Figure 8.

ATL is required for NE fusion. (A) Interphase cytosol and light membranes were mixed with buffer, 2 µM of the cytoplasmic fragment of X. laevis ATL2 (cytATL), or 2 µM of mutant fragment (cytATL(R232Q)). Demembranated sperm and an energy regenerating system were added. The samples were incubated at room temperature for 1.5 h and mixed with fluorescently labeled 70-kD dextran for 5 min on ice. After fixation with a solution containing Hoechst to stain chromatin and DHCC to stain membranes, the samples were analyzed by confocal microscopy. Bar, 20 µm. The number of closed nuclei (CNE) was counted based on membrane continuity and exclusion of dextran. Fusion is expressed as the percentage of the control. At least 100 nuclei were counted for each sample. The data plotted are the mean ± SD of three independent experiments. ***, P < 0.001; NS (not significant), P > 0.05; Student’s t test. (B) Interphase cytosol, light membranes, sperm, and an energy regenerating system were mixed. After 10, 30, or 50 min of incubation at room temperature, buffer or 2 µM cytATL was added. Samples were fixed after 1.5 h and analyzed by confocal microscopy. Bar, 20 µm. NS, not significant (P > 0.05, Student’s t test).