrin light chain; the scatter plot displays 2000 structures from n = 4 cells surveyed in one experiment; error bars indicate SD from the mean.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230618&req=5

Figure 2: A superresolution microscopy survey of clathrin heterogeneity. Whole fixed cells were permeabilized and labeled with a mouse anti–clathrin heavy chain mAb, followed by secondary anti-mouse Alexa Fluor 647. We imaged CCSs on the ventral cell surface by dSTORM using TIRF illumination. (A) dSTORM superresolution images of CCSs in HEK-293T and HeLa cells. Images were reconstructed using a 25-nm pixel size; scale bar, 4 μm. Insets display standard diffraction-limited TIRF images of the same area. (B) Enlarged areas from dSTORM images in A, displaying small, round CCPs in HEK-293T and large, pleomorphic FCLs in HeLa cells; scale bar, 1 μm. Heat map intensity scale indicates density of localizations/μm2. (C) Morphometric analysis of clathrin heterogeneity was performed by automated image segmentation (see Materials and Methods). Scatter plots display the circularity and two-dimensional surface area of individual CCSs from HEK-293T and HeLa cells; each plot displays 2000 representative structures. Color coding indicates CCSs defined as small, <30,000 nm2 (white); medium, 30,000–100,000 nm2 (light gray); or large, >100,000 nm2 (dark gray). (D) Stacked histograms displaying the relative frequency of small, medium, and large structures in HEK-293T and HeLa cells from n = 8 and 13 cells, respectively, surveyed across three independent experiments; error bars indicate SEM. (E) HeLa cells were stained with rabbit anti–clathrin light chain polyclonal serum, followed by secondary anti-rabbit Alexa Fluor 647. Representative dSTORM image (i). The image was reconstructed with a 25-nm pixel size; scale bar, 4 μm. An enlarged area is shown in ii; scale bar, 1 μm. (F, G) Morphometric analysis of CCSs stained with clathrin light chain; the scatter plot displays 2000 structures from n = 4 cells surveyed in one experiment; error bars indicate SD from the mean.

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.


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)

A superresolution microscopy survey of clathrin heterogeneity. Whole fixed cells were permeabilized and labeled with a mouse anti–clathrin heavy chain mAb, followed by secondary anti-mouse Alexa Fluor 647. We imaged CCSs on the ventral cell surface by dSTORM using TIRF illumination. (A) dSTORM superresolution images of CCSs in HEK-293T and HeLa cells. Images were reconstructed using a 25-nm pixel size; scale bar, 4 μm. Insets display standard diffraction-limited TIRF images of the same area. (B) Enlarged areas from dSTORM images in A, displaying small, round CCPs in HEK-293T and large, pleomorphic FCLs in HeLa cells; scale bar, 1 μm. Heat map intensity scale indicates density of localizations/μm2. (C) Morphometric analysis of clathrin heterogeneity was performed by automated image segmentation (see Materials and Methods). Scatter plots display the circularity and two-dimensional surface area of individual CCSs from HEK-293T and HeLa cells; each plot displays 2000 representative structures. Color coding indicates CCSs defined as small, <30,000 nm2 (white); medium, 30,000–100,000 nm2 (light gray); or large, >100,000 nm2 (dark gray). (D) Stacked histograms displaying the relative frequency of small, medium, and large structures in HEK-293T and HeLa cells from n = 8 and 13 cells, respectively, surveyed across three independent experiments; error bars indicate SEM. (E) HeLa cells were stained with rabbit anti–clathrin light chain polyclonal serum, followed by secondary anti-rabbit Alexa Fluor 647. Representative dSTORM image (i). The image was reconstructed with a 25-nm pixel size; scale bar, 4 μm. An enlarged area is shown in ii; scale bar, 1 μm. (F, G) Morphometric analysis of CCSs stained with clathrin light chain; the scatter plot displays 2000 structures from n = 4 cells surveyed in one experiment; error bars indicate SD from the mean.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230618&req=5

Figure 2: A superresolution microscopy survey of clathrin heterogeneity. Whole fixed cells were permeabilized and labeled with a mouse anti–clathrin heavy chain mAb, followed by secondary anti-mouse Alexa Fluor 647. We imaged CCSs on the ventral cell surface by dSTORM using TIRF illumination. (A) dSTORM superresolution images of CCSs in HEK-293T and HeLa cells. Images were reconstructed using a 25-nm pixel size; scale bar, 4 μm. Insets display standard diffraction-limited TIRF images of the same area. (B) Enlarged areas from dSTORM images in A, displaying small, round CCPs in HEK-293T and large, pleomorphic FCLs in HeLa cells; scale bar, 1 μm. Heat map intensity scale indicates density of localizations/μm2. (C) Morphometric analysis of clathrin heterogeneity was performed by automated image segmentation (see Materials and Methods). Scatter plots display the circularity and two-dimensional surface area of individual CCSs from HEK-293T and HeLa cells; each plot displays 2000 representative structures. Color coding indicates CCSs defined as small, <30,000 nm2 (white); medium, 30,000–100,000 nm2 (light gray); or large, >100,000 nm2 (dark gray). (D) Stacked histograms displaying the relative frequency of small, medium, and large structures in HEK-293T and HeLa cells from n = 8 and 13 cells, respectively, surveyed across three independent experiments; error bars indicate SEM. (E) HeLa cells were stained with rabbit anti–clathrin light chain polyclonal serum, followed by secondary anti-rabbit Alexa Fluor 647. Representative dSTORM image (i). The image was reconstructed with a 25-nm pixel size; scale bar, 4 μm. An enlarged area is shown in ii; scale bar, 1 μm. (F, G) Morphometric analysis of CCSs stained with clathrin light chain; the scatter plot displays 2000 structures from n = 4 cells surveyed in one experiment; error bars indicate SD from the mean.
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.

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