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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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Flat clathrin lattices partition CCR5 at the plasma membrane. CHO-CCR5 cells expressing LCa-PAGFP and prelabeled with mouse anti-CCR5 mAb directly conjugated to Alexa Fluor 647 were incubated for 1 h in the presence or absence of 125 nM CCL5. Cells were fixed and imaged by two-color PALM/dSTORM. (A) Representative superresolution images of the cell surface distribution of clathrin (magenta) and CCR5 (green) in control or CCL5-treated CHO-CCR5 cells. Images were reconstructed with a 25-nm pixel size; scale bar, 1 μm. (B) The molecular codistribution of FCLs and CCR5 was quantified using Hopkins spatial statistics in multiple arbitrarily chosen regions of interest (see Materials and Methods). A Hopkins value of 0.5 demonstrates a random codistribution, whereas values significantly above or below 0.5 indicate association or segregation, respectively. In untreated cells, a Hopkins value of ∼0.4 indicates that FCLs and CCR5 were segregated. Stimulation with CCL5 resulted in a Hopkins index of ∼0.7, demonstrating association of CCR5 with FCLs. As a control, we compared the distribution of CCR5 to simulated random points, which gave a value of 0.5, indicating that the algorithm is functioning correctly. n = 11 (untreated) and 10 (CCL5) regions of interest, taken from three fields across two independent experiments for each condition; error bars indicate SEM; **p = 0.003, ****p ≥ 0.0001, unpaired t test (Prism; GraphPad, La Jolla, CA).
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Figure 6: Flat clathrin lattices partition CCR5 at the plasma membrane. CHO-CCR5 cells expressing LCa-PAGFP and prelabeled with mouse anti-CCR5 mAb directly conjugated to Alexa Fluor 647 were incubated for 1 h in the presence or absence of 125 nM CCL5. Cells were fixed and imaged by two-color PALM/dSTORM. (A) Representative superresolution images of the cell surface distribution of clathrin (magenta) and CCR5 (green) in control or CCL5-treated CHO-CCR5 cells. Images were reconstructed with a 25-nm pixel size; scale bar, 1 μm. (B) The molecular codistribution of FCLs and CCR5 was quantified using Hopkins spatial statistics in multiple arbitrarily chosen regions of interest (see Materials and Methods). A Hopkins value of 0.5 demonstrates a random codistribution, whereas values significantly above or below 0.5 indicate association or segregation, respectively. In untreated cells, a Hopkins value of ∼0.4 indicates that FCLs and CCR5 were segregated. Stimulation with CCL5 resulted in a Hopkins index of ∼0.7, demonstrating association of CCR5 with FCLs. As a control, we compared the distribution of CCR5 to simulated random points, which gave a value of 0.5, indicating that the algorithm is functioning correctly. n = 11 (untreated) and 10 (CCL5) regions of interest, taken from three fields across two independent experiments for each condition; error bars indicate SEM; **p = 0.003, ****p ≥ 0.0001, unpaired t test (Prism; GraphPad, La Jolla, CA).

Mentions: A number of reports suggest that FCLs may organize the cell surface distribution of both endocytic and nonendocytic receptors (Pumplin and Bloch, 1990; Miller et al., 1991; Sanan and Anderson, 1991; De Deyne et al., 1998; Lamaze et al., 2001; Kim et al., 2013). Therefore we evaluated the molecular codistribution of clathrin and CCR5 in our experimental system. To achieve this, we performed dual-color PALM/dSTORM superresolution imaging (Betzig et al., 2006; Bates et al., 2007; Heilemann et al., 2008) of photoactivatable, GFP-tagged clathrin light chain (PAGFP-LCa) and CCR5 labeled with mAb directly conjugated to Alexa Fluor 647. In untreated cells, CCR5 was distributed throughout the plasma membrane, organized into abundant puncta that likely represent individual or small clusters of receptors (Figure 6A). Critically, there was no ostensible association with FCLs. After 60 min of treatment with CCL5, the receptors had reorganized into larger clusters often arranged in and around FCLs, leaving the remaining plasma membrane predominantly devoid of receptor (Figure 6A).


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)

Flat clathrin lattices partition CCR5 at the plasma membrane. CHO-CCR5 cells expressing LCa-PAGFP and prelabeled with mouse anti-CCR5 mAb directly conjugated to Alexa Fluor 647 were incubated for 1 h in the presence or absence of 125 nM CCL5. Cells were fixed and imaged by two-color PALM/dSTORM. (A) Representative superresolution images of the cell surface distribution of clathrin (magenta) and CCR5 (green) in control or CCL5-treated CHO-CCR5 cells. Images were reconstructed with a 25-nm pixel size; scale bar, 1 μm. (B) The molecular codistribution of FCLs and CCR5 was quantified using Hopkins spatial statistics in multiple arbitrarily chosen regions of interest (see Materials and Methods). A Hopkins value of 0.5 demonstrates a random codistribution, whereas values significantly above or below 0.5 indicate association or segregation, respectively. In untreated cells, a Hopkins value of ∼0.4 indicates that FCLs and CCR5 were segregated. Stimulation with CCL5 resulted in a Hopkins index of ∼0.7, demonstrating association of CCR5 with FCLs. As a control, we compared the distribution of CCR5 to simulated random points, which gave a value of 0.5, indicating that the algorithm is functioning correctly. n = 11 (untreated) and 10 (CCL5) regions of interest, taken from three fields across two independent experiments for each condition; error bars indicate SEM; **p = 0.003, ****p ≥ 0.0001, unpaired t test (Prism; GraphPad, La Jolla, CA).
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Figure 6: Flat clathrin lattices partition CCR5 at the plasma membrane. CHO-CCR5 cells expressing LCa-PAGFP and prelabeled with mouse anti-CCR5 mAb directly conjugated to Alexa Fluor 647 were incubated for 1 h in the presence or absence of 125 nM CCL5. Cells were fixed and imaged by two-color PALM/dSTORM. (A) Representative superresolution images of the cell surface distribution of clathrin (magenta) and CCR5 (green) in control or CCL5-treated CHO-CCR5 cells. Images were reconstructed with a 25-nm pixel size; scale bar, 1 μm. (B) The molecular codistribution of FCLs and CCR5 was quantified using Hopkins spatial statistics in multiple arbitrarily chosen regions of interest (see Materials and Methods). A Hopkins value of 0.5 demonstrates a random codistribution, whereas values significantly above or below 0.5 indicate association or segregation, respectively. In untreated cells, a Hopkins value of ∼0.4 indicates that FCLs and CCR5 were segregated. Stimulation with CCL5 resulted in a Hopkins index of ∼0.7, demonstrating association of CCR5 with FCLs. As a control, we compared the distribution of CCR5 to simulated random points, which gave a value of 0.5, indicating that the algorithm is functioning correctly. n = 11 (untreated) and 10 (CCL5) regions of interest, taken from three fields across two independent experiments for each condition; error bars indicate SEM; **p = 0.003, ****p ≥ 0.0001, unpaired t test (Prism; GraphPad, La Jolla, CA).
Mentions: A number of reports suggest that FCLs may organize the cell surface distribution of both endocytic and nonendocytic receptors (Pumplin and Bloch, 1990; Miller et al., 1991; Sanan and Anderson, 1991; De Deyne et al., 1998; Lamaze et al., 2001; Kim et al., 2013). Therefore we evaluated the molecular codistribution of clathrin and CCR5 in our experimental system. To achieve this, we performed dual-color PALM/dSTORM superresolution imaging (Betzig et al., 2006; Bates et al., 2007; Heilemann et al., 2008) of photoactivatable, GFP-tagged clathrin light chain (PAGFP-LCa) and CCR5 labeled with mAb directly conjugated to Alexa Fluor 647. In untreated cells, CCR5 was distributed throughout the plasma membrane, organized into abundant puncta that likely represent individual or small clusters of receptors (Figure 6A). Critically, there was no ostensible association with FCLs. After 60 min of treatment with CCL5, the receptors had reorganized into larger clusters often arranged in and around FCLs, leaving the remaining plasma membrane predominantly devoid of receptor (Figure 6A).

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