Figure 5.

Binding of PIP2 to the µ2 subunit is required downstream of initial AP2 activation for later stages of CCP maturation. (A) Schematic representation of the possible early roles of α–PIP2 interactions. The question mark and double arrowheads point to potential roles in enhancing the rates and or extents of clathrin polymerization, CCP nucleation, cargo recruitment, and CCP maturation. (B) Initiation density of all subthreshold CLSs and bona fide CCPs for the indicated wt or mutant cells (≥22 cells per condition, #CCPs µ2^wt 27,943, #CCPs µ2^PIP2− 36,523). Box plots show medians, 25th and 75th percentiles, and outermost data points. ***, P ≤ 0.0005, unpaired t test. n.s., not significant. (C) Mean clathrin fluorescence intensity traces in lifetime cohorts of CCPs from µ2^wt (gray) and µ2^PIP2− (blue) reconstituted cells. Intensities are shown as mean ± SE calculated from 17 cells per condition. (D) Slope of intensity trace (averaged in the time interval 3–8 s of the elapsed lifetime) in µ2^wt (gray) and µ2^PIP2− (blue) cells. (E) Fraction of CCPs found in short-lived versus longer-lived lifetime cohorts in µ2^WT (gray) and µ2^PIP2− (blue) cells. ***, P < 0.001. (F) Lifetime distributions of all bona fide CCPs (black lines), dynamin-2 (DYN2)-positive CCPs (green lines), and Dyn2-negative CCPs (blue lines) in µ2^PIP2− cells.