Figure 4.

TIM mutants affect nuclear expression of and feedback repression by PER. A, Mutant TIM proteins anchor PER in the cytoplasm. S2 cells transfected with hs-YFP tagged wild-type tim (wt) or mutant tim (PL) and hs-CFP tagged per were fixed 8 h after heat shock induction of TIM and PER. Scale bar, 10 μm. Quantification shows PER localization in TIM-expressing cells, scored as mostly nuclear (green), mostly cytoplasmic (blue), or both nuclear and cytoplasmic (red). The top and bottom panels of the hs-tim(WT) images show nuclear PER and PER in both the nucleus and cytoplasm, respectively. At least 100 cells were counted for each condition in three independent experiments. B, TIM mutants inhibit feedback repression by PER on CLK-CYC-mediated transcription. Drosophila S2 cells were transiently transfected with two reporter genes (E-box-luc and actin-Rluc), and various combinations of pAc-dClk, wt, or mutant (TA or PL) pIZ-s-tim, and pAc-per, as indicated. After 48 h, cells were harvested and assayed for luciferase. Data were normalized to Rluc expression to control for transfection efficiency. Results are presented as the percentage of full activation seen in the dClk alone condition and expressed as averages of three independent experiments. Error bars indicate SEM. *p < 0.05 by t test.