(A–D) Temporal pattern of the asymmetric index (A), fluorescence intensity of Par3-mKate2 (B), its rate of change (B) and the ratio of Par6-GFP/Par3-mKate2 (D) of S2 cells that were transfected with pMT-Par3-mKate2 and pAct-Par6-GFP, followed by induction by CuSO4 addition 2 days after plasmid transfection. Time 0 is the timing of CuSO4 addition (2 days following plasmid transfection). Measurements were taken every 30 min. The blue and red line respectively indicates the averaged values of 10 cells showing a non-polarized Par6-GFP distribution (ASI around 0.2), and that of 13 cells with a polarized Par6-GFP distribution at 8 hr after induction (ASI around 0.4). Fluorescence intensity reached the steady level around 8 hr after induction (B, D). Bars indicate s.d. The temporal pattern of the fluorescence intensity/cell of Par3-mKate2 and Par6-GFP/Par3-mKate2 ratio are not significantly different between the polarized cell group (red line) and non-polarized cell group (blue line). The ASI value began to increase immediately after the rise of Par3-mKate2 levels (approximately 2 hr after induction) in the polarized cells (blue line) in (A), and maintained a high level afterwards, while the non-polarized cells (red line) initially showed a slight increase in the ASI value and subsequently a decrease from 5 hr after induction onwards. The timing of the increase in ASI roughly corresponded to that of Par-dot emergence (2–4 hr after induction; see Figure 4A–C), and the timing of a decrease in ASI value in non-polarized cells roughly corresponds to the late period of Par-island formation (4–6 hr after induction), although there are cell-to-cell variations in these timings. in (A), t test, p=5.6×10−5, 0.04, 5.5 × 10−4, 1.1 × 10−6, 5.5 × 10−4, and 4.3 × 10−7 for every 30 min time point from 6 hr after induction. (E) Comparison of Par3-mKate2 expression level induced by the Metallothionein promoter, pMT-Par3-mKate2, and that promoted by the pAct-Gal4xUAS system. The mKate2 fluorescence intensity of the individual S2 cells was measured 2 days after transfection of pAct-Gal4 and UAS-Par3-mKate2, or at 8 hr post-CuSO4 induction of pMT-Par3-mKate2, 2 days after transfection of the plasmid. The expression level of Par3-mKate2 per cell was approximately 16-fold higher when it was driven by the UAS-Gal4 system (Mean 5.5 × 104 ± 7.1 × 104) than that of the steady state level induced by the Metallothionein promoter (3.4 × 103 ± 9.8 × 102). We also estimated the ratio of overexpressed Par3 protein level to endogenous Par3 in S2 cells to be approximately 300-fold and 20-fold for the Gal4-UAS system and Metallothionein promoter, respectively (Figure 6—figure supplement 1). (F) Schematic presentation of S2 cell polarization process from Par-dot formation to clustering of Par-islands.