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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 are long lived. HeLa cells expressing LCb-RFP and Dyn-2–EGFP were imaged by TIRF microscopy for 10 min at 0.33 frame/s. FCLs appeared as large, intense clathrin structures that persisted beyond the period of acquisition. (A) Stills taken from the beginning (T = 0 s) and end (T = 600 s) of a representative time course. Images display the cell surface distribution of clathrin (magenta) and dynamin (green); scale, 10 μm. (B) The lifetime of clathrin events was quantified using an automated tracking algorithm (see Materials and Methods). The histogram displays the distribution of CCS lifetimes using 9-s-wide bins, based on 6628 events from three acquisitions across two independent experiments. (C) Representative kymographs displaying fluorescent traces from CCPs, including “hot-spot”-type dynamics (ii), and FCLs. Line plots display normalized fluorescence intensity from the lowermost kymographs (iii, vi). Examples were chosen from transient and long-lived structures identified using the tracking algorithm. (D) Whole fixed HeLa cells were permeabilized and labeled with mouse anti–clathrin heavy chain mAb and goat anti–dynamin-2 polyclonal IgG, followed by secondary anti-mouse Alexa Fluor 488 and anti-goat Alexa Fluor 647. (i) Images of the cell surface distribution of clathrin (magenta) and dynamin (green) were acquired using standard TIRF microscopy; scale bar, 5 μm. The distribution of dynamin was also examined by superresolution microscopy. (ii) A dSTORM reconstruction of the boxed area from i; the image was reconstructed using a 25-nm pixel size; scale bar, 2 μm. Heat map intensity scale indicates density of localizations/μm2.
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Figure 3: Flat clathrin lattices are long lived. HeLa cells expressing LCb-RFP and Dyn-2–EGFP were imaged by TIRF microscopy for 10 min at 0.33 frame/s. FCLs appeared as large, intense clathrin structures that persisted beyond the period of acquisition. (A) Stills taken from the beginning (T = 0 s) and end (T = 600 s) of a representative time course. Images display the cell surface distribution of clathrin (magenta) and dynamin (green); scale, 10 μm. (B) The lifetime of clathrin events was quantified using an automated tracking algorithm (see Materials and Methods). The histogram displays the distribution of CCS lifetimes using 9-s-wide bins, based on 6628 events from three acquisitions across two independent experiments. (C) Representative kymographs displaying fluorescent traces from CCPs, including “hot-spot”-type dynamics (ii), and FCLs. Line plots display normalized fluorescence intensity from the lowermost kymographs (iii, vi). Examples were chosen from transient and long-lived structures identified using the tracking algorithm. (D) Whole fixed HeLa cells were permeabilized and labeled with mouse anti–clathrin heavy chain mAb and goat anti–dynamin-2 polyclonal IgG, followed by secondary anti-mouse Alexa Fluor 488 and anti-goat Alexa Fluor 647. (i) Images of the cell surface distribution of clathrin (magenta) and dynamin (green) were acquired using standard TIRF microscopy; scale bar, 5 μm. The distribution of dynamin was also examined by superresolution microscopy. (ii) A dSTORM reconstruction of the boxed area from i; the image was reconstructed using a 25-nm pixel size; scale bar, 2 μm. Heat map intensity scale indicates density of localizations/μm2.

Mentions: As expected following our observations by EM and dSTORM, large, intense CCSs representing FCLs were easily discernible by live TIRF imaging of HeLa cells expressing LCb-RFP. FCLs were notable for their stability, displaying no lateral mobility, very little variation in morphology, and typically persisting beyond the time frame of our experiments (10-min acquisition at 0.33 frame/s; Figure 3A and Figure 3 Video 1). On closer examination of the data, numerous transient CCP-type events were also apparent (highlighted with arrows in Figure 3 Video 1). We quantified the dynamics of CCSs in HeLa cells using an unbiased tracking algorithm previously validated for clathrin studies (Jaqaman et al., 2008; Loerke et al., 2009; Cocucci et al., 2012; Aguet et al., 2013; Figure 3B). A large proportion of events were very short lived (<20s) and are likely to represent small, abortive CCPs and endosomal structures that transiently move into the TIRF field (Ehrlich et al., 2004; Loerke et al., 2009). A further population of short-lived events (20–300 s) principally consisted of diffraction-limited CCP-type structures. Very few events displayed an intermediate lifetime of 300–600 s; however, stable FCL events with lifetimes beyond the duration of our experiments (>600 s) accounted for ∼12% of all clathrin events. Representative kymographs of transient pits and persistent lattice events are shown in Figure 3C.


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 are long lived. HeLa cells expressing LCb-RFP and Dyn-2–EGFP were imaged by TIRF microscopy for 10 min at 0.33 frame/s. FCLs appeared as large, intense clathrin structures that persisted beyond the period of acquisition. (A) Stills taken from the beginning (T = 0 s) and end (T = 600 s) of a representative time course. Images display the cell surface distribution of clathrin (magenta) and dynamin (green); scale, 10 μm. (B) The lifetime of clathrin events was quantified using an automated tracking algorithm (see Materials and Methods). The histogram displays the distribution of CCS lifetimes using 9-s-wide bins, based on 6628 events from three acquisitions across two independent experiments. (C) Representative kymographs displaying fluorescent traces from CCPs, including “hot-spot”-type dynamics (ii), and FCLs. Line plots display normalized fluorescence intensity from the lowermost kymographs (iii, vi). Examples were chosen from transient and long-lived structures identified using the tracking algorithm. (D) Whole fixed HeLa cells were permeabilized and labeled with mouse anti–clathrin heavy chain mAb and goat anti–dynamin-2 polyclonal IgG, followed by secondary anti-mouse Alexa Fluor 488 and anti-goat Alexa Fluor 647. (i) Images of the cell surface distribution of clathrin (magenta) and dynamin (green) were acquired using standard TIRF microscopy; scale bar, 5 μm. The distribution of dynamin was also examined by superresolution microscopy. (ii) A dSTORM reconstruction of the boxed area from i; the image was reconstructed using a 25-nm pixel size; scale bar, 2 μm. Heat map intensity scale indicates density of localizations/μm2.
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Figure 3: Flat clathrin lattices are long lived. HeLa cells expressing LCb-RFP and Dyn-2–EGFP were imaged by TIRF microscopy for 10 min at 0.33 frame/s. FCLs appeared as large, intense clathrin structures that persisted beyond the period of acquisition. (A) Stills taken from the beginning (T = 0 s) and end (T = 600 s) of a representative time course. Images display the cell surface distribution of clathrin (magenta) and dynamin (green); scale, 10 μm. (B) The lifetime of clathrin events was quantified using an automated tracking algorithm (see Materials and Methods). The histogram displays the distribution of CCS lifetimes using 9-s-wide bins, based on 6628 events from three acquisitions across two independent experiments. (C) Representative kymographs displaying fluorescent traces from CCPs, including “hot-spot”-type dynamics (ii), and FCLs. Line plots display normalized fluorescence intensity from the lowermost kymographs (iii, vi). Examples were chosen from transient and long-lived structures identified using the tracking algorithm. (D) Whole fixed HeLa cells were permeabilized and labeled with mouse anti–clathrin heavy chain mAb and goat anti–dynamin-2 polyclonal IgG, followed by secondary anti-mouse Alexa Fluor 488 and anti-goat Alexa Fluor 647. (i) Images of the cell surface distribution of clathrin (magenta) and dynamin (green) were acquired using standard TIRF microscopy; scale bar, 5 μm. The distribution of dynamin was also examined by superresolution microscopy. (ii) A dSTORM reconstruction of the boxed area from i; the image was reconstructed using a 25-nm pixel size; scale bar, 2 μm. Heat map intensity scale indicates density of localizations/μm2.
Mentions: As expected following our observations by EM and dSTORM, large, intense CCSs representing FCLs were easily discernible by live TIRF imaging of HeLa cells expressing LCb-RFP. FCLs were notable for their stability, displaying no lateral mobility, very little variation in morphology, and typically persisting beyond the time frame of our experiments (10-min acquisition at 0.33 frame/s; Figure 3A and Figure 3 Video 1). On closer examination of the data, numerous transient CCP-type events were also apparent (highlighted with arrows in Figure 3 Video 1). We quantified the dynamics of CCSs in HeLa cells using an unbiased tracking algorithm previously validated for clathrin studies (Jaqaman et al., 2008; Loerke et al., 2009; Cocucci et al., 2012; Aguet et al., 2013; Figure 3B). A large proportion of events were very short lived (<20s) and are likely to represent small, abortive CCPs and endosomal structures that transiently move into the TIRF field (Ehrlich et al., 2004; Loerke et al., 2009). A further population of short-lived events (20–300 s) principally consisted of diffraction-limited CCP-type structures. Very few events displayed an intermediate lifetime of 300–600 s; however, stable FCL events with lifetimes beyond the duration of our experiments (>600 s) accounted for ∼12% of all clathrin events. Representative kymographs of transient pits and persistent lattice events are shown in Figure 3C.

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