Fig. 3.

Inactivation of SOCE mediated by constitutive active STIM1 mutants during meiosis. (A) The distribution of STIM1 and Orai1 following the expression of the constitutively active STIM1 mutants, STIM1^D76A and the C terminus (CT-STIM1). Orthogonal views across the z-stack are also shown. Bars on orthogonal views indicate the planes at which the images where taken (P1, P2). (Scale bar, 2 μm.) (B) Estimates of total cell membrane GFP–Orai1 at different time points during oocyte maturation. GFP–Orai1 was estimated by quantifying membrane GFP fluorescence and multiplying it by the average cell capacitance at the equivalent time point. The data were normalized to the oocyte values (n = 4, mean ± SE). (C) SOCE levels during maturation were measured using the endogenous Ca²⁺-activated Cl⁻ current (I_Cl-T) as a reporter. I_Cl-T faithfully reports Ca²⁺ influx through SOCE channels (27, 28). (D and E) FRAP analysis of STIM1 and STIM1^D76A diffusion. (D) Kinetics of the fluorescence recovery following photobleaching in cells expressing STIM1 before and after store depletion, and STIM1^D76A in both oocytes and eggs (n = 12–26; mean ± SE). Data were fitted with a monoexponential decay function. (E) Recovery half-time (T_1/2) (n = 12–26; mean ± SE). The asterisk donates significantly different groups (P ≤ 0.00064).