Figure 6. β- or γ-actin knockout inhibits endocytosis at hippocampal synapses.

(A) Western blot of β-actin, AP-2, Clathrin heavy chain (CLT), dynamin (DMN) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase, loading control) from Actb^LoxP/LoxP hippocampal cultures transfected with nothing (Ctrl) or with lentivirus containing a Cre enzyme (Actb^−/−).

(B) Similar to A, except from Actg1^LoxP/LoxP cultures.

(C) Upper: western blot of β-actin, γ-actin and GAPDH from Cre-ER^TM;Actb^LoxP/LoxP culture at 0, 2 and 4 days (d) after addition of 4-OH-tamoxifen (1 μM).

Lower: β- (left) and γ-actin (right) western blot intensity from Cre-ER^TM;Actb^LoxP/LoxP culture at 0, 2 and 4 days after addition of 4-OH-tamoxifen (mean + s.e.m., normalized to day 0, n = 4). ***, p < 0.001 (ANOVA).

(D) SypH and mCherry images of a neuron transfected with SypH and a plasmid containing Cre-mCherry. mCherry is localized to nucleus (superimposed image), due to a nuclear localization sequence tagged at the Cre N-terminal. The box region is enlarged (right) to indicate a place for SypH imaging.

(E) F_SypH (mean + s.e.m.) induced by Train_10s (left, n = 8 experiments) or Train_2s (n = 5) in control boutons, and F_SypH induced by Train_10s in Actb^−/− boutons (Actb^LoxP/LoxP boutons transfected with SypH and a Cre plasmid, n = 11) or in Actg1^−/− boutons (Actg1^LoxP/LoxP boutons transfected with SypH and a Cre plasmid, n = 10). F_SypH is normalized to baseline, s.e.m. is plotted every 10 s, temperature was 22–24°C (applies to Figs. 6–8 if not mentioned otherwise).

(F) Traces in E (same color coding) scaled to the same amplitude and superimposed. Train_2s was aligned to the end of Train_10s.

(G) Rate_decay and ΔF (mean + s.e.m.) induced by Train_10s and Train_2s in control boutons (Train_10s, n = 8; Train_2s, n = 5) and by Train_10s in Actb^−/− (n = 11) and Actg1^−/− boutons (n = 10 experiments). ΔF was normalized to baseline F (ΔF/F, applies to all panels in Figs. 6–7). **: p < 0.01, t test (compared to Train_10s data in Ctrl).

(H) Applying MES solution (pH:5.5, bars) quenched F_SypH (mean + s.e.m.) to a similar level (dotted line) before and after Train_10s in Actb^−/− boutons (n = 6 experiments). ΔS represents pre-existing SypH molecules at the plasma membrane that can be quenched.

(I) F_SypH traces, Rate_decay and ΔF (mean + s.e.m.) induced by Train_10s in Ctrl (n = 4, black) or Actb^−/− boutons (n = 5 experiments, red) at 34–37°C. Mean F_SypH traces were also scaled and superimposed (right). *: p < 0.05; **: p < 0.01, t test.

(J) F_SypH traces, Rate_decay and ΔF (mean + s.e.m.) induced by a 10 s train at 80 Hz at 22–24°C in Ctrl (n = 4, black) or Actb^−/− boutons (n = 5 experiments, red). Mean F_SypH traces were also scaled and superimposed (right). **: p < 0.01, t test.