Flat clathrin lattices: stable features of the plasma membrane.
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.
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 email@example.com firstname.lastname@example.org.Show MeSH
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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.
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 email@example.com firstname.lastname@example.org.