Now Reading: Far-red chemigenetic kinase biosensors enable multiplexed and super-resolved imaging of signaling networks
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Far-red chemigenetic kinase biosensors enable multiplexed and super-resolved imaging of signaling networks
Far-red chemigenetic kinase biosensors enable multiplexed and super-resolved imaging of signaling networks
Data availability
Plasmids encoding pcDNA3.1-HaloAKAR2.0-T2A-EGFP, pcDNA3.1-HaloAKAR2.1-T2A-EGFP, pcDNA3.1-HaloAKAR2.2-T2A-EGFP, pcDNA3.1-HaloAKAR2.1-NES-T2A-EGFP-CAAX, pcDNA3.1-HaloAKAR2.1-CLC, pcDNA3.1-HaloCKAR2.2-T2A-EGFP, pcDNA3.1-HaloAktKAR2.2-T2A-EGFP and pcDNA3.1-HaloEKAR2.2-T2A-EGFP are deposited on Addgene. The stable cell line generated is available upon request. Source data are provided with this paper. Further data supporting the findings of this study are available upon request.
Code availability
Custom MATLAB scripts (R2023a 9.14.0.2254940) for clathrin-coated pit analysis, custom ImageJ macros and R code used to analyze imaging data, as well as bash and R scripts used to analyze sequencing data, are available via GitHub at https://github.com/jinzhanglab-ucsd (ref. 30). Python code (Jupyter Notebook 6.5.4 with python 3.11.5) used to analyze lattice light-sheet data is available via GitHub at https://github.com/pylattice/livelattice (ref. 73) and https://github.com/schoeneberglab/Segmentation/blob/main/3D_spheroid_cell_segmentation.ipynb (ref. 74).
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Acknowledgements
We thank L. Lavis and J. Grimm (Janelia Research Campus) for providing JF fluorophores. We thank Irving Garcia (USC) for assistance with cell culture. We thank C. Alvarez (UCSD) for assistance with cloning. We thank J. Bogomolovas and J. Chen (UCSD) for access and assistance to their fluorescence polarization plate reader. We thank J. Chung and J. Yang (UCSD) for access and assistance to their fluorescence and absorbance spectrometer. We thank Z. Liang (UCSD) for help with processing of sequencing results. We thank H. Farrants (Janelia Research Campus) for discussions on chemigenetic biosensors. We thank A. Linnemann (Indiana University) for providing the pAAV-Ins-GCaMP plasmid map. We thank Q. Su (UCSD) for providing the ExRai-CKAR2 plasmid. We thank Y. Kwon (UCSD) for cloning β1AR. We thank N. Bergkamp (UCSD) for critical reading of the manuscript. We thank Q. Ni (UCSD) for support in material acquisition and tissue culture. We thank P. Guo from the UC San Diego Nikon Imaging Center, as well as J. Santini and M. Erb from the UC San Diego School of Medicine Microscopy Core, for assistance with microscopy. We thank the Translational Imaging Center, A. Shwartz and J. Junge, at USC for their help with STED microscopy. This publication includes data generated at the UC San Diego IGM Genomics Center utilizing an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (no. S10 OD026929). The Leica SP8 in the UC San Diego School of Medicine Microscopy Core was funded by NINDS P30NS04710. This research was further supported by the Swiss National Science Foundation (SNSF) grants P2ELP3_199834 and P500PN_214234 to M.S.F.; the Charles Lee Powell Foundation Powell Fellowship at the University of California, San Diego (UCSD) and the UCSD Interfaces Graduate Training Program National Institutes of Health (NIH)/National Institute of Biomedical Imaging and Bioengineering (NIBIB) T32 Training in Multi-scale Analysis of Biological Structures and Function Training Grant 5T32EB009380-15 to S.A.S.; an NIH-K01EB035649 grant to L.L.; an EMBO (ALTF 849-2020) and HFSP (LT000404/2021-L) fellowship to F.S.; an American Heart Association Predoctoral Fellowship (24PRE1196243) to Z.W.; the National Science Foundation (NSF) Graduate Research Fellowship (DGE-2038238) and American Heart Association Predoctoral Fellowship (24PRE1186687) to A.C.L. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF; the Swiss National Science Foundation (P2BSP3_200177) and the Larry L. Hillblom Foundation (2023-D-012-FEL) to T.V.R.; the US National Institute of Diabetes and Digestive and Kidney Diseases (P30DK063491 and R01DK101395) and a grant from Janssen Pharmaceuticals to J.M.O.; NIH R01GM149976, NIH U01AI167892, NIH 5R01NS111039, NIH R21NS125395, NIHU54DK134301, NIHU54 HL165443 and UCSD Startup funds to L.S.; NIH Grants DP2 GM150022 and R01 GM148765 to J.S.; NIH grant R01 CA262815 to J.Z. and Y.W.; NIH grants EB029122, R35 GM140929, R01 HL121365 and HD107206 to Y.W.; and NIH grants R35 CA197622, R01 DK073368, R01 DE030497, R01 HL162302 and RF1 MH126707 to J.Z.
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Extended data
Extended Data Fig. 1 Brightness measurements.
a,b, Basal (a) and activated brightness (b) of HaloAKAR-JF635 biosensors. In b, the brightness of HaloTag7-JF635 is given for comparison. Individual data points are shown from three independent repeats along with mean and 95% confidence intervals. Numbers of cells (n) can be found in Supplementary Table 6. Note that experiment a and b were measured using different microscopy settings given in the methods section.
Extended Data Fig. 2 Subcellularly targeted HaloAKAR biosensors.
a,b, Plasma membrane targeting via fusion to the CAAX sequence of KRAS (a) or Lyn11 (b). c, Outer mitochondrial membrane targeting by fusing to Tomm20. d, Actin targeting via fusion with β-actin. e, Microtubule targeting via microtubule binding protein CEP41. f, Clathrin targeting via fusion with clathrin light chain. Average time courses of HeLa cells expressing targeted HaloAKAR2.0, HaloAKAR2.1, or HaloAKAR2.2 labeled with JF635-CA and stimulated with 50 μM Fsk and 100 μM IBMX (Fsk/IBMX) are shown along with representative images after stimulation. Time courses and images are representative of three replicates. Thick and thin solid lines indicate mean and single-cell responses, respectively; shaded areas correspond to standard deviation (a: n = 8, 8, 7 cells; b: n = 7, 6, 4 cells; c: n = 7, 7, 5 cells; d: n = 5, 7, 4 cells; e: n = 6, 4, 5 cells; f: n = 5, 8, 6 cells). Scale bars, 20 µm.
Extended Data Fig. 3 Characterization of the HaloAKAR series in HEK293T cells in 2D and 3D culture.
a, Characterization of HEK293T cells stably expressing cytosolic HaloAKAR2.1 (HaloAKAR2.1-NES) and plasma membrane EGFP (EGFP-CAAX) labeled with JF635-CA in 2D culture. A representative image overlay (EGFP: green, HaloAKAR: magenta) after Fsk/IBMX stimulation is given. b,c, HEK293T cells expressing cytosolic HaloAKAR2.0-NES, HaloAKAR2.1-NES, or HaloAKAR2.2-NES and plasma membrane EGFP (EGFP-CAAX) labeled with JF635-CA in 2D culture. The dynamic range (ΔF/F0) after Fsk/IBMX stimulation (b, n = 70, 75, 88 cells from three replicates) and time courses after stimulation with Fsk/IBMX (c, n = 29, 25, 29 cells). The dynamic range in HEK293T cells are ΔF/F0 = 0.85 ± 0.04, 6.46 ± 0.24, and 11.21 ± 0.92. d, Time course-measurements of HEK293T cells stably expressing HaloAKAR2.1-NES and EGFP-CAAX labeled with JF635-CA in 2D culture stimulated with 1 µM isoproterenol (iso) followed by Fsk/IBMX (n = 28 cells). e-h, Characterization of the same cell line in 3D spheroid culture. 4-d-old spheroids were labeled with JF635-CA and imaged by spinning-disc confocal microscopy, acquiring images of the entire spheroid (e and f) or a single plane (g and h). Representative overlay images of a single plane (e and g) and the corresponding time course quantification (f: n = 27 cells, h: n = 20 cells) is given. Measuring z-stacks over the entire spheroid height led to some bleaching, in contrast to the single-plane measurements (g and h). Time courses and images are representative of three replicates. Thick solid lines indicate mean response; shaded areas and error bars correspond to 95% confidence interval; single-cell traces are shown if n < 10 (c). Scale bars, 20 μm.
Supplementary information
Supplementary Information
Supplementary Figs. 1–23, Tables 1–9, Video 1, methods and references.
Reporting Summary
Supplementary Video 1
PKA activity in isolated mouse islets upon acute glucose stimulation. Video of isolated mouse islets expressing HaloAKAR2.2-T2A-EGFP labeled with JF635-CA responding to actuate stimulation with glucose (25 mM) at 2 min. Intensity increases corresponding to increased PKA activity can be seen at 5 min (3 min after addition). Scale bar, 50 μm.
Supplementary Data 1
Statistical source data.
Source data
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Frei, M.S., Sanchez, S.A., He, X. et al. Far-red chemigenetic kinase biosensors enable multiplexed and super-resolved imaging of signaling networks.
Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02642-8
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DOI: https://doi.org/10.1038/s41587-025-02642-8
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