ABSTRACT
Alpha subunits of heterotrimeric G proteins (Gα) are involved in a variety of cellular functions. Here we report an optogenetic strategy to spatially and temporally manipulate Gα in living cells. More specifically, we applied the blue light-induced dimerization system, known as the Magnet system, and an alternative red light-induced dimerization system consisting of Arabidopsis thaliana phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) to optically control the activation of two different classes of Gα (Gαq and Gαs). By utilizing this strategy, we demonstrate successful regulation of Ca2+ and cAMP using light in mammalian cells. The present strategy is generally applicable to different kinds of Gα and could contribute to expanding possibilities of spatiotemporal regulation of Gα in mammalian cells.
Subject(s)
GTP-Binding Protein alpha Subunits/radiation effects , Animals , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/radiation effects , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/radiation effects , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/radiation effects , COS Cells , Calcium Signaling/radiation effects , Chlorocebus aethiops , Cyclic AMP/metabolism , Dimerization , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits/metabolism , HEK293 Cells , HeLa Cells , Humans , Light , Optogenetics , Phytochrome B/genetics , Phytochrome B/metabolism , Phytochrome B/radiation effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/radiation effectsABSTRACT
PURPOSE: Uveal melanoma (UM) tumors require large doses of radiation therapy (RT) to achieve tumor ablation, which frequently results in damage to adjacent normal tissues, leading to vision-threatening complications. Approximately 50% of UM patients present with activating somatic mutations in the gene encoding for G protein αq-subunit (GNAQ), which lead to constitutive activation of downstream pathways, including protein kinase C (PKC). In this study, we investigated the impact of small-molecule PKC inhibitors bisindolylmaleimide I (BIM) and sotrastaurin (AEB071), combined with ionizing radiation (IR), on survival in melanoma cell lines. METHODS: Cellular radiosensitivity was determined by using a combination of proliferation, viability, and clonogenic assays. Cell-cycle effects were measured by flow cytometry. Transcriptomic and proteomic profiling were performed by quantitative real-time PCR, reverse-phase protein array analysis, and immunofluorescence. RESULTS: We found that the PKC inhibitors combined with IR significantly decreased the viability, proliferation, and clonogenic potential of GNAQ(mt), but not GNAQ(wt)/BRAF(mt) cells, compared with IR alone. Combined treatment increased the antiproliferative and proapoptotic effects of IR in GNAQ(mt) cells through delayed DNA-damage resolution and enhanced induction of proteins involved in cell-cycle arrest, cell-growth arrest, and apoptosis. CONCLUSIONS: Our preclinical results suggest that combined modality treatment may allow for reductions in the total RT dose and/or fraction size, which may lead to better functional organ preservation in the treatment of primary GNAQ(mt) UM. These findings suggest future clinical trials combining PKC inhibitors with RT in GNAQ(mt) UM warrant consideration.
Subject(s)
DNA, Neoplasm/genetics , GTP-Binding Protein alpha Subunits/genetics , Melanoma/enzymology , Mutation , Protein Kinase Inhibitors/pharmacology , Uveal Neoplasms/enzymology , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis/radiation effects , Cell Cycle/drug effects , Cell Cycle/radiation effects , Cell Line, Tumor , Cell Proliferation , Cell Survival , Combined Modality Therapy , Flow Cytometry , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein alpha Subunits/radiation effects , GTP-Binding Protein alpha Subunits, Gq-G11 , Humans , Melanoma/genetics , Melanoma/therapy , Radiation, Ionizing , Real-Time Polymerase Chain Reaction , Uveal Neoplasms/genetics , Uveal Neoplasms/therapyABSTRACT
Visual thresholds of mice for the detection of small, brief targets were measured with a novel behavioral methodology in the dark and in the presence of adapting lights spanning â¼8 log(10) units of intensity. To help dissect the contributions of rod and cone pathways, both wild-type mice and mice lacking rod (Gnat1(-/-)) or cone (Gnat2(cpfl3)) function were studied. Overall, the visual sensitivity of mice was found to be remarkably similar to that of the human peripheral retina. Rod absolute threshold corresponded to 12-15 isomerized pigment molecules (R*) in image fields of 800 to 3000 rods. Rod "dark light" (intrinsic retinal noise in darkness) corresponded to that estimated previously from single-cell recordings, 0.012 R* s(-1) rod(-1), indicating that spontaneous thermal isomerizations are responsible. Psychophysical rod saturation was measured for the first time in a nonhuman species and found to be very similar to that of the human rod monochromat. Cone threshold corresponded to â¼5 R* cone(-1) in an image field of 280 cones. Cone dark light was equivalent to â¼5000 R* s(-1) cone(-1), consistent with primate single-cell data but 100-fold higher than predicted by recent measurements of the rate of thermal isomerization of mouse cone opsins, indicating that nonopsin sources of noise determine cone threshold. The new, fully automated behavioral method is based on the ability of mice to learn to interrupt spontaneous wheel running on the presentation of a visual cue and provides an efficient and highly reliable means of examining visual function in naturally behaving normal and mutant mice.
