![]() So far, a few single-FP cAMP sensors (Flamindo2, cAMPr, Pink Flamindo and R-FlincA) based on different mammalian cyclic nucleotide-binding domains (CNBDs) and green/red FPs have been created 11, 12, 13, 14. The latter is much more sensitive and, because they only require a single-color channel, can be more easily used together with other spectrally compatible sensors and actuators 10. Current GEFIs for cAMP were developed based on two strategies: fluorescence resonance energy transfer (FRET) between two fluorescent proteins (FPs) or circular permutation/splitting of a single FP 7, 8, 9. Genetically encoded fluorescent indicators (GEFIs) with advanced optical imaging have emerged as a powerful tool for real-time monitoring the spatiotemporal dynamics of signaling molecules including calcium in intact model organisms 6. However, it is largely unclear how cAMP signaling is regulated under physiological and pathological conditions in vivo 3, 4, 5. A growing body of evidence has shown that cAMP is precisely controlled in space and time in living cells and its abnormal dynamics are associated with many diseases 3. cAMP plays critical roles in regulating numerous cellular physiological processes, including neuronal plasticity and innate and adaptive immune cell activities, through its effector proteins such as protein kinase A (PKA), exchange protein directly activated by cAMP (EPAC), and cyclic nucleotide-activated ion channels (CNG and HCN channels) 2. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.Ĭyclic adenosine 3′,5′-monophosphate (cAMP), which is produced from adenosine triphosphate (ATP) by adenylyl cyclase (AC), acts as a key second messenger downstream of many cell surface receptors, especially G-protein-coupled receptors (GPCRs) 1. Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted β-strand conformation that may serve as a fluorescence modulation switch. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum Δ F/ F 0 of 1100% in HEK293T cells), decent brightness, appropriate affinity (a K d of 2.17 μM) and fast response kinetics (an association and dissociation half-time of 0.20 and 0.087 s, respectively). However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Nature Communications volume 13, Article number: 5363 ( 2022)ĬAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. Scroll down to the Account section where the number of meeting participants is displayed.A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging.In the navigation panel, click Profile.If you are not sure of your account and user type, you can find what your meeting participant limit is by following these steps: How to find your meeting participant limit Once a meeting reach capacity, joining participants will get a dialog box stating the meeting has reached the maximum number of allowed participants and joining is only possible if some participants leave.If end-to-end encryption is enabled for a meeting, participants are limited to 200 regardless of the account's default capacity and large meeting add-on.Meeting capacity from multiple large meeting add-ons cannot be combined.The meeting participant limit can be increased by purchasing a license (for free/basic accounts), upgrading the current plan, or purchasing a large meeting add-on.A basic user within a Business plan or above inherits the account's default meeting capacity, such as a Business account's default meeting capacity of 300.(Optional) Add-ons: 500 participants 1,000 Participants.Basic (free or free with credit card): 100 participants.How to find your meeting participant limitīy default, these are the meeting participant limit per account type:.The maximum number of participants in your meeting depends on your Zoom plan/account type and user type.
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