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Molecular recording of calcium signals via calcium-dependent proximity labeling

Nature Chemical Biology (2024)Cite this article

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Abstract

Calcium ions serve as key intracellular signals. Local, transient increases in calcium concentrations can activate calcium sensor proteins that in turn trigger downstream effectors. In neurons, calcium transients play a central role in regulating neurotransmitter release and synaptic plasticity. However, it is challenging to capture the molecular events associated with these localized and ephemeral calcium signals. Here we present an engineered biotin ligase that generates permanent molecular traces in a calcium-dependent manner. The enzyme, calcium-dependent BioID (Cal-ID), biotinylates nearby proteins within minutes in response to elevated local calcium levels. The biotinylated proteins can be identified via mass spectrometry and visualized using microscopy. In neurons, Cal-ID labeling is triggered by neuronal activity, leading to prominent protein biotinylation that enables transcription-independent activity labeling in the brain. In summary, Cal-ID produces a biochemical record of calcium signals and neuronal activity with high spatial resolution and molecular specificity.

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Fig. 1: Cal-ID: a calcium-dependent proximal labeling enzyme.
Fig. 2: Identification and visualization of calcium signaling microdomains in HEK293T cells.
Fig. 3: Cal-ID activation is associated with neuronal activity in neurons.
Fig. 4: PMCA1/2 are associated with calcium signaling microdomains in neurons.
Fig. 5: In vivo Cal-ID biotinylation labels active neurons in the brain.
Fig. 6: In vivo Cal-ID activity labeling with D1R activation in the dorsal striatum.

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Data availability

The data generated in this study are provided in Supplementary Information. Data are also available from the corresponding author upon request. Plasmids and libraries will be available through Addgene. Raw mass spectrometry data are available via ProteomeXchange/PRIDE with identifier PXD033244. Source data are provided with this paper.

Code availability

R codes were generated for statistical analysis and visualization. All source codes will be available from the corresponding author upon request.

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Acknowledgements

We thank S. Upadhyayula, S. Eacker and members of the Ingolia lab for discussion, and M. Farhan for mouse work. Mass spectrometry was conducted at the UC Davis Proteomics Core. Confocal imaging was conducted at the CRL Molecular Imaging Center, RRID:SCR_017852, supported by Gordon and Betty Moore Foundation. We thank Baltimore, Izsvak, Lipscombe, Roux, Ting and Zhang labs for plasmids. Figures 5a and 6a, and Extended Data Fig. 9a were created with BioRender.com. This work was supported by FRAXA Fragile X Foundation Postdoctoral Fellowship (J.W.K.), National Institutes of Health NCI DP2CA195768 (N.T.I.), NINDS R21NS112842 (N.T.I.), NINDS R35NS097227 (Y.N.J), and the Weill Neurohub Next Great Ideas Program (N.T.I., H.S.B., Y.N.J.). Y.N.J. is a Howard Hughes Medical Institute Investigator. H.S.B. is a Chan Zuckerberg Biohub Investigator and a Weill Neurohub Investigator. R.G. acknowledges funding support from Scialog grant no. 28707, sponsored jointly by Research Corporation for Science Advancement and the Walder Foundation.

Author information

Author notes

  1. These authors contributed equally: Adeline J. H. Yong, Erin E. Aisenberg.

Authors and Affiliations

  1. Department of Molecular and Cell Biology at the University of California, Berkeley, Berkeley, CA, USA

    J. Wren Kim, Joseph H. Lobel, Helen S. Bateup & Nicholas T. Ingolia

  2. Department of Physiology at the University of California, San Francisco, San Francisco, CA, USA

    Adeline J. H. Yong & Yuh Nung Jan

  3. Howard Hughes Medical Institute at the University of California, San Francisco, San Francisco, CA, USA

    Adeline J. H. Yong & Yuh Nung Jan

  4. Helen Wills Neuroscience Institute at the University of California, Berkeley, Berkeley, CA, USA

    Erin E. Aisenberg & Helen S. Bateup

  5. Department of Chemistry at the University of Illinois, Chicago, Chicago, IL, USA

    Wei Wang & Ruixuan Gao

  6. Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Ted M. Dawson & Valina L. Dawson

  7. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Helen S. Bateup

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  1. J. Wren Kim
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Contributions

J.W.K. and N.T.I. conceived the study and designed the experiments. J.W.K., A.J.H.Y., E.E.A., J.H.L conducted the experiments. J.W.K. and N.T.I. analyzed proteomics data. J.W.K. performed ExM experiments with help from W.W. and R.G. Y.N.J. supervised biochemistry and microscopy experiments with neurons. H.S.B. oversaw in vivo experiments. T.M.D. and V.L.D. oversaw conceptualization of the study. J.W.K. and N.T.I. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Nicholas T. Ingolia.

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Extended data

Extended Data Fig. 1 Development and optimization of Cal-ID in HEK293T cells.

(a) A schematic diagram of the Cal-ID design used to screen BioID split sites (this early version used split BioID rather than TurboID). CaM: calmodulin, RS20: CaM-binding peptide. (b) Cal-ID activation results in HEK293T cells with 1 µM thapsigargin. Representative blot, n = 3 (independent). Biotin: 100 µM, 30 min incubation (Cal-ID), 1 hour incubation (BioID). Cal-ID constructs were transfected transiently. *: endogenous biotin carrier proteins, #: self-biotinylation of Cal-ID or BioID. Numbers: molecular weight (kDa). (c) Representative blots testing Cal-ID (T195/G196 split) with a self-inhibitory AviTag* motif in HEK293T cells with 3 µM thapsigargin and prolonged incubation. Representative blot, n = 3 (independent). Biotin: 100 µM, 1 hour incubation, transient transfection. (d) Representative blots comparing Cal-ID split site variants T195/G196 and L73/G74 for short Incubation. Both variants contain a self-inhibitory motif. Representative blot, n = 3 (independent). Transient transfection in HEK293T cells, 3 µM thapsigargin, biotin: 50 µM, 15 min incubation.

Source data

Extended Data Fig. 2 Validation of Cal-ID and quantitative mass spectrometry in HEK293T cells.

(a) Cal-ID biotinylation and immunoprecipitation of CaV1.2. of Cal-ID-PM: a 20-aa C-terminal CAAX motif from KRAS was added to Cal-ID for plasma membrane localization. Exogenous CaV1.2 was cotransfected with alpha-2/delta and beta subunits in HEK293T cells. Representative blot, n = 3 (independent). Biotin: 100 µM, KCl: 100 mM. 30 min incubation. Immunoprecipitation was performed with antibodies against HA epitope. *: endogenous biotin carrier proteins, #: self-biotinylation of Cal-ID. Numbers on the left: molecular weight (kDa), numbers at the bottom: densitometry results, * indicates control condition. (b) Representative blots showing that Ca2+ chelator BAPTA-AM and EGTA treatment does not affect cpTurboID biotinylation. Representative blot, n = 4 (independent). Transient transfection in HEK293T cells, BAPTA-AM: 40 µM, EGTA: 2 mM, biotin: 100 µM, serum: replacement with fresh culture media. Culture media was replaced, EGTA was treated, incubated for 1 minute, then BAPTA-AM was treated and incubated for 3 min, then biotin was added and labeled for 30 min. Numbers on the left: molecular weight (kDa), *: endogenous biotin carrier proteins, #: self-biotinylation of Cal-ID or BioID. (c and d) Scatterplots showing mass spectrometry reproducibility between biological replicates (c) Cal-ID, (d) cpTurboID, prepared from HEK293T stable cell lines.

Source data

Extended Data Fig. 3 Visualization of Cal-ID target proteins identified by mass spectrometry and ER-localized Cal-ID.

(a to c) Confocal microscope images visualizing biotinylated proteins and mass spectrometry-identified target proteins in Cal-ID expressing HEK293T cells. Co-localization of streptavidin signals and (a) β-tubulin found in mitotic cells, (b) SURF6 in mitotic cells, (c) FKBP7 at the ER. (b) SURF6 did not show co-localization with streptavidin signals during interphase (top) but showed co-localization during mitosis (bottom). Tet-inducible stable HEK293T cells, scale bars = 5 µm. Yellow bar indicates the reference line for the co-localization plot. Representative images, independent measurements: (a) n = 3, (b) n = 3, (c) n = 3.

Extended Data Fig. 4 ER membrane-localized Cal-ID labels calcium release from the ER.

(a) A schematic of Cal-ID-ERM, an ER outer membrane-localized Cal-ID. CYB5 ER transmembrane motif is 45 aa long. (b and c) Ca2+ chelation or IP3R/RyR inhibition blocks Cal-ID-ERM activity. Biotin: 100 µM, serum: fresh media containing 10 % FBS. EGTA: 2 mM. BAPTA-AM: 20 µM. Ryanodine: 100 µM. Xestospongin C: 3 µM. Calcium channel inhibitors or calcium chelators were treated first, incubated for 3 minutes, then biotin was added. Total 15 min incubation. HEK293T cells. (b) Numbers on the left: molecular weight (kDa). Independent measurements: n = 3. *: endogenous biotin carrier proteins, #: self-biotinylation of Cal-ID. Numbers at the bottom: densitometry results, *: control condition. (c) 20 minutes incubation with biotin. Independent measurements: n = 4. Scale bars: 50 µm. (d) Time-course Cal-ID-ERM activation and inactivation experiment with HEK293T cells. Top panel: an experimental schematic, orange bar indicates incubation time. Independent measurements: n = 3. Biotin: 100 µM, serum: fresh media containing 10 % FBS. Total 15 min incubation. *: endogenous biotin carrier proteins, #: self-biotinylation of Cal-ID. Numbers: molecular weight (kDa). EGTA: 2 mM. BAPTA-AM: 20 µM. Numbers on the left: molecular weight (kDa), numbers at the bottom: densitometry results, * indicates control condition. (e and f) Representative images from Cal-ID-ERM-labeled and expanded HEK293T cells with V5/IP3R co-staining. Independent measurements: n = 5. Biotin: 100 µM, 20 min incubation post serum replenishment. (f) is a high magnification image from the same cell of (e) (see white box) but from a different plane. Cal-ID-ERM was transiently expressed. All scale bars: 2.2 µm (10 µm). For ExM images, the scale bar sizes are provided at the pre-expansion scale (the biological scale) with the corresponding post-expansion size (the physical size) indicated in parentheses.

Source data

Extended Data Fig. 5 Cal-ID/cpTurboID biotinylation and neuronal activity manipulation.

(a) cpTurboID biotinylation levels are not associated with neuronal activity. Primary hippocampal neurons at DIV 21. Independent measurements: n = 2. Silenced: 50 µM APV (NMDAR antagonist), 10 µM NBQX (AMPAR antagonist). APV + NBQX: treated overnight. Stimulated: 8 mM CaCl2 was added along with biotin. Labeling: 100 µM biotin was added and labeled for 1 hour. P-S6 is neuronal activity marker. Scale bars: 50 µm. (b) Chemical LTP (cLTP) increases Cal-ID biotinylation. At DIV 21, mouse primary cortical neurons were transferred to aCSF (artificial cerebrospinal fluid) with 500 nM TTX, 20 µM bicuculline, and 1 µM strychnine, equilibrated for 20 minutes. For cLTP condition, Mg2+ was removed and 200 µM glycine was added. 30 minutes incubation with 100 µM biotin for labeling. Independent measurements: n = 2. Numbers on the left: molecular weight (kDa). *: endogenous biotin carrier proteins, #: self-biotinylation of Cal-ID. Numbers at the bottom: densitometry results, * indicates control condition.

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Extended Data Fig. 6 Prominent soma biotinylation by Cal-ID.

(a) Subcellular fractionation of Cal-ID-biotinylated primary cortical neurons at DIV 21. T: cpTurboID, C: Cal-ID. Independent measurements: n = 3. Numbers on the left: molecular weight in kDa. *: endogenous biotin carrier proteins, #: self-biotinylation of Cal-ID or cpTurboID. PSD-95, GluA1/2: postsynaptic markers, Synaptophysin: presynaptic marker, V5: Cal-ID/cpTurboID epitope. biotin: 100 µM, 1 hour incubation. (b) Representative images of biotinylation patterns in neurons by Cal-ID or cpTurboID. Independent measurements: n = 6. Color map: signal intensity. Mouse primary hippocampal neurons at DIV 21. Scale bars: 100 µm. (c and d), Scatterplots showing mass spectrometry reproducibility between biological replicates (c) Cal-ID, (d) cpTurboID, prepared from mouse primary cortical neurons at DIV 21.

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Extended Data Fig. 7 Proximity ligation assay from mouse primary neurons.

(a) A schematic diagram of proximity ligation assay (PLA) to visualize self-biotinylation of Cal-ID. (b) PLA was performed with V5 (Cal-ID) and biotin. Independent measurements: n = 3. Activated: 50 µM APV + 10 µM NBQX for overnight silencing, followed by inhibitor wash-off and addition of 4 mM CaCl2 along with 100 µM biotin for 1 hour. Mouse primary hippocampal neurons at DIV 21, lentiviral delivery, scale bar: 100 µm. (c) PLA experiments with PMCA1 or PMCA2 antibodies with biotin antibodies were conducted without Cal-ID expression (V5) in mouse primary hippocampal neurons at DIV 21. Independent measurements: n = 2. Scale bars: 50 µm.

Extended Data Fig. 8 Immunoblotting of biotinylated proteins from the mouse brain.

(a) In vivo biotinylation time course results. Unilateral (left), intracortical injection of cpTurboID-encoding AAV was performed at P0/1. Biotin injection was performed at 4 weeks of age. Biotin: 24 mg/kg in PBS (i.p.). 1 to 3 hours of post injection incubation time before euthanasia. N = 1 per time point, independent measurements: n = 3. *: endogenously biotinylated proteins. Arrow: cpTurboID-mediated biotinylation. (b) In vivo Cal-ID biotinylation with kainic acid-mediated activation in the cortex. Unilateral (left), intracortical injection of Cal-ID-encoding AAV was performed at P0/1. Biotin injection was performed at 4 weeks of age. Biotin: 24 mg/kg, kainic acid: 15 mg/kg. Kainic acid was injected 45 minutes after biotin injection. 3 hours of incubation post kainic acid injection. Independent measurements: n = 2. Arrow: Cal-ID-mediated biotinylation. Increased phosphorylation of CREB indicates activation of neurons. Numbers on the left: molecular weight in kDa. *: endogenous biotin carrier proteins, #: self-biotinylation of Cal-ID. Numbers at the bottom: densitometry results, *: control condition.

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Extended Data Fig. 9 Cal-ID neuronal activity labeling in the cortex.

(a) A schematic of in vivo Cal-ID activity labeling of activated cortical neurons by kainic acid-induced seizure. AAV was injected to the cortex of postnatal day 0 or 1 CD1 mouse pups. Labeling was performed at the 6 weeks of age. i.p: intraperitoneal. Biotin: 24 mg/kg, kainic acid: 15 mg/kg. IHC: immunohistochemistry. (b) Representative images from brain slices of saline or kainic acid-injected and Cal-ID-labeled mouse brain. Single measurement with 6 littermates (3 for saline, 3 for kainic acid). Coronal section with 40 µm thickness. Scale bar: 200 µm. (c and d) Quantification of (c) streptavidin (d) c-Fos mean signal intensity from Cal-ID expressing (V5 positive) neurons. Statistical test: two-way ANOVA, [saline vs kainic acid] ((c) F = 66.25, p = 1.9e-15, (d) F = 861.96, p < 2.2e-16), error bar: mean ± SD. Boxplot visualizes the median, the first and the third quartile (whiskers: 1.5 × IQR, error bar: mean ± SD). (e) Correlation between c-Fos and Streptavidin signals. Spearman’s rank correlation test (ρ = 0.3297383). (Data points: 442 neurons from 3 mice (saline), 241 neurons from 3 mice (kainic acid). AU: arbitrary unit. KA: kainic acid. Raw intensity values were obtained using the identical acquisition conditions. ***p < 0.001.

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Extended Data Table. 10 In vivo Cal-ID neuronal activity labeling.

(a) A representative image from the mouse cortex without AAV injection; this image is from the right hemisphere (uninjected side) from a mouse received Cal-ID AAV injection to the left hemisphere and was treated with biotin and kainic acid i.p. injections. Scale bar: 500 µm. Biotin: 24 mg/kg, kainic acid: 15 mg/kg. 40 µm thickness sagittal section. Single measurement, 2 mice. (b) Low magnification representative images of the mouse dorsal striatum from Cal-ID injected and labeled mice with saline or SKF-81297 injection, D1/D5R agonist. AAV was stereotactically injected to the striatum at 6 weeks of age. i.p: intraperitoneal. Biotin: 24 mg/kg, SKF-81297: 10 mg/kg. IHC: immunohistochemistry. SKF-81297 was injected 45 minutes after biotin injection and incubated with 3 hours before euthanasia. Single measurement with littermates: 3 mice for saline, 5 mice for SKF-81297. Coronal section with 40 µm thickness. Scale bar: 200 µm.

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Tables 1–6.

Supplementary Video 1

Three-dimensional projection of Cal-ID-ERM/ExM, HEK293T cell. A three-dimensional projection movie from a Cal-ID-ERM-labeled and expanded HEK293T cell (biotin 100 µM, 20 min incubation post serum replenishment; streptavidin (green) and V5 (Cal-ID-ERM) (red); 17 z-stack images, 3 µm interval).

Supplementary Video 2

Three-dimensional projection of Cal-ID-ERM/ExM, HEK293T cell. A three-dimensional projection movie from a Cal-ID-ERM-labeled and expanded HEK293T cell (biotin 100 µM, 20 min incubation post serum replenishment; streptavidin (green), V5 (Cal-ID-ERM, red) and IP3R (magenta); nine z-stack images, 3 µm interval).

Source data

Source Data Fig. 1

Unprocessed western blots and western blot quantification.

Source Data Fig. 2

Unprocessed western blots and mass spectrometry top hits (HEK293T cells).

Source Data Fig. 3

Unprocessed western blots and western blot quantification.

Source Data Fig. 4

Immunofluorescence quantification and mass spectrometry top hits (mouse cortical neurons).

Source Data Fig. 5

Immunohistochemistry quantification.

Source Data Fig. 6

Immunohistochemistry quantification.

Source Data Extended Data Fig. 1

Unprocessed western blots and western blot quantification.

Source Data Extended Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 4

Unprocessed western blots and western blot quantification.

Source Data Extended Data Fig. 5

Unprocessed western blots and western blot quantification.

Source Data Extended Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 8

Unprocessed western blots and western blot quantification.

Source Data Extended Data Fig. 9

Immunohistochemistry quantification.

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Kim, J.W., Yong, A.J.H., Aisenberg, E.E. et al. Molecular recording of calcium signals via calcium-dependent proximity labeling. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01603-7

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