Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments

Abstract

Second messenger cAMP regulates many cellular functions through its effectors, such as cAMP-dependent protein kinase (PKA) and Epac (exchange proteins directly activated by cAMP). Spatial and temporal control of cAMP signaling is crucial to differential regulation of cellular targets involved in various signaling cascades. To investigate the compartmentalized cAMP signaling, we constructed fluorescent indicators that report intracellular cAMP dynamics and Epac activation by sandwiching the full-length Epac1 between cyan and yellow mutants of GFP. Elevations of cAMP decreased FRET and increased the ratio of cyan-to-yellow emissions by 10-30% in living mammalian cells. This response can be reversed by removing cAMP-elevating agents and abolished by mutating the critical residue responsible for cAMP binding. Targeting of the reporter to the plasma membrane, where cAMP is produced in response to the activation of beta-adrenergic receptor, revealed a faster cAMP response at the membrane than in the cytoplasm and mitochondria. Simultaneous imaging with targeted cAMP indicator and PKA activity reporter allowed the detection of a much delayed PKA response in the nucleus after the rapid accumulation of cAMP at the plasma membrane of the same cell, despite the immediate presence of a pool of cAMP in the nucleus. Thus, cAMP dynamics and the activation of its effectors are precisely controlled spatiotemporally in vivo.

Fig. 1.

Domain structure and comparison of FRET responses for Epac-based cAMP reporters. Sandwiched between ECFP and citrine are truncated forms of Epac2, full-length Epac1 with or without R522E mutation, with R522 corresponding to R279 in Epac1. The construct that generated the biggest FRET response was designated as ICUE1.

Fig. 2.

Responses of ICUE1 to changes in cellular cAMP levels. (A) FRET response of HEK-293 cells transfected with ICUE1. The first image is a YFP-only image. Pseudocolor images depict the FRET response of the reporter to ISO stimulation at various time points. (Scale bar, 10 μm.) (B) Representative emission ratio time courses of ICUE1 and the R522E mutant stimulated with 10 μM ISO followed by 10 μM propranolol and 50 μM forskolin (FsK). (C) Representative emission ratio time courses of ICUE1 stimulated with 10 μM ISO, 50 μM forskolin, 10 μM PGE₁, 300 μM 8-pCPT-2′-O-Me-cAMP, and 100 μM P-(4,5-dimethoxy-2-nitrobenzyl) cAMP followed by UV uncaging. The flash signs indicate 5-s UV flashes at two different time points.

Fig. 3.

Fusions of ICUE1 targeted to various subcellular locations. (A) Domain structures of the fusion constructs. (B) YFP-only images showing plasma membrane and nuclear distributions of various fusions. (Scale bars, 10 μm.) Merged pseudocolor images show colocalization of nuclear localized ICUE1 with Hoechst 33342 cell-permeable dye in nucleus and mitochondria-targeted ICUE1 with MitoTracker at mitochondria. (C) Representative emission ratio time courses for the untagged (ICUE1), plasma membrane-targeted (pm ICUE1), mitochondria-targeted (MitoCOX- and MitoDAKAP1-ICUE1), and nuclear localized reporters (NLS-ICUE1) stimulated with 10 μM ISO. (D) Representative emission ratio time courses for pm ICUE1 stimulated with 10 μM PGE₁ followed by the removal of PGE₁ and the addition of 10 μM ISO. (E) Representative emission ratio time courses for NLS-ICUE1 in response to 10 μM PGE₁ and 10 μM ISO separated by a washing step.

Fig. 4.

Simultaneous imaging of FRET reporters targeted to different sub-cellular locations. (A) Cellular distribution of different fusions. (B) Representative emission ratio time courses for the pm ICUE1 and nuclear localized PKA activity reporter (NLS-AKAR) in the same cell stimulated with 10 μM ISO. Identical results were found in four different cells. The AKAR response was plotted by using a normalized ratio of yellow-to-cyan emissions. (C) Representative emission ratio time courses for pm ICUE1 and NLS-ICUE1 in the same cell stimulated with 10 μM ISO followed by 10 μM propranolol (n = 4).