Fast manipulation of cellular cAMP level by light in vivo

Schröder-Lang, Saskia; Schwärzel, Martin; Seifert, Reinhard; Strünker, Timo; Kateriya, Suneel; Looser, Jens; Watanabe, Masakatsu; Kaupp, U. Benjamin; Hegemann, Peter; Nagel, Georg

doi:10.1038/nmeth975

Cited by 236 publications

(204 citation statements)

References 16 publications

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“…OaPAC also requires longer illumination times to give the same rise in cAMP level, allowing finer control of the degree of stimulation. The lower light sensitivity allows much more precise control of cAMP level in human cells than achievable with Euglena PAC proteins (28). The ability of OaPAC to work in different cells types in demonstrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

Ohki

Sugiyama

Kawai

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

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Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.optogenetics | X-ray crystallography | blue light | allostery

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Section: Resultsmentioning

confidence: 99%

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

Ohki

Sugiyama

Kawai

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

106

View full text Add to dashboard Cite

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“…To investigate further the spatial code by which cAMP regulates axon guidance, we used mCherry-tagged photoactivated adenylyl cyclase (27) (mCherry-PACα; Fig. S5A and Movie S4) to generate localized elevations of cAMP mimicking the duration of those stimulated by Netrin-1 (27). Exposure of quadrants of growth cones expressing mCherry-PACα to blue light for 3 min every 20 min induced turning of axons toward the stimulated side ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Spatial and temporal second messenger codes for growth cone turning

Nicol

Hong

Spitzer

2011

Proc. Natl. Acad. Sci. U.S.A.

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Cyclic AMP (cAMP) and calcium are ubiquitous, interdependent second messengers that regulate a wide range of cellular processes. During development of neuronal networks they are critical for the first step of circuit formation, transducing signals required for axon pathfinding. Surprisingly, the spatial and temporal cAMP and calcium codes used by axon guidance molecules are unknown. Here, we identify characteristics of cAMP and calcium transients generated in growth cones during Netrin-1-dependent axon guidance. In filopodia, Netrin-1-dependent Deleted in Colorectal Cancer (DCC) receptor activation induces a transient increase in cAMP that causes a brief increase in calcium transient frequency. In contrast, activation of DCC in growth cone centers leads to a transient calciumdependent cAMP increase and a sustained increase in frequency of calcium transients. We show that filopodial cAMP transients regulate spinal axon guidance in vitro and commissural axon pathfinding in vivo. These growth cone codes provide a basis for selective activation of specific downstream effectors.cyclic AMP dynamics | calcium dynamics | compartmentalization | photoactivated adenylyl cyclase alpha | cyclic AMP oscillations C yclic AMP (cAMP) is a major cellular second messenger that activates and integrates multiple intracellular signaling pathways. Microdomains of cAMP are generated in cardiac myocytes (1), regional domains of cAMP are present in neurons (2, 3), and cAMP diffusion is restricted by rapid degradation by phosphodiesterases (4) that limit the duration of cAMP signaling (5, 6). However, little is known about spatial compartmentalization and temporal dynamics of cAMP in neuronal growth cones, despite the importance of cAMP for axon pathfinding in response to a wide range of molecular guidance cues, including Netrin-1 (7-11), semaphorins (12), Slits, and ephrins (13).Calcium is another ubiquitous second messenger involved in axon pathfinding, mediating responses to axon guidance molecules. In addition to axon steering, calcium modulates axon outgrowth and retraction (14,15). A sustained gradient of calcium across the growth cone is thought to be generated by asymmetric activation of axon guidance receptors, and to be the relevant calcium signal for axon pathfinding (16). Spontaneous fast and spatially restricted filopodial calcium transients are also sufficient to steer axons (17, 18). The frequency of slower transients in the entire growth cone controls the rate of axon extension in vivo and in vitro (19). The regulation of these transients by axon guidance cues has not been investigated.Filopodia are critical for axon pathfinding (20), and clues to their operation are provided by subcellular localization of signaling components. Filopodial enrichment of regulatory subunit II of protein kinase A, a major effector of cAMP, is required for growth cone attraction mediated by intracellular gradients of cAMP or PACAP (21). Modulation of spontaneous calcium transients in filopodia regulates axon turning (17). Temporal regulation o...

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“…d Light-regulation of DNA binding activity of the dimeric tryptophan repressor protein TrpR using LOV-Jα; in the dark, binding of LOV to a shared helix of TrpR populates an inactive conformation of the TrpR domain; unfolding of Jα allows for the release of the LOV domain and, thus, activation of the TrpR repressor (modified after Strickland et al 2010). e Utilization of naturally occurring, light-sensitive adenylyl cyclases from the alga Euglena gracilis (euPAC) or the bacterium Beggiatoa (bPAC) to manipulate the cellular cAMP level by light (Ryu et al 2010;Schröder-Lang et al 2007;Stierl et al 2011). f Construction of a light-activated transcription system based on the (rapid) interaction of the cryptochrome CRY2 with CIB1 upon blue light irradiation (modified after Kennedy et al 2010;Liu et al 2012) of neuronal spiking (Boyden et al 2005).…”

Section: Application Of Light-sensitive Modules In Synthetic Biology mentioning

confidence: 99%

“…Heterologous expression of euPACα allows for fine spatiotemporal control of the cAMP level in amphibian cells, i.e., Xenopus laevis oocytes, and in mammalian cells, i.e., human embryonic kidney (HEK293) cells (Schröder-Lang et al 2007). Also insect cells have been modified: transgenic fruit flies expressing euPACα show changes in their behavior after blue light illumination (Schröder-Lang et al 2007). In nematodes, i.e., cholinergic neurons from transgenic Caenorhabditis elegans, expression of euPACα allows manipulation of the behavior in a light-dependent manner.…”

Section: Application Of Light-sensitive Modules In Synthetic Biology mentioning

confidence: 99%