Luminescence-activated nucleotide cyclase regulates spatial and temporal cAMP synthesis

Abstract

Edited by Henrik G. Dohlman 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 lightactivated 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. cAMP was first described over 60 years ago (1). This second messenger is ubiquitous in all eukaryotic cells and regulates multiple cell processes. A wide variety of diseases are linked to aberrant cAMP signaling, including cancer, heart disease, muscular hypertrophy, diabetes, mood disorders, and thyroid hyperproliferative disorders (2-9). cAMP research has a long and rich history due to its clinical relevance; yet, we are still discovering new information encoded in spatial and temporal signaling. Recent studies using Förster resonance energy transfer (FRET) (10-14), other biosensors, and mathematic modeling (15-18) have advanced the cAMP signaling paradigm by providing strong evidence that cAMP signaling requires tight spatio-temporal regulation. The resulting models propose that cAMP is regulated in distinct subcellular compartments containing adenylyl cyclases (ACs), 4 cAMP effector proteins, phosphodiesterases, and scaffolding proteins. These regions create local areas of high and low cAMP concentrations that co-localize with cAMP effectors to regulate unique biological responses. Mounting evidence shows non-uniform cAMP accumulation throughout subcellular regions (i.e. plasma membrane versus bulk cytosol versus nucleus) (11, 13, 19-23). More recently, smaller scale compartmentalization or microdomains have been shown to exist and regulate cell function (10, 12, 13, 24-26). To identify bona fide compartments and define their function, new tools for localized cAMP synthesis are needed. Distinct kinetic profiles characterize cAMP signaling. Transient cAMP production followed by signal termination is a classic pattern found in G protein-coupled receptor (GPCR) pathways (27-30). Sustained cAMP production can occur in several GPCR pathways (31-36) or from activating mutations that lead to constitutive cAMP synthesis (37-40). Finally, cAMP oscillations have been reported in pancreatic ␤, neuronal, pituitary, and myocardiac cells ...

PACmn for improved optogenetic control of intracellular cAMP

“…Our interpretation of the observed increase in dark activity of Lyn-bPAC is that when bPAC is directly fused to the membrane anchor, the orientation between the two bPAC molecules is altered changing the enzymatic activity. NanoLuc-bPAC, which is particularly interesting for in vivo applications where chemiluminescent bPAC activation is less invasive than direct light delivery, also has dark activity [57]. We suspect that adjusting/increasing the linkers and introducing either the F198Y, R278A (or other) mutations as we did for Lyn-bPAC may reduce the dark activity reported for not only NanoLuc-bPAC but also when bPAC is attached to other proteins such as cyclic nucleotide dependent channels [39,58].…”

Tuning the Consonance of Microscopic Neuro-Cardiac Interactions Allows the Heart Beats to Play Countless Genres

“…It is now well appreciated that specifically distributed Adenylyl Cyclases (that make cAMP) and specific phosphodiesterase isoforms (that degrade cAMP) are selectively localized in different cellular domains ( Bers et al, 2019 ), including the plasma membrane, SR, nucleus and mitochondria ( Mongillo and Zaccolo, 2006 ; Leroy et al, 2008 ; Mika et al, 2012 ; Stangherlin and Zaccolo, 2012 ; Bedioune et al, 2018 ; Ghigo and Mika, 2019 ). Such molecular arrangement is an uttermost factor allowing the same signaling molecule (cAMP) to control both the thundering effect of the “fight-or-flight” reaction and, independently, gene expression, mitochondrial dynamics or other subdued homeostatic processes ( Mongillo et al, 2004 , 2006 ; Saucerman et al, 2006 ; Nikolaev et al, 2010 ; Sample et al, 2012 ; Tsvetanova and von Zastrow, 2014 ; Li et al, 2015 ; Bers et al, 2019 ; Naim et al, 2019 ; Zaccolo et al, 2021 ; Figure 1 ).…”

CAP1 binds and activates adenylyl cyclase in mammalian cells

“…Next, we assessed whether CAP1-cyclase interaction has functional consequences over cAMP dynamics in mammalian cells. We performed these experiments using PCCL3 thyroid follicular cells, a cAMP-responsive cell line we extensively studied (39)(40)(41)(42). Real-time cAMP was monitored using the fluorescence resonance energy transfer (FRET)-based cAMP sensor, H188 (43), under conditions where CAP1 expression levels were positively (CAP1 overexpression) or negatively (sh-CAP1) modulated.…”

“…Exploiting that both sh-Vector and sh-CAP1 plasmids express an independent dsRed unit, we performed a BrdU incorporation assay monitoring % BrdU/dsRed at a single-cell level (SI Appendix, Fig. S4), as reported before (39,41). As shown in Fig.…”