Rationally designed fluorogenic protease reporter visualizes spatiotemporal dynamics of apoptosis in vivo

Abstract

Fluorescence resonance energy transfer-based reporters have been widely used in imaging cell signaling; however, their in vivo application has been handicapped because of poor signal. Although fluorogenic reporters overcome this problem, no such reporter of proteases has been demonstrated for in vivo imaging. Now we have redesigned an infrared fluorescent protein so that its chromophore incorporation is regulated by protease activity. Upon protease activation, the infrared fluorogenic protease reporter becomes fluorescent with no requirement of exogenous cofactor. To demonstrate biological applications, we have designed an infrared fluorogenic executioner-caspase reporter, which reveals spatiotemporal coordination between cell apoptosis and embryonic morphogenesis, as well as dynamics of apoptosis during tumorigenesis in Drosophila. The designed scaffold may be used to engineer reporters of other proteases with specific cleavage sequence.

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

“…By fusing one fragment on a target protein and detecting its association with the other fragment, these constructs have demonstrated powerful applications in the visualization of subcellular protein localization [1][2][3] , quantification of protein aggregation 4 , detection of cytosolic peptide delivery 5,6 , identification of cell contacts and synapses 7,8 , as well as scaffolding protein assembly 3,9,10 . Recently, they have also enabled the generation of large-scale human cell line libraries with fluorescently tagged endogenous proteins through CRISPR/Cas9-based gene editing 11 .…”

“…With the splitting point between the tenth and eleventh β-strands, the resulting GFP 11 fragment is a 16-amino acid (a.a.) short peptide. The corresponding GFP 1-10 fragment remains almost non-fluorescent until complementation, making GFP 1-10/11 well suited for protein labeling by fusing GFP 11 to the target protein and over-expressing GFP [1][2][3][4][5][6][7][8][9][10] in the corresponding subcellular compartments. However, there lacks a second, orthogonal split FP system with comparable complementation performance for multicolor imaging and multiplexed scaffolding of protein assembly.…”

“…2 Protein labeling through transient expression or endogenous knock-in using mNG2 11 . a Fluorescence images of HeLa cells co-expressing either mNG2 11 or GFP 11 labeled H2B or CLTA with the corresponding FP [1][2][3][4][5][6][7][8][9][10] . Scale bars are 10 μm.…”

“…b FACS backgroud from wild-type HEK 293T cells, or HEK 293T cells stably expressing GFP [1][2][3][4][5][6][7][8][9][10] (using the stong SFFV promoter or the weak PGK promoter) or mNG2 [1][2][3][4][5][6][7][8][9][10] (using SFFV). c Comparison of tagging endogenous LMNA, RAB11A, and CLTA using mNG2 11 or GFP 11 knock-in in SFFV-mNG2 [1][2][3][4][5][6][7][8][9][10] and PGK-GFP 1-10 cells, respectively, (flow cytometry histograms) and the corresponding confocal images of mNG2 11 knock-in cells. Scale bars are 10 μm.…”

“…d Comparison of tagging a low-abundance endogenous protein SPTLC1 through mNG2 11 or GFP 11 knock-in (flow cytometry histograms). e Whole cell fluorescence intensity of full length mNG2 and mNG2 11 -CLTA/mNG2 [1][2][3][4][5][6][7][8][9][10] ; histone H2B c, nuclear); zyxin d, focal adhesion); TOMM20 e, mitochondria outer membrane); β-actin f, actin cytoskeleton); heterochromatin protein 1 g, nuclear); clathrin light chain A h, clathrin-coated pits); vimentin i, intermediate filament). j Confocal image of endogenous Sec61B (ER) labeled by sfCherry2 11 in HEK 293T cells stably expressing sfCherry2 [1][2][3][4][5][6][7][8][9][10] .…”