Figure 6.

PI(4,5)P₂ induced actin polymerization is reduced with amph (−/−) testis cytosol. (A) Actin polymerization was monitored by a visual assay in which 1 μM rhodamine-actin was used to follow actin polymerization by fluorescent confocal microscopy. The cytosol of amph 1 (+/+) and amph 1 (−/−) testis at a concentration of 20 mg/ml was prepared. Control cytosol (top) or cytosol pretreated with 10 μM (Cyt D) for 5 min (bottom) were mixed with liposomes composed of 30% PS, 46% PC, 20% cholesterol, and 4% PI(4,5)P₂. To determine the recovery of activity, 2 μM recombinant amphiphysin 1 was added to the cytosol before mixing. After 15 min of incubation at room temperature, the mixtures were analyzed by fluorescent confocal microscopy. Bar, 10 μm. (B) The liposomes-induced actin polymerization was measured using pyrene fluorescence. Cytosol from amph 1 (+/+) testis (red line) or amph 1 (−/−) testis (black line) were treated with 50 μM PS/PC/Chol liposomes containing 4% of PI(4,5)P₂. As controls, the incubation was carried out in presence of 10 μM Cyt D (green line), or in absence of liposomes (blue line). The arrow indicates the time when the liposomes and cytosol were added. (C) Comparison of the actin polymerization activity between amph 1 (+/+) and amph 1 (−/−) cytosol is shown. To avoid effect of the initial peak, activity of F-actin formation at 100 s was measured by pyrene-actin fluorescent intensity. All activities are normalized to that of the amph 1 (+/+) testis cytosol. The mean ± SEM of three independent experiments is plotted. Note the reduced actin polymerization in amph 1 (−/−) cytosol compared with the polymerization in the amph 1 (+/+) cytosol. Statistically significant differences from the value of Amph (+/+) cytosol are indicated by (**p < 0.01) (Student's t test). (D) The liposomes-induced actin polymerization measured by pyrene fluorescence demonstrated the recovery of F-actin formation by recombinant amphiphysin. Amph 1 (−/−) cytosol (black line) was supplemented with 2 μM recombinant full-length amphiphysin 1 (red line), with ΔSH3 mutant (blue line) or with ΔBAR mutant (green line). The incubation and the measurement of activity were carried out as described in B. The arrow indicates the time when the lipids and the cytosol were added. (E) F-actin formation after 100 s induction in amph 1 (−/−) cytosol treated with liposomes containing PI(4,5)P₂ alone, with recombinant full-length amphiphysin 1, with ΔSH3 mutant or with ΔBAR mutant was measured by pyrene-actin fluorescence. The recombinant mutant proteins in the mixture were at a concentration of 2 μM. The incubation with 2 μM recombinant full-length amphiphysin 1 was carried out in presence of 10 μM Cyt D as a control. All activities are normalized to that in amph (+/+) cytosol. The mean ± SEM of three independent experiments is plotted. Statistically significant differences from the value of amph (+/+) cytosol are indicated by (**p < 0.01) (Student's t test).