PUBLICATION

A cAMP Sensor Based on Ligand-Dependent Protein Stabilization

Authors

Sidoli, M., Chen, L.C., Lu, A.J., Wandless, T.J., Talbot, W.S.

ID

ZDB-PUB-220716-8

Date

2022

Source

ACS Chemical Biology 17(8): 2024-2030 (Journal)

Registered Authors

Talbot, William S.

Keywords

none

MeSH Terms

Cyclic AMP*/metabolism
Biosensing Techniques*
Animals
Ligands
Zebrafish/metabolism
Cyclic AMP-Dependent Protein Kinases/metabolism
Fluorescence Resonance Energy Transfer

(all 7)

PubMed

35839076 Full text @ ACS Chem. Biol.

Abstract

cAMP is a ubiquitous second messenger with many functions in diverse organisms. Current cAMP sensors, including Föster resonance energy transfer (FRET)-based and single-wavelength-based sensors, allow for real time visualization of this small molecule in cultured cells and in some cases in vivo. Nonetheless the observation of cAMP in living animals is still difficult, typically requiring specialized microscopes and ex vivo tissue processing. Here we used ligand-dependent protein stabilization to create a new cAMP sensor. This sensor allows specific and sensitive detection of cAMP in living zebrafish embryos, which may enable new understanding of the functions of cAMP in living vertebrates.

Genes / Markers

Figures

Show all Figures

Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
GFP	gpr161b^st128/st128; gpr161a^st129/st129; st1002Tg/st1002Tg; zf3899Tg/zf3899Tg	standard conditions	Prim-5	somite	IFL	Fig. 4
GFP	st1002Tg/st1002Tg; zf3899Tg/zf3899Tg	standard conditions	Prim-5	somite	IFL	Fig. 4
GFP	zf3899Tg/zf3899Tg	chemical treatment by environment: 8-(4-chlorophenylthio)-cAMP	Prim-25	caudal fin	IFL	Fig. 3
GFP	zf3899Tg/zf3899Tg	standard conditions	Prim-5	horizontal myoseptum	IFL	Fig. 3
GFP	zf3899Tg/zf3899Tg	chemical treatment by environment: forskolin	Prim-5	horizontal myoseptum	IFL	Fig. 3
GFP	zf3899Tg/zf3899Tg	standard conditions	Prim-5	horizontal myoseptum	IFL	Fig. 2
GFP	zf3899Tg/zf3899Tg	standard conditions	10-13 somites to 14-19 somites	somite adaxial cell	IFL	Fig. 3
GFP	zf3899Tg/zf3899Tg	chemical treatment by environment: forskolin	10-13 somites to 14-19 somites	somite adaxial cell	IFL	Fig. 3
GFP	zf3900Tg/zf3900Tg	standard conditions	Prim-5	horizontal myoseptum	IFL	Fig. 2
GFP	zf3900Tg/zf3900Tg	standard conditions	Prim-5	horizontal myoseptum	IFL	Fig. 3

1 - 10 of 16

Show

Phenotype

Fish	Conditions	Stage	Phenotype	Figure
gpr161b^st128/st128; gpr161a^st129/st129; st1002Tg/st1002Tg; zf3899Tg/zf3899Tg	standard conditions	Prim-5	somite adaxial cell mCherry expression increased amount, abnormal	Fig. 4
gpr161b^st128/st128; gpr161a^st129/st129; st1002Tg/st1002Tg; zf3899Tg/zf3899Tg	standard conditions	Prim-5	somite GFP expression decreased amount, abnormal	Fig. 4
zf3899Tg/zf3899Tg	standard conditions	Prim-5	detection of cAMP normal process quality, normal	Fig. 2
zf3899Tg/zf3899Tg	chemical treatment by environment: forskolin	10-13 somites to 14-19 somites	somite adaxial cell GFP expression increased amount, abnormal	Fig. 3
zf3899Tg/zf3899Tg	chemical treatment by environment: forskolin	Prim-5	horizontal myoseptum GFP expression increased amount, abnormal	Fig. 3
zf3900Tg/zf3900Tg	standard conditions	Prim-5	detection of cAMP decreased process quality, abnormal	Fig. 2
zf3900Tg/zf3900Tg	standard conditions	Prim-5	horizontal myoseptum GFP expression decreased amount, abnormal	Fig. 2
zf3900Tg/zf3900Tg	standard conditions	Prim-5	horizontal myoseptum GFP expression decreased amount, abnormal	Fig. 3
zf3900Tg/zf3900Tg	chemical treatment by environment: forskolin	Prim-5	horizontal myoseptum GFP expression decreased amount, abnormal	Fig. 3

1 - 9 of 9

Show

Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
st128		Small Deletion	gpr161b
st129		Indel	gpr161a
st1002Tg	Tg(8xgli-Xla.Cryaa:NLS-d1mCherry)	Transgenic Insertion
zf3899Tg	Tg(ubb:DDcAMP)	Transgenic Insertion
zf3900Tg	Tg(ubb:DDcAMP_R307Q,myl7:mCherry)	Transgenic Insertion
zf3901Tg	Tg(ubb:GFP)	Transgenic Insertion
zf3902Tg	Tg(ubb:cAMPr)	Transgenic Insertion
zf3903Tg	Tg(ubb:N49-GFP)	Transgenic Insertion

1 - 8 of 8

Show

Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
gpr161a	CRISPR2-gpr161a	CRISPR
gpr161b	CRISPR2-gpr161b	CRISPR

Fish

Fish
gpr161b^st128/st128; gpr161a^st129/st129; st1002Tg/st1002Tg; zf3899Tg/zf3899Tg
st1002Tg/st1002Tg; zf3899Tg/zf3899Tg
zf3899Tg/zf3899Tg
zf3900Tg/zf3900Tg
zf3901Tg/zf3901Tg
zf3902Tg/zf3902Tg

1 - 6 of 6

Show

Antibodies

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
DDcAMP	EFG	DDcAMP
GFP	EFG	GFP
mCherry	EFG	mCherry

Mapping

Sidoli et al., 2022 ZDB-PUB-220716-8 PMID:35839076

A cAMP Sensor Based on Ligand-Dependent Protein Stabilization

Sidoli et al., 2022

ZDB-PUB-220716-8
PMID:35839076