Fig. 4. NE formation is SNARE-dependent. (A) Nuclear formation reactions were performed in egg extracts. Reactions contained either buffer or 37.5 µM or 100 µM exogenous, bacterially expressed -SNAP_wt, -SNAP_L294A or the -SNAP_N80 variant that lacks the SNARE-binding site. Samples were processed and imaged as in Fig. 1A. Note that only the full-length variants with SNARE-binding activity blocked NE formation. Bar, 5 µm. (B) Quantification of three experiments performed as in Fig. 1B. Results are presented as percentage of control. (C) Maximum intensity projections of confocal sections taken as in Fig. 1A. Note that excess -SNAP_L294A efficiently blocked the formation of tubules or cisternae on the chromatin surface. (D) Nuclear formation reactions were performed in the presence of 50 µM wild-type or L294A -SNAP for 45 minutes. Samples were taken and fixed (0 minutes). The remaining reaction was split and either diluted 1:2 in fresh cytosol (+ dilution) or not (– dilution) and incubated for a further 120 minutes, followed by fixation. NE membranes were imaged as before. Note that the effect of both -SNAP variants was equally well restored with fresh cytosol, which was confirmed by quantification (data not shown). (E,F) ER formation was performed for 60 minutes in the presence of 70 µM -SNAP_L294A or buffer and quantified as in Fig. 1E. Bar, 10 µm. All results are presented as means (n=3, error bars indicate ± s.e.m.).