Interrogating cyclic AMP signaling using optical approaches

Abstract

Optical reporters for cAMP represent a fundamental advancement in our ability to investigate the dynamics of cAMP signaling. These fluorescent sensors can measure changes in cAMP in single cells or in microdomains within cells as opposed to whole populations of cells required for other methods of measuring cAMP. The first optical cAMP reporters were FRET-based sensors utilizing dissociation of purified regulatory and catalytic subunits of PKA, introduced by Roger Tsien in the early 1990s. The utility of these sensors was vastly improved by creating genetically encoded versions that could be introduced into cells with transfection, the first of which was published in the year 2000. Subsequently, improved sensors have been developed using different cAMP binding platforms, optimized fluorescent proteins, and targeting motifs that localize to specific microdomains. The most common sensors in use today are FRET-based sensors designed around an Epac backbone. These rely on the significant conformational changes in Epac when it binds cAMP, altering the signal between FRET pairs flanking Epac. Several other strategies for optically interrogating cAMP have been developed, including fluorescent translocation reporters, dimerization-dependent FP based biosensors, BRET (bioluminescence resonance energy transfer)-based sensors, non-FRET single wavelength reporters, and sensors based on bacterial cAMP-binding domains. Other newly described mammalian cAMP-binding proteins such as Popdc and CRIS may someday be exploited in sensor design. With the proliferation of engineered fluorescent proteins and the abundance of cAMP binding targets in nature, the field of optical reporters for cAMP should continue to see rapid refinement in the coming years.

Aspects of astrocytic cAMP signaling with an emphasis on the putative power of compartmentalized signals in health and disease

“…This timeline gives an overview of the cAMP and Ca 2+ methodological progresses from discovery to present including some representative references. Data for timeline as reported in literature (for detailed cAMP reporters see Jiang, Falcone, Curci, and Hofer (); Musheshe, Schmidt, and Zaccolo (); Paramonov, Mamaeva, Sahlgren, and Rivero‐Muller (); Schleicher and Zaccolo ()) (for Ca2+ see Campbell (); Grienberger and Konnerth (); Looger and Griesbeck (); Suzuki, Kanemaru, and Iino ()). ∧ , Salomon, Londos, and Rodbell (); Г , Takeda, Kuno, Shuntoh, and Tanaka (); ᶳ, Nordstedt and Fredholm (); °, Adams, Harootunian, Buechler, Taylor, and Tsien (); ¤ , Zaccolo et al (); Zaccolo and Pozzan (); ¥ , DiPilato et al (); Nikolaev, Bunemann, Hein, Hannawacker, and Lohse (); Ponsioen et al (); ₽ , Nikolaev et al ()ᶤ, Everett and Cooper (); + , Sprenger et al (); £ , Vergara and Delay (); *, Grynkiewicz, Poenie, and Tsien (); ҍ , Tsien, Rink, and Poenie (); ᶬ, Kendall, Sala‐Newby, Ghalaut, Dormer, and Campbell (); ᶣ, Romoser, Hinkle, and Persechini (); ᶲ, Miyawaki et al (); ᶿ, Nakai, Ohkura, and Imoto (); Sawano and Miyawaki (); Ω , Wang, Wong, Flores, Vosshall, and Axel (); Ⅎ , Ji et al (); ⋔ , Tallini et al (); ℓ , Willoughby, Wachten, Masada, and Cooper (); †, Dana et al ().…”

“…This timeline gives an overview of the cAMP and Ca 2+ methodological progresses from discovery to present including some representative references. Data for timeline as reported in literature (for detailed cAMP reporters see Jiang, Falcone, Curci, and Hofer (2017) Everett and Cooper (2013); + , Sprenger et al (2015); £ , Vergara and Delay (1985); *, Grynkiewicz, Poenie, and Tsien (1985); ҍ , Tsien, Rink, and Poenie (1985); ᶬ, Kendall, Sala-Newby, Ghalaut, Dormer, and Campbell (1992); ᶣ, Romoser, Hinkle, and Persechini (1997); ᶲ, Miyawaki et al (1997); ᶿ, Nakai, Ohkura, and Imoto (2001); Sawano and Miyawaki (2000); Ω , Wang, Wong, Flores, Vosshall, and Axel (2010); †, Dana et al (2018). Abbreviations: AC, adenylyl cyclase; Epac, exchange protein directly activated by cAMP; GECI, genetically encoded Ca 2+ indicators; HCN2, hyperpolarizationactivated cyclic nucleotide-gated channel 2; PEI, polyethyleneimine; PKA, protein kinase A; PLN, phospholamban; PM, plasma membrane; smGC, smooth muscle isoform of myosin heavy chain-GCaMP1 expressing mice expressing a targeted cAMP biosensor have been produced and successfully employed to study a cAMP microdomain in proximity to SERCA pumps in cardiac myocytes (Sprenger et al, 2015).…”

Hedgehog and Gpr161: Regulating cAMP Signaling in the Primary Cilium

“…Different PDEs mediate the breakdown of cAMP by hydrolyzing it to AMP [31]. Using visualization technologies, the existence of cell-and context-specific cAMP nanodomains has been demonstrated [32][33][34][35][36][37][38][39][40]. Spatially separated pools of cAMP are likely to be engaged in different functional outputs.…”

Sensing Senses: Optical Biosensors to Study Gustation

“…The first genetically encoded cAMP sensor was generated tagging the PKA with two FRET-suited GFP mutants and this was used to monitor cAMP fluctuation in cardiac myocytes [252,253]. Since then, cAMP sensors have been continuously improved including newly discovered cAMP binding sequences and fluorescent proteins (for reviewed see [254]). Furthermore, subcellular targeting of such sensors permits to study changes in cAMP levels in microand nanodomains [255] and the genetic approach renders them suitable for studies in recombinant cells lines, primary cells and in vivo [256][257][258].…”