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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 dynamic molecular assemblies. HeLa cells expressing LCb-RFP and Dyn-2–EGFP were imaged by spinning disk microscopy. The fluorescence signals associated with selected groups of FCLs were photobleached, and fluorescence recovery was monitored for 10 min at 0.33 frame/s. (A) Stills from a representative experiment displaying fluorescence recovery of LCb-RFP (magenta) and Dyn-2–EGFP (green) in a selected group of FCLs (highlighted by dashed line); scale bar, 10 μm. Clathrin turnover was relatively slow, whereas dynamin exchange occurred very rapidly. (B) Mean fluorescence recovery profiles for clathrin and dynamin, n = 32 and 9 selected groups of FCLs, respectively; error bars indicate SEM. (C) Time to 50% recovery of clathrin fluorescence in control or ATP-depleted HeLa cells; n = 32 and 10 selected groups of FCLs, respectively; error bars indicate SEM.
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Figure 4: Flat clathrin lattices are dynamic molecular assemblies. HeLa cells expressing LCb-RFP and Dyn-2–EGFP were imaged by spinning disk microscopy. The fluorescence signals associated with selected groups of FCLs were photobleached, and fluorescence recovery was monitored for 10 min at 0.33 frame/s. (A) Stills from a representative experiment displaying fluorescence recovery of LCb-RFP (magenta) and Dyn-2–EGFP (green) in a selected group of FCLs (highlighted by dashed line); scale bar, 10 μm. Clathrin turnover was relatively slow, whereas dynamin exchange occurred very rapidly. (B) Mean fluorescence recovery profiles for clathrin and dynamin, n = 32 and 9 selected groups of FCLs, respectively; error bars indicate SEM. (C) Time to 50% recovery of clathrin fluorescence in control or ATP-depleted HeLa cells; n = 32 and 10 selected groups of FCLs, respectively; error bars indicate SEM.

Mentions: The molecular exchange of clathrin triskelia between the cytosolic pool and CCSs at the plasma membrane remains a contentious topic. Various reports used fluorescence recovery after photobleaching (FRAP) to demonstrate turnover of the constituents of CCSs (Wu et al., 2001; 2003; Mettlen et al., 2010; Taylor et al., 2012), although interpretation of these results is confounded by the intrinsic transitory nature of CCPs. Others studies of pharmacologically arrested CCPs found no fluorescence recovery (Macia et al., 2006; Kleist et al., 2011). Furthermore, completion of CME requires the active disassembly of clathrin from CCVs by Hsc70 and auxillin/GAK (Schlossman et al., 1984; Greene and Eisenberg, 1990; Ungewickell et al., 1995; Böcking et al., 2011). The latter observations suggest that the basketwork of maturing CCPs does not undergo molecular exchange. The long lifetimes of FCLs make them particularly well suited to FRAP analysis. Therefore we examined clathrin and dynamin turnover by simultaneous photobleaching of LCb-RFP and Dyn-2–EGFP within selected groups of FCLs. Of importance, the interaction between clathrin light and heavy chain is very stable, such that the molecular dynamics of LCb-RFP faithfully represents the behavior of clathrin triskelia subunits (Hoffmann et al., 2010; Brodsky, 2012). Clathrin fluorescence displayed gradual but sustained recovery, reaching 50% after ∼115 s (Figure 4 and Figure 4 Video 1), demonstrating that subunits of the FCL basketwork are being exchanged with the cytosolic pool. Dynamin recovery also indicated turnover, although with much faster kinetics; the signal recovered to 50% in <9 s. This transitory recruitment may be analogous to the “flickering” association of dynamin with maturing CCPs that precedes the stable assembly of the scission machinery, as proposed by Taylor et al. (2012) or the abortive recruitment of dynamin to endocytically immature CCSs recently suggested by Grassart et al. (2014). Mean fluorescence recovery profiles (Figure 4B) show that LCb-RFP signal recovers to only ∼65%, indicating that a significant proportion of clathrin within FCLs is not available for exchange; on the contrary, the vast majority of dynamin was mobile, with fluorescence reaching >90% recovery within ∼80 s.


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 dynamic molecular assemblies. HeLa cells expressing LCb-RFP and Dyn-2–EGFP were imaged by spinning disk microscopy. The fluorescence signals associated with selected groups of FCLs were photobleached, and fluorescence recovery was monitored for 10 min at 0.33 frame/s. (A) Stills from a representative experiment displaying fluorescence recovery of LCb-RFP (magenta) and Dyn-2–EGFP (green) in a selected group of FCLs (highlighted by dashed line); scale bar, 10 μm. Clathrin turnover was relatively slow, whereas dynamin exchange occurred very rapidly. (B) Mean fluorescence recovery profiles for clathrin and dynamin, n = 32 and 9 selected groups of FCLs, respectively; error bars indicate SEM. (C) Time to 50% recovery of clathrin fluorescence in control or ATP-depleted HeLa cells; n = 32 and 10 selected groups of FCLs, respectively; error bars indicate SEM.
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Figure 4: Flat clathrin lattices are dynamic molecular assemblies. HeLa cells expressing LCb-RFP and Dyn-2–EGFP were imaged by spinning disk microscopy. The fluorescence signals associated with selected groups of FCLs were photobleached, and fluorescence recovery was monitored for 10 min at 0.33 frame/s. (A) Stills from a representative experiment displaying fluorescence recovery of LCb-RFP (magenta) and Dyn-2–EGFP (green) in a selected group of FCLs (highlighted by dashed line); scale bar, 10 μm. Clathrin turnover was relatively slow, whereas dynamin exchange occurred very rapidly. (B) Mean fluorescence recovery profiles for clathrin and dynamin, n = 32 and 9 selected groups of FCLs, respectively; error bars indicate SEM. (C) Time to 50% recovery of clathrin fluorescence in control or ATP-depleted HeLa cells; n = 32 and 10 selected groups of FCLs, respectively; error bars indicate SEM.
Mentions: The molecular exchange of clathrin triskelia between the cytosolic pool and CCSs at the plasma membrane remains a contentious topic. Various reports used fluorescence recovery after photobleaching (FRAP) to demonstrate turnover of the constituents of CCSs (Wu et al., 2001; 2003; Mettlen et al., 2010; Taylor et al., 2012), although interpretation of these results is confounded by the intrinsic transitory nature of CCPs. Others studies of pharmacologically arrested CCPs found no fluorescence recovery (Macia et al., 2006; Kleist et al., 2011). Furthermore, completion of CME requires the active disassembly of clathrin from CCVs by Hsc70 and auxillin/GAK (Schlossman et al., 1984; Greene and Eisenberg, 1990; Ungewickell et al., 1995; Böcking et al., 2011). The latter observations suggest that the basketwork of maturing CCPs does not undergo molecular exchange. The long lifetimes of FCLs make them particularly well suited to FRAP analysis. Therefore we examined clathrin and dynamin turnover by simultaneous photobleaching of LCb-RFP and Dyn-2–EGFP within selected groups of FCLs. Of importance, the interaction between clathrin light and heavy chain is very stable, such that the molecular dynamics of LCb-RFP faithfully represents the behavior of clathrin triskelia subunits (Hoffmann et al., 2010; Brodsky, 2012). Clathrin fluorescence displayed gradual but sustained recovery, reaching 50% after ∼115 s (Figure 4 and Figure 4 Video 1), demonstrating that subunits of the FCL basketwork are being exchanged with the cytosolic pool. Dynamin recovery also indicated turnover, although with much faster kinetics; the signal recovered to 50% in <9 s. This transitory recruitment may be analogous to the “flickering” association of dynamin with maturing CCPs that precedes the stable assembly of the scission machinery, as proposed by Taylor et al. (2012) or the abortive recruitment of dynamin to endocytically immature CCSs recently suggested by Grassart et al. (2014). Mean fluorescence recovery profiles (Figure 4B) show that LCb-RFP signal recovers to only ∼65%, indicating that a significant proportion of clathrin within FCLs is not available for exchange; on the contrary, the vast majority of dynamin was mobile, with fluorescence reaching >90% recovery within ∼80 s.

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