SUMMARYThe transcriptional repressor Capicua (Cic) controls multiple aspects of Drosophila embryogenesis and has been implicated in vertebrate development and human diseases. Receptor tyrosine kinases (RTKs) can antagonize Cic-dependent gene repression, but the mechanisms responsible for this effect are not fully understood. Based on genetic and imaging studies in the early Drosophila embryo, we found that Torso RTK signaling can increase the rate of Cic degradation by changing its subcellular localization. We propose that Cic is degraded predominantly in the cytoplasm and show that Torso reduces the stability of Cic by controlling the rates of its nucleocytoplasmic transport. This model accounts for the experimentally observed spatiotemporal dynamics of Cic in the early embryo and might explain RTK-dependent control of Cic in other developmental contexts. DEVELOPMENTprotein Dronpa (Ando et al., 2004) or the fluorescent protein Venus (Nagai et al., 2002) and controlled by the regulatory sequences of the cic gene (Fig. 1A). The spatial distributions of Cic resulting from either of these constructs mimic the distribution of endogenous Cic (supplementary material Fig. S1).Cic is first detected after the 9th nuclear division . At this stage, Cic predominantly localizes to nuclei and levels in the middle region of the embryo are already significantly higher than those at the poles. Levels of Cic in the central region drop during mitoses, reflecting protein dispersal into the cytoplasm. During the subsequent interphase, nuclear levels recover and eventually exceed those observed in the preceding mitosis. The levels within individual nuclei increase ~2.5-fold between cycles 10 and the beginning of cycle 14. By contrast, levels at the poles remain low from the first time Cic is detected and until the embryo reaches the cellularization stage. Torso increases the rate of Cic degradationStudies in mice suggest that Cic-target gene derepression depends on RTK-dependent reduction of Cic levels (Fryer et al., 2011). To explore this mechanism in the embryo, we examined the degradation rates of Cic at different levels of RTK signaling. Based on the pattern of double-phosphorylated ERK (dpERK), which serves as a reporter of Torso signaling, the central region and poles of the wild-type embryo correspond to zero and maximal levels of Torso activation, respectively. To generate an intermediate level of Torso activation, we used a gain-of-function allele of torso (Szabad et al., 1989) (Fig. 2A,B).If Torso reduces the stability of Cic, its lifetime should be inversely correlated with the level of ERK activation. To test this prediction, we measured the lifetime of Cic using an optical pulselabeling method, which relies on the photo-switchable fluorescent protein Dronpa. Dronpa is synthesized in a bright state, which can be reversibly converted to the dark state by brief illumination at 3963 RESEARCH REPORT RTK-dependent control of Capicua 496 nm. The dark state is optically stable, but it can be converted back to the bright state ...

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