Luminescence-activated nucleotide cyclase regulates spatial and temporal cAMP synthesis

Abstract

cAMP is a ubiquitous second messenger that regulates cellular proliferation, differentiation, attachment, migration, and several other processes. It has become increasingly evident that tight regulation of cAMP accumulation and localization confers divergent yet specific signaling to downstream pathways. Currently, few tools are available that have sufficient spatial and temporal resolution to study location-biased cAMP signaling. Here, we introduce a new fusion protein consisting of a light-activated adenylyl cyclase (bPAC) and luciferase (nLuc). This construct allows dual activation of cAMP production through temporally precise photostimulation or chronic chemical stimulation that can be fine-tuned to mimic physiological levels and duration of cAMP synthesis to trigger downstream events. By targeting this construct to different compartments, we show that cAMP produced in the cytosol and nucleus stimulates proliferation in thyroid cells. The bPAC-nLuc fusion construct adds a new reagent to the available toolkit to study cAMP-regulated processes in living cells.

Keywords: bPAC; bioluminescence; cell proliferation; compartmentalization; cyclic AMP (cAMP); fluorescence resonance energy transfer (FRET); microdomains; nano-luciferase; optogenetics; second messenger; signaling; thyroid.

Figure 1.

Luminescence-activated cyclase characterization. A, design of dual photo and chemical activator of cAMP production by fusion of bPAC and nLuc. B, transiently transfected HEK293 cells treated with 100 μm h-CTZ luminesce at a maximum of 455 nm (mean ± S.D. of n = 4). C, this effect occurs in a dose-dependent manner (mean ± S.D. of n = 4). D, h-CTZ increases cAMP in HC1 cells expressing bPAC–nLuc in a dose-dependent manner as shown by ELISA analysis (mean ± S.D. of n = 4). E, accumulation of cytosolic cAMP in HEK293 co-expressing H208 and MYR–bPAC–nLuc stimulated with pulses of 4.4 μW/mm² light (mean ± S.D. of n = 8 cells). F, stimulation with 1:1000 Fz promotes sustained cAMP production well over 30 min (mean ± S.D. of n = 4 cells). RLU, relative luminescence units.

Figure 2.

Light activation of bPAC–nLuc promotes thyroid cell proliferation. A, real-time cAMP recordings in PCCL3 cells co-expressing red dimerization-dependent cAMP sensor and bPAC–nLuc or myc-empty vector (Myc-eV) control. A decrease in fluorescence represents increased cAMP levels (representative single cell traces shown, 250 μm IBMX shows sensor saturation). B, BrdU incorporation in PCCL3 transiently expressing bPAC–nLuc or bPAC-mCherry upon light exposure (mean ± S.D. of n = 3–4, >500 cells each, two-way analysis of variance with Tukey's multiple comparisons test, *, p < 0.05; **, p < 0.01).

Figure 3.

Targeted bPAC–nLuc exhibits different levels of activity and effects on thyroid cell proliferation. A, transfected PCCL3 cells stained with anti-Myc (red) and DAPI (blue) expressing bPAC–nLuc constructs throughout the cell (unmodified), NLS, cytosol (NES), and ER. B, PCCL3 co-expressing H208 or NLS-H208 with targeted bPAC–nLuc treated with 1:3000 Fz (mean of >45 cell traces from three independent experiments). The area under the curve from time 0 to 13.5 min normalized between basal and maximum response to 100 μm IBMX (one-way ANOVA with Tukey's multiple comparisons test). C, EdU incorporation in lentiviral-infected stable PCCL3 cell lines expressing targeted bPAC–nLuc constructs treated with 1:1000 Fz-4377. The dotted line shows basal levels of proliferation (mean ± S.D. of n = 5–6, >20,000 cells each, two-way analysis of variance with Dunnett's multiple comparisons test). *, p < 0.05; **, p < 0.01; ****, p < 0.0001.