A chemogenetic approach for dopamine imaging with tunable sensitivity.

Dopamine / metabolism Animals Humans Optogenetics / methods Mice Male Corpus Striatum / metabolism Receptors, Dopamine / metabolism Mice, Inbred C57BL Allosteric Regulation Photometry / methods HEK293 Cells

Journal

Nature communications

ISSN: 2041-1723

Titre abrégé: Nat Commun

Pays: England

ID NLM: 101528555

Informations de publication

Date de publication:
02 Jul 2024

Historique:

received: 07 11 2023

accepted: 05 06 2024

medline: 3 7 2024

pubmed: 3 7 2024

entrez: 2 7 2024

Statut: epublish

Résumé

Genetically-encoded dopamine (DA) sensors enable high-resolution imaging of DA release, but their ability to detect a wide range of extracellular DA levels, especially tonic versus phasic DA release, is limited by their intrinsic affinity. Here we show that a human-selective dopamine receptor positive allosteric modulator (PAM) can be used to boost sensor affinity on-demand. The PAM enhances DA detection sensitivity across experimental preparations (in vitro, ex vivo and in vivo) via one-photon or two-photon imaging. In vivo photometry-based detection of optogenetically-evoked DA release revealed that DETQ administration produces a stable 31 minutes window of potentiation without effects on animal behavior. The use of the PAM revealed region-specific and metabolic state-dependent differences in tonic DA levels and enhanced single-trial detection of behavior-evoked phasic DA release in cortex and striatum. Our chemogenetic strategy can potently and flexibly tune DA imaging sensitivity and reveal multi-modal (tonic/phasic) DA signaling across preparations and imaging approaches.

Identifiants

DOI: 10.1038/s41467-024-49442-3 PMID: 38956067

pubmed: 38956067

doi: 10.1038/s41467-024-49442-3

pii: 10.1038/s41467-024-49442-3

doi:

Substances chimiques

Dopamine VTD58H1Z2X

Receptors, Dopamine 0

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

Pagination

5551

Subventions

Organisme : EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)

ID : 101016787

Organisme : Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)

ID : 310030_196455

Organisme : Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)

ID : 310030L_212508

Informations de copyright

Références

Berke, J. D. What does dopamine mean? Nat. Neurosci. 21, 787–793 (2018).

pubmed: 29760524 pmcid: 6358212 doi: 10.1038/s41593-018-0152-y

Björklund, A. & Dunnett, S. B. Dopamine neuron systems in the brain: an update. Trends Neurosci. 30, 194–202 (2007).

pubmed: 17408759 doi: 10.1016/j.tins.2007.03.006

Zhuang, Y. et al. Structural insights into the human D1 and D2 dopamine receptor signaling complexes. Cell 184, 931–942.e18 (2021).

pubmed: 33571431 pmcid: 8215686 doi: 10.1016/j.cell.2021.01.027

Jaquins-Gerstl, A. & Michael, A. C. A review of the effects of FSCV and microdialysis measurements on dopamine release in the surrounding tissue. Analyst 140, 3696–3708 (2015).

pubmed: 25876757 pmcid: 4437820 doi: 10.1039/C4AN02065K

Sabatini, B. L. & Tian, L. Imaging Neurotransmitter and Neuromodulator Dynamics In Vivo with Genetically Encoded Indicators. Neuron 108, 17–32 (2020).

pubmed: 33058762 doi: 10.1016/j.neuron.2020.09.036

Patriarchi, T. et al. Imaging neuromodulators with high spatiotemporal resolution using genetically encoded indicators. Nat. Protoc. 14, 3471–3505 (2019).

pubmed: 31732722 doi: 10.1038/s41596-019-0239-2

Wu, Z., Lin, D. & Li, Y. Pushing the frontiers: tools for monitoring neurotransmitters and neuromodulators. Nat. Rev. Neurosci. 23, 257–274 (2022).

pubmed: 35361961 pmcid: 11163306 doi: 10.1038/s41583-022-00577-6

Labouesse, M. A. & Patriarchi, T. A versatile GPCR toolkit to track in vivo neuromodulation: not a one-size-fits-all sensor. Neuropsychopharmacology 46, 2043–2047 (2021).

Patriarchi, T. et al. Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors. Science 360, eaat4422 (2018).

pubmed: 29853555 pmcid: 6287765 doi: 10.1126/science.aat4422

Sun, F. et al. A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice. Cell 174, 481–496.e19 (2018).

pubmed: 30007419 pmcid: 6092020 doi: 10.1016/j.cell.2018.06.042

Warne, T., Edwards, P. C., Doré, A. S., Leslie, A. G. W. & Tate, C. G. Molecular basis for high affinity agonist binding in GPCRs. Science 364, 775–778 (2019).

pubmed: 31072904 pmcid: 6586556 doi: 10.1126/science.aau5595

Labouesse, M. A., Cola, R. B. & Patriarchi, T. GPCR-Based Dopamine Sensors-A Detailed Guide to Inform Sensor Choice for In vivo Imaging. Int. J. Mol. Sci. 21, 8048 (2020).

Grace, A. A. Phasic versus tonic dopamine release and the modulation of dopamine system responsivity: a hypothesis for the etiology of schizophrenia. Neuroscience 41, 1–24 (1991).

pubmed: 1676137 doi: 10.1016/0306-4522(91)90196-U

Dreyer, J. K., Herrik, K. F., Berg, R. W. & Hounsgaard, J. D. Influence of Phasic and Tonic Dopamine Release on Receptor Activation. J. Neurosci. 30, 14273–14283 (2010).

pubmed: 20962248 pmcid: 6634758 doi: 10.1523/JNEUROSCI.1894-10.2010

Liu, C. et al. An action potential initiation mechanism in distal axons for the control of dopamine release. Science 375, 1378–1385 (2022).

pubmed: 35324301 pmcid: 9081985 doi: 10.1126/science.abn0532

Mohebi, A., Collins, V. L. & Berke, J. D. Accumbens cholinergic interneurons dynamically promote dopamine release and enable motivation. Elife. 12, e85011 (2023).

Lewis, M. A. et al. Discovery of D1 Dopamine Receptor Positive Allosteric Modulators: Characterization of Pharmacology and Identification of Residues that Regulate Species Selectivity. J. Pharm. Exp. Ther. 354, 340–349 (2015).

doi: 10.1124/jpet.115.224071

Svensson, K. A., Hao, J. & Bruns, R. F. Positive allosteric modulators of the dopamine D1 receptor: A new mechanism for the treatment of neuropsychiatric disorders. Adv. Pharm. 86, 273–305 (2019).

doi: 10.1016/bs.apha.2019.06.001

Luderman, K. D. et al. Identification of Positive Allosteric Modulators of the D1 Dopamine Receptor That Act at Diverse Binding Sites. Mol. Pharm. 94, 1197–1209 (2018).

doi: 10.1124/mol.118.113175

Bruns, R. F. et al. Preclinical profile of a dopamine D1 potentiator suggests therapeutic utility in neurological and psychiatric disorders. Neuropharmacology 128, 351–365 (2018).

pubmed: 29102759 doi: 10.1016/j.neuropharm.2017.10.032

Wang, X. et al. Intracellular Binding Site for a Positive Allosteric Modulator of the Dopamine D1 Receptor. Mol. Pharm. 94, 1232–1245 (2018).

doi: 10.1124/mol.118.112649

Zhuang, Y. et al. Mechanism of dopamine binding and allosteric modulation of the human D1 dopamine receptor. Cell Res 31, 593–596 (2021).

pubmed: 33750903 pmcid: 8089099 doi: 10.1038/s41422-021-00482-0

Xiao, P. et al. Ligand recognition and allosteric regulation of DRD1-Gs signaling complexes. Cell 184, 943–956.e18 (2021).

pubmed: 33571432 pmcid: 11005940 doi: 10.1016/j.cell.2021.01.028

Svensson, K. A. et al. An Allosteric Potentiator of the Dopamine D1 Receptor Increases Locomotor Activity in Human D1 Knock-In Mice without Causing Stereotypy or Tachyphylaxis. J. Pharm. Exp. Ther. 360, 117–128 (2017).

doi: 10.1124/jpet.116.236372

Hao, J. et al. Synthesis and Pharmacological Characterization of 2-(2,6-Dichlorophenyl)-1-((1S,3R)-5-(3-hydroxy-3-methylbutyl)-3-(hydroxymethyl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one (LY3154207), a Potent, Subtype Selective, and Orally Available Positive Allosteric Modulator of the Human Dopamine D1 Receptor. J. Med. Chem. 62, 8711–8732 (2019).

pubmed: 31532644 doi: 10.1021/acs.jmedchem.9b01234

Dixon, A. S. et al. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem. Biol. 11, 400–408 (2016).

pubmed: 26569370 doi: 10.1021/acschembio.5b00753

Kagiampaki, Z. et al. Sensitive multicolor indicators for monitoring norepinephrine in vivo. Nat Methods 20, 1426–1436 (2023).

Elizarova, S. et al. A fluorescent nanosensor paint detects dopamine release at axonal varicosities with high spatiotemporal resolution. Proc. Natl Acad. Sci. USA 119, e2202842119 (2022).

pubmed: 35613050 pmcid: 9295782 doi: 10.1073/pnas.2202842119

Bulumulla, C. et al. Visualizing synaptic dopamine efflux with a 2D composite nanofilm. eLife 11, e78773 (2022).

pubmed: 35786443 pmcid: 9363124 doi: 10.7554/eLife.78773

Klein Herenbrink, C. et al. Multimodal detection of dopamine by sniffer cells expressing genetically encoded fluorescent sensors. Commun. Biol. 5, 1–9 (2022).

doi: 10.1038/s42003-022-03488-5

Bäckman, C. M. et al. Characterization of a mouse strain expressing Cre recombinase from the 3’ untranslated region of the dopamine transporter locus. Genesis 44, 383–390 (2006).

pubmed: 16865686 doi: 10.1002/dvg.20228

Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

pubmed: 20023653 doi: 10.1038/nn.2467

Klapoetke, N. C. et al. Independent Optical Excitation of Distinct Neural Populations. Nat. Methods 11, 338–346 (2014).

pubmed: 24509633 pmcid: 3943671 doi: 10.1038/nmeth.2836

Podda, M. V., Riccardi, E., D’Ascenzo, M., Azzena, G. B. & Grassi, C. Dopamine D1-like receptor activation depolarizes medium spiny neurons of the mouse nucleus accumbens by inhibiting inwardly rectifying K+ currents through a cAMP-dependent protein kinase A-independent mechanism. Neuroscience 167, 678–690 (2010).

pubmed: 20211700 doi: 10.1016/j.neuroscience.2010.02.075

Meltzer, H. Y. et al. The allosteric dopamine D1 receptor potentiator, DETQ, ameliorates subchronic phencyclidine-induced object recognition memory deficits and enhances cortical acetylcholine efflux in male humanized D1 receptor knock-in mice. Behav. Brain Res 361, 139–150 (2019).

pubmed: 30521930 doi: 10.1016/j.bbr.2018.12.006

Rajagopal, L. et al. The dopamine D1 receptor positive allosteric modulator, DETQ, improves cognition and social interaction in aged mice and enhances cortical and hippocampal acetylcholine efflux. Behav. Brain Res 459, 114766 (2024).

pubmed: 38048913 doi: 10.1016/j.bbr.2023.114766

de Jong, J. W. et al. A Neural Circuit Mechanism for Encoding Aversive Stimuli in the Mesolimbic Dopamine System. Neuron 101, 133–151.e7 (2019).

pubmed: 30503173 doi: 10.1016/j.neuron.2018.11.005

Liu, Z. et al. A distinct D1-MSN subpopulation down-regulates dopamine to promote negative emotional state. Cell Res 32, 139–156 (2022).

pubmed: 34848869 doi: 10.1038/s41422-021-00588-5

Salinas, A. G. et al. Distinct sub-second dopamine signaling in dorsolateral striatum measured by a genetically-encoded fluorescent sensor. Nat. Commun. 14, 5915 (2023).

pubmed: 37739964 pmcid: 10517008 doi: 10.1038/s41467-023-41581-3

Mohebi, A. et al. Dissociable dopamine dynamics for learning and motivation. Nature 570, 65–70 (2019).

pubmed: 31118513 pmcid: 6555489 doi: 10.1038/s41586-019-1235-y

Roth, B. L. DREADDs for Neuroscientists. Neuron 89, 683–694 (2016).

pubmed: 26889809 pmcid: 4759656 doi: 10.1016/j.neuron.2016.01.040

Bonaventura, J. et al. High-potency ligands for DREADD imaging and activation in rodents and monkeys. Nat. Commun. 10, 4627 (2019).

pubmed: 31604917 pmcid: 6788984 doi: 10.1038/s41467-019-12236-z

Gradinaru, V., Thompson, K. R. & Deisseroth, K. eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications. Brain Cell Biol. 36, 129–139 (2008).

pubmed: 18677566 pmcid: 2588488 doi: 10.1007/s11068-008-9027-6

Wallace, C. W., Loudermilt, M. C. & Fordahl, S. C. Effect of fasting on dopamine neurotransmission in subregions of the nucleus accumbens in male and female mice. Nutr. Neurosci. 25, 1338–1349 (2022).

pubmed: 33297887 doi: 10.1080/1028415X.2020.1853419

Sych, Y., Chernysheva, M., Sumanovski, L. T. & Helmchen, F. High-density multi-fiber photometry for studying large-scale brain circuit dynamics. Nat. Methods 16, 553–560 (2019).

pubmed: 31086339 doi: 10.1038/s41592-019-0400-4

Sych, Y., Fomins, A., Novelli, L. & Helmchen, F. Dynamic reorganization of the cortico-basal ganglia-thalamo-cortical network during task learning. Cell Rep. 40, 111394 (2022).

pubmed: 36130513 pmcid: 9513804 doi: 10.1016/j.celrep.2022.111394

Vander Weele, C. M. et al. Dopamine enhances signal-to-noise ratio in cortical-brainstem encoding of aversive stimuli. Nature 563, 397–401 (2018).

doi: 10.1038/s41586-018-0682-1

Herenbrink, C. K. et al. Multimodal detection of dopamine by sniffer cells expressing genetically encoded fluorescent sensors. Commun Biol. 5, 578 (2022).

Condon, A. F. et al. The residence of synaptically released dopamine on D2 autoreceptors. Cell Rep. 36, 109465 (2021).

pubmed: 34348146 pmcid: 8351352 doi: 10.1016/j.celrep.2021.109465

Jørgensen, S. H. et al. Behavioral encoding across timescales by region-specific dopamine dynamics. Proc. Natl Acad. Sci. USA 120, e2215230120 (2023).

pubmed: 36749722 pmcid: 9963838 doi: 10.1073/pnas.2215230120

Li, J. & Mooney, D. J. Designing hydrogels for controlled drug delivery. Nat. Rev. Mater. 1, 1–17 (2016).

doi: 10.1038/natrevmats.2016.71

Kedir, W. M., Deresa, E. M. & Diriba, T. F. Pharmaceutical and drug delivery applications of pectin and its modified nanocomposites. Heliyon 8, e10654 (2022).

pubmed: 36164543 pmcid: 9508417 doi: 10.1016/j.heliyon.2022.e10654

Bernhard, S. & Tibbitt, M. W. Supramolecular engineering of hydrogels for drug delivery. Adv. Drug Deliv. Rev. 171, 240–256 (2021).

pubmed: 33561451 doi: 10.1016/j.addr.2021.02.002

Cunningham, M. G., O’Connor, R. P. & Wong, S. E. Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents. J Vis Exp 17, 716 (2008).

Zhuo, Y. et al. Improved green and red GRAB sensors for monitoring dopaminergic activity in vivo. Nat. Methods 21, 680–691 (2024).

Liu, C., Goel, P. & Kaeser, P. S. Spatial and temporal scales of dopamine transmission. Nat. Rev. Neurosci. 22, 345–358 (2021).

pubmed: 33837376 pmcid: 8220193 doi: 10.1038/s41583-021-00455-7

Sulzer, D., Cragg, S. J. & Rice, M. E. Striatal dopamine neurotransmission: regulation of release and uptake. Basal Ganglia 6, 123–148 (2016).

pubmed: 27141430 pmcid: 4850498 doi: 10.1016/j.baga.2016.02.001

Owesson-White, C. A. et al. Sources contributing to the average extracellular concentration of dopamine in the nucleus accumbens: Extracellular dopamine concentration. J. Neurochem. 121, 252–262 (2012).

pubmed: 22296263 pmcid: 3323736 doi: 10.1111/j.1471-4159.2012.07677.x

Kim, H. R. et al. A Unified Framework for Dopamine Signals across Timescales. Cell 183, 1600–1616.e25 (2020).

pubmed: 33248024 pmcid: 7736562 doi: 10.1016/j.cell.2020.11.013

Wang, Y., Toyoshima, O., Kunimatsu, J., Yamada, H. & Matsumoto, M. Tonic firing mode of midbrain dopamine neurons continuously tracks reward values changing moment-by-moment. eLife 10, e63166 (2021).

Mikhael, J. G., Lai, L. & Gershman, S. J. Rational inattention and tonic dopamine. PLOS Comput. Biol. 17, e1008659 (2021).

pubmed: 33760806 pmcid: 7990190 doi: 10.1371/journal.pcbi.1008659

Delignat-Lavaud, B. et al. Synaptotagmin-1-dependent phasic axonal dopamine release is dispensable for basic motor behaviors in mice. Nat. Commun. 14, 4120 (2023).

pubmed: 37433762 pmcid: 10336101 doi: 10.1038/s41467-023-39805-7

Iino, Y. et al. Dopamine D2 receptors in discrimination learning and spine enlargement. Nature 579, 555–560 (2020).

pubmed: 32214250 doi: 10.1038/s41586-020-2115-1

Ellwood, I. T. et al. Tonic or Phasic Stimulation of Dopaminergic Projections to Prefrontal Cortex Causes Mice to Maintain or Deviate from Previously Learned Behavioral Strategies. J. Neurosci. 37, 8315–8329 (2017).

pubmed: 28739583 pmcid: 5577850 doi: 10.1523/JNEUROSCI.1221-17.2017

Grieder, T. E. et al. Phasic D1 and tonic D2 dopamine receptor signaling double dissociate the motivational effects of acute nicotine and chronic nicotine withdrawal. Proc. Natl Acad. Sci. 109, 3101–3106 (2012).

pubmed: 22308372 pmcid: 3286981 doi: 10.1073/pnas.1114422109

Schultz, W., Dayan, P. & Montague, P. R. A neural substrate of prediction and reward. Science 275, 1593–1599 (1997).

pubmed: 9054347 doi: 10.1126/science.275.5306.1593

Cohen, J. Y., Haesler, S., Vong, L., Lowell, B. B. & Uchida, N. Neuron-type-specific signals for reward and punishment in the ventral tegmental area. Nature 482, 85–88 (2012).

pubmed: 22258508 pmcid: 3271183 doi: 10.1038/nature10754

Hart, A. S., Rutledge, R. B., Glimcher, P. W. & Phillips, P. E. M. Phasic dopamine release in the rat nucleus accumbens symmetrically encodes a reward prediction error term. J. Neurosci. 34, 698–704 (2014).

pubmed: 24431428 pmcid: 3891951 doi: 10.1523/JNEUROSCI.2489-13.2014

Steinberg, E. E. et al. A causal link between prediction errors, dopamine neurons and learning. Nat. Neurosci. 16, 966–973 (2013).

pubmed: 23708143 pmcid: 3705924 doi: 10.1038/nn.3413

Jeong, H. et al. Mesolimbic dopamine release conveys causal associations. Science 378, eabq6740 (2022).

pubmed: 36480599 pmcid: 9910357 doi: 10.1126/science.abq6740

Robinson, B. G. et al. RIM is essential for stimulated but not spontaneous somatodendritic dopamine release in the midbrain. eLife 8, e47972 (2019).

pubmed: 31486769 pmcid: 6754207 doi: 10.7554/eLife.47972

Yorgason, J. T., Zeppenfeld, D. M. & Williams, J. T. Cholinergic Interneurons Underlie Spontaneous Dopamine Release in Nucleus Accumbens. J. Neurosci. 37, 2086 (2017).

pubmed: 28115487 pmcid: 5338756 doi: 10.1523/JNEUROSCI.3064-16.2017

Liu, C. & Kaeser, P. S. Mechanisms and regulation of dopamine release. Curr. Opin. Neurobiol. 57, 46–53 (2019).

pubmed: 30769276 pmcid: 6629510 doi: 10.1016/j.conb.2019.01.001

Massengill, C. I. et al. Sensitive genetically encoded sensors for population and subcellular imaging of cAMP in vivo. Nat. Methods 19, 1461–1471 (2022).

pubmed: 36303019 pmcid: 10171401 doi: 10.1038/s41592-022-01646-5

Ma, L. et al. A Highly Sensitive A-Kinase Activity Reporter for Imaging Neuromodulatory Events in Awake Mice. Neuron 99, 665–679.e5 (2018).

pubmed: 30100256 pmcid: 6152931 doi: 10.1016/j.neuron.2018.07.020

Zhang, J.-F. et al. An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice. Nat. Chem. Biol. 17, 39–46 (2021).

pubmed: 32989297 doi: 10.1038/s41589-020-00660-y

Foster, D. J. & Conn, P. J. Allosteric modulation of GPCRs: new insights and potential utility for treatment of schizophrenia and other CNS disorders. Neuron 94, 431–446 (2017).

pubmed: 28472649 pmcid: 5482176 doi: 10.1016/j.neuron.2017.03.016

Wan, Q. et al. Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells. J. Biol. Chem. 293, 7466–7473 (2018).

pubmed: 29523687 pmcid: 5949987 doi: 10.1074/jbc.RA118.001975

Fasano, C., Thibault, D. & Trudeau, L.-E. Culture of postnatal mesencephalic dopamine neurons on an astrocyte monolayer. Curr. Protoc. Neurosci. Chapter 3, Unit 3.21 (2008).

pubmed: 18633997

van Brakel, J. P. G. Robust peak detection algorithm using z-scores. Stack Overflow 36, 2787–2795 (2014).

Thoeni, S., Loureiro, M., O’Connor, E. C. & Lüscher, C. Depression of Accumbal to Lateral Hypothalamic Synapses Gates Overeating. Neuron 107, 158–172.e4 (2020).

pubmed: 32333845 doi: 10.1016/j.neuron.2020.03.029

Kalvass, J. C., Maurer, T. S. & Pollack, G. M. Use of plasma and brain unbound fractions to assess the extent of brain distribution of 34 drugs: comparison of unbound concentration ratios to in vivo p-glycoprotein efflux ratios. Drug Metab. Dispos. 35, 660–666 (2007).

pubmed: 17237155 doi: 10.1124/dmd.106.012294

Duffet, L. et al. A photocaged orexin-B for spatiotemporally precise control of orexin signaling. Cell Chem. Biol. 29, 1729–1738.e8 (2022).

Mathis, A. et al. DeepLabCut: markerless pose estimation of user-defined body parts with deep learning. Nat. Neurosci. 21, 1281–1289 (2018).

pubmed: 30127430 doi: 10.1038/s41593-018-0209-y

Labouesse, M. A. et al. A non-canonical triatopallidal Go pathway that supports motor control. Nat Commun 14, 6712 (2023).

Salinas, A. G. et al. Distinct sub-second dopamine signaling in dorsolateral striatum measured by a genetically-encoded fluorescent sensor. Nat Commun. 14, 5915 (2023).

Patriarchi Lab. PatriarchiLab/InVivoPhotometry: 1. [object Object] https://doi.org/10.5281/ZENODO.11262981 (2024).

Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat Methods 9, 679–682 (2022).