Elucidating cyclic AMP signaling in subcellular domains with optogenetic tools and fluorescent biosensors

Klausen, Christina; Kaiser, Fabian; Stüven, Birthe; Hansen, Jan N.; Wachten, Dagmar

doi:10.1042/bst20190246

Cited by 9 publications

(5 citation statements)

References 314 publications

(388 reference statements)

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“…To scrutinize bPAC and LAPD activity during nanobody-binding in a ciliary context, we aimed to use cAMP biosensors, which can be targeted to the primary cilium. However, all reported biosensors spectrally overlap with the LAPD activation spectrum to some extend ( Klausen et al, 2019 ). For bPAC, one biosensor is well suited to simultaneously activate bPAC and measure changes in cAMP levels: the red-shifted cAMP biosensor R-FlincA ( Ohta et al, 2018 ).…”

Section: Resultsmentioning

confidence: 99%

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

Hansen

Kaiser

Klausen

et al. 2020

eLife

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Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary cilium constitutes a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling has been challenging due to the lack of tools to investigate ciliary signaling. Here, we describe a nanobody-based targeting approach for optogenetic tools in mammalian cells and in vivo in zebrafish to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after fusion to ciliary targeting sequences. We functionally localized modifiers of cAMP signaling, the photo-activated adenylyl cyclase bPAC and the light-activated phosphodiesterase LAPD, and the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we studied the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.

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Section: Resultsmentioning

confidence: 99%

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

Hansen

Kaiser

Klausen

et al. 2020

eLife

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“…Recently, the single-FP cAMP indicator cADDis was used to detect cAMP level changes upon the stimulation of the ligand of Gs or Gi protein-coupled GPCRs using automated microscope methods ( Tewson et al, 2016 ; Clayton, 2018 ), suggesting its potential capabilities in HCS. Since G-Flamp2 has a higher affinity for cAMP ( K d of 1.9 μM vs. 10–100 μM) and larger fluorescence change (ΔF/F 0 of 13 vs. −0.55) than cADDis ( Klausen et al, 2019 ), and a relatively high brightness, G-Flamp2 would be more suitable for HCS to identify and evaluate the effect of candidate compounds targeting GPCR-cAMP signaling pathways in drug discovery.…”

Section: Discussionmentioning

confidence: 99%

An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening

Liu

Ren

et al. 2022

Front. Pharmacol.

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Cyclic adenosine 3′,5′-monophosphate (cAMP) is an important intracellular second messenger molecule downstream of many G protein-coupled receptors (GPCRs). Fluorescence imaging with bright and sensitive cAMP indicators allows not only dissecting the spatiotemporal dynamics of intracellular cAMP, but also high-content screening of compounds against GPCRs. We previously reported the high-performance circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1. Here, we developed improved G-Flamp1 variants G-Flamp2 and G-Flamp2b. Compared to G-Flamp1, G-Flamp2 exhibited increased baseline fluorescence (1.6-fold) and larger fluorescence change (ΔF/F0) (1,300% vs. 1,100%) in HEK293T cells, while G-Flamp2b showed increased baseline fluorescence (3.1-fold) and smaller ΔF/F0 (400% vs. 1,100%). Furthermore, live cell imaging of mitochondrial matrix–targeted G-Flamp2 confirmed cytosolic cAMP was able to enter the mitochondrial matrix. G-Flamp2 imaging also showed that adipose tissue extract activated the Gi protein-coupled orphan GPCR GPR50 in HEK293T cells. Taken together, our results showed that the high-performance of G-Flamp2 would facilitate sensitive intracellular cAMP imaging and activity measurement of compounds targeting GPCR-cAMP signaling pathway during early drug development.

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“…Taking advantage of the progress reached in animal systems, many other strategies and their combination may help in elucidating cAMP signalling in plant systems. Indeed, optogenetic approaches and genetically encoded fluorescent biosensors are effectively used to monitor and modulate cAMP levels [ 141 , 142 ]. Photoactivated ACs and light-regulated PDEs, or even their association, are successfully used in animal cells [ 143 , 144 ].…”

Section: Discussionmentioning

confidence: 99%

Cyclic AMP: A Polyhedral Signalling Molecule in Plants

Blanco

Fortunato

Viggiano

et al. 2020

IJMS

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The cyclic nucleotide cAMP (3′,5′-cyclic adenosine monophosphate) is nowadays recognised as an important signalling molecule in plants, involved in many molecular processes, including sensing and response to biotic and abiotic environmental stresses. The validation of a functional cAMP-dependent signalling system in higher plants has spurred a great scientific interest on the polyhedral role of cAMP, as it actively participates in plant adaptation to external stimuli, in addition to the regulation of physiological processes. The complex architecture of cAMP-dependent pathways is far from being fully understood, because the actors of these pathways and their downstream target proteins remain largely unidentified. Recently, a genetic strategy was effectively used to lower cAMP cytosolic levels and hence shed light on the consequences of cAMP deficiency in plant cells. This review aims to provide an integrated overview of the current state of knowledge on cAMP’s role in plant growth and response to environmental stress. Current knowledge of the molecular components and the mechanisms of cAMP signalling events is summarised.

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Elucidating cyclic AMP signaling in subcellular domains with optogenetic tools and fluorescent biosensors

Cited by 9 publications

References 314 publications

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium

An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening

Cyclic AMP: A Polyhedral Signalling Molecule in Plants

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