FIGURE 5.

Endocytosis following stimulation in single μ2 domain mutants shows only subtle defects that are more severe when combined. A, representative ensemble average traces of endocytosis from neurons expressing either WT or various μ2 mutants. Post-stimulus vG-pH fluorescence decays were fit to single exponential decays. Individual domain mutants slow endocytosis only marginally (rescue WT, 18.1 ± 1.2 s, n = 9; YXXϕ, 20.1 ± 1.0 s, n = 12; T156A, 22.3 ± 0.8 s, n = 16; Y334A,K335A (YK), 19.7 ± 1.0 s, n = 19; K341E/K343E/K345E (KKKEEE), 21.4 ± 0.8 s, n = 15), whereas combined mutants slow endocytosis more significantly (3M, 24.7 ± 2.3 s, n = 14; All-in-one, 33.8 ± 2.6 s, n = 14). Inset, the distribution of single exponential time constants for individual bouton analysis from rescue WT or mutants derived from between 139 and 324 boutons. B, time constants of endocytosis after stimulation for different number of stimuli (25, 50, 100, and 300 action potentials at 10 Hz, respectively, n = 8–18 cells) in μ2 mutant-expressing neurons. Error bars indicate S.E. C, comparison of post-stimulus endocytosis time constant with degree of AP-2 expression for each rescue construct used. After live cell imaging, cells were fixed and labeled with anti-α-adaptin antibody and quantified as in Fig. 4. The solid line indicates the time constant of cells that did not receive shRNA (control) (∼15 s), and the dotted line shows the time constant of AP-2KD without rescue (∼42 s) from (12).