Fig. 6.

Addition of ATP to semi-intact cells prevents Sec body formation. (A) Visualization of S2 cells permeabilization using the non-membrane permeant TO-PRO-3 in intact cells in Schneider's medium (Sch) and in semi-intact cells (SICs;+10 µg/ml digitonin,+1% dextran) incubated in Sch and KRB for 2 h at 26°C. Note that TO-PRO-3 stains the nucleus only in the SIC system. (B) Effect of buffer composition in the formation of Sec bodies in SICs for 2 h at 26°C. Decreasing the pH of KRB from 7.4 to 6 decreases the efficiency of Sec body formation. Replacing Cl⁻ by acetate in the KRB and replacing KRB by the import buffer (20 mM HEPES, 110 mM KAc, 2 mM MgAc and 0.5 mM EGTA) abolishes Sec body formation. (C,C′) Immunofluorescence visualization of Sec16 (red, C) and quantification of Sec body formation (C′) (marked by Sec16) in the SIC system for cells incubated in Sch, in KRB and in KRB supplemented with 0.5 mM ATP, 0.5 mM AMP and 0.5 mM Adenosine. Cells in the white box are magnified 2.5 times. Scale bars: 10 µm. Errors bars: s.d. ns, not significant; *P<0.05, ***P<0.001.