RESUMEN
A grand challenge in biosensor design is to develop a single-molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Here, we created a family of adaptable, turn-on maturation (ATOM) biosensors consisting of a monobody (circularly permuted at one of two positions) or a nanobody (circularly permuted at one of three positions) inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells coexpressing cyan, yellow and red ATOM sensors detected biosensor targets that were specifically localized to various subcellular compartments. Fluorescence activation involved ligand-dependent chromophore maturation with turn-on ratios of up to 62-fold in cells and 100-fold in vitro. Endoplasmic reticulum- and mitochondria-localized ATOM sensors detected ligands that were targeted to those organelles. The ATOM design was validated with three monobodies and one nanobody inserted into distinct fluorescent proteins, suggesting that customized ATOM sensors can be generated quickly.
Asunto(s)
Técnicas Biosensibles , Proteínas , Humanos , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/metabolismo , Técnicas Biosensibles/métodosRESUMEN
It has been well established that cardiovascular diseases exhibit significant differences between sexes in both preclinical models and humans. In addition, there is growing recognition that disrupted circadian rhythms can contribute to the onset and progression of cardiovascular diseases. However little is known about sex differences between the cardiac circadian clock and circadian transcriptomes in mice. Here, we show that the the core clock genes are expressed in common in both sexes but the circadian transcriptome of the mouse heart is very sex-specific. Hearts from female mice expressed significantly more rhythmically expressed genes (REGs) than male hearts and the temporal pattern of REGs was distinctly different between sexes. We next used a cardiomyocyte-specific knock out of the core clock gene, Bmal1, to investigate its role in sex-specific gene expression in the heart. All sex differences in the circadian transcriptomes were significantly diminished with cardiomyocyte-specific loss of Bmal1. Surprisingly, loss of cardiomyocyte Bmal1 also resulted in a roughly 8-fold reduction in the number of all the differentially expressed genes between male and female hearts. We conclude that cardiomyocyte-specific Bmal1, and potentially the core clock mechanism, is vital in conferring sex-specific gene expression in the adult mouse heart.
RESUMEN
A grand challenge in biosensor design is to develop a single molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Conceptually, this can be achieved by fusing a small, antibody-like binding domain to a fluorescent protein in such a way that target binding activates fluorescence. Although this design is simple to envision, its execution is not obvious. Here, we created a family of adaptable, turn-on monobody (ATOM) biosensors consisting of a monobody, circularly permuted at one of two positions, inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells co-expressing cyan, yellow, and red ATOM sensors detected the biosensor targets (WDR5, SH2, and hRAS proteins) that were localized to the nucleus, cytoplasm, and plasma membrane, respectively, with high specificity. ER- and mitochondria-localized ATOM sensors also detected ligands that were targeted to those organelles. Fluorescence activation involved ligand-dependent chromophore maturation with fluorescence turn-on ratios of >20-fold in cells and up to 100-fold in vitro . The sensing mechanism was validated with three arbitrarily chosen monobodies inserted into jellyfish as well as anemone lineages of fluorescent proteins, suggesting that ATOM sensors with different binding specificities and additional colors can be generated relatively quickly.