Heterogeneity of Ca2+ handling among and within Golgi compartments.

The Golgi apparatus (GA) is a dynamic intracellular Ca(2+) store endowed with complex Ca(2+) homeostatic mechanisms in part distinct from those of the endoplasmic reticulum (ER). We describe the generation of a novel fluorescent Ca(2+) probe selectively targeted to the medial-Golgi. We demonstrate that in the medial-Golgi: (i) Ca(2+) accumulation takes advantage of two distinct pumps, the sarco/endoplasmic reticulum Ca(2+) ATPase and the secretory pathway Ca(2+) ATPase1; (ii) activation of IP3 or ryanodine receptors causes Ca(2+) release, while no functional two-pore channel was found; (iii) luminal Ca(2+) concentration appears higher than that of the trans-Golgi, but lower than that of the ER, suggesting the existence of a cis- to trans-Golgi Ca(2+) concentration gradient. Thus, the GA represents a Ca(2+) store of high complexity where, despite the continuous flow of membranes and luminal contents, each sub-compartment maintains its Ca(2+) identity with specific Ca(2+) homeostatic characteristics. The functional role of such micro-heterogeneity in GA Ca(2+) handling is discussed.

Ca(2+) signalling in the Golgi apparatus.

The Golgi apparatus plays a central role in lipid and protein post-translational modification and sorting. Morphologically the organelle is heterogeneous and it is possible to distinguish stacks of flat cysternae (cis- and medial Golgi), tubular-reticular networks and vesicles (trans-Golgi). These morphological differences parallel a distinct functionality with a selective distribution and complementary roles of the enzymes found in the different compartments. The Golgi apparatus has been also shown to be involved in Ca(2+) signalling: it is indeed endowed with Ca(2+) pumps, Ca(2+) release channels and Ca(2+) binding proteins and is thought to participate in determining the spatio-temporal complexity of the Ca(2+) signal within the cell, though this role is still poorly understood. Recently, it has been demonstrated that the organelle is heterogeneous in terms of Ca(2+) handling and selective reduction of Ca(2+) concentration, both in vitro and in a genetic human disease, within one of its sub-compartment results in alterations of protein trafficking within the secretory pathway and of the entire Golgi morphology. In this paper we review the available information on the Ca(2+) toolkit within the Golgi, its heterogeneous distribution in the organelle sub-compartments and discuss the implications of these characteristics for the physiopathology of the Golgi apparatus.

The trans-golgi compartment: A new distinct intracellular Ca store.

The Golgi apparatus (GA) is an intracellular organelle that plays a central role in lipid and protein posttranslational modification and sorting. In addition, the GA has been also shown to be involved in Ca(2+) signalling, as: (i) it accumulates Ca(2+) within its lumen in an ATP-dependent process catalyzed by two enzymes, the sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) and the secretory pathway Ca(2+) ATPase1 (SPCA1), and (ii) it releases Ca(2+) during cell stimulation in response to inositol 1,4,5-trisphosphate (IP(3)) receptor activation. Therefore, on this aspect, the GA appears to behave similarly to the major intracellular Ca(2+) store, the endoplasmic reticulum (ER). By using a new FRET-based Ca(2+) probe, specifically targeted to the trans-compartment of the GA, we demonstrate that the organelle is heterogeneous in terms of Ca(2+) handling, the trans-Golgi being insensitive to IP(3) and capable of accumulating Ca(2+) solely through the activity of SPCA1. The SERCA and the IP(3) receptor appear to be restricted to the cis- and intermediate GA compartments. Moreover, selective reduction of Ca(2+) concentration within the trans-Golgi, obtained by reducing the level of SPCA1 by RNAi, results in major alterations of protein trafficking within the secretory pathway and induces the collapse of the entire GA morphology.

Unique characteristics of Ca2+ homeostasis of the trans-Golgi compartment.

Taking advantage of a fluorescent Ca(2+) indicator selectively targeted to the trans-Golgi lumen, we here demonstrate that its Ca(2+) homeostatic mechanisms are distinct from those of the other Golgi subcompartments: (i) Ca(2+) uptake depends exclusively on the activity of the secretory pathway Ca(2+) ATPase1 (SPCA1), whereas the sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) is excluded; (ii) IP(3) generated by receptor stimulation causes Ca(2+) uptake rather than release; (iii) Ca(2+) release can be triggered by activation of ryanodine receptors in cells endowed with robust expression of the latter channels (e.g., in neonatal cardiac myocyte). Finally, we show that, knocking down the SPCA1, and thus altering the trans-Golgi Ca(2+) content, specific functions associated with this subcompartment, such as sorting of proteins to the plasma membrane through the secretory pathway, and the structure of the entire Golgi apparatus are dramatically altered.

PKA microdomain organisation and cAMP handling in healthy and dystrophic muscle in vivo.

Signalling through protein kinase A (PKA) triggers a multitude of intracellular effects in response to a variety of extracellular stimuli. To guarantee signal specificity, different PKA isoforms are compartmentalised by Akinase anchoring proteins (AKAPs) into functional microdomains. By using genetically encoded fluorescent reporters of cAMP concentration that are targeted to the intracellular sites where PKA type I and PKA type II isoforms normally reside, we directly show for the first time spatially and functionally separate PKA microdomains in mouse skeletal muscle in vivo. The reporters localised into clearly distinct patterns within sarcomers, from where they could be displaced by means of AKAP disruptor peptides indicating the presence of disparate PKA type I and PKA type II anchor sites within skeletal muscle fibres. The functional relevance of such differential localisation was underscored by the finding of mutually exclusive and AKAP-dependent increases in [cAMP] in the PKA type I and PKA type II microdomains upon application of different cAMP agonists. Specifically, the sensors targeted to the PKA type II compartment responded only to norepinephrine, whereas those targeted to the PKA type I compartment responded only to alpha-calcitonin gene-related peptide. Notably, in dystrophic mdx mice the localisation pattern of the reporters was altered and the functional separation of the cAMP microdomains was abolished. In summary, our data indicate that an efficient organisation in microdomains of the cAMP/PKA pathway exists in the healthy skeletal muscle and that such organisation is subverted in dystrophic skeletal muscle.

Protein kinase A type I and type II define distinct intracellular signaling compartments.

Protein kinase A (PKA) is a key regulatory enzyme that, on activation by cAMP, modulates a wide variety of cellular functions. PKA isoforms type I and type II possess different structural features and biochemical characteristics, resulting in nonredundant function. However, how different PKA isoforms expressed in the same cell manage to perform distinct functions on activation by the same soluble intracellular messenger, cAMP, remains to be established. Here, we provide a mechanism for the different function of PKA isoforms subsets in cardiac myocytes and demonstrate that PKA-RI and PKA-RII, by binding to AKAPs (A kinase anchoring proteins), are tethered to different subcellular locales, thus defining distinct intracellular signaling compartments. Within such compartments, PKA-RI and PKA-RII respond to distinct, spatially restricted cAMP signals generated in response to specific G protein-coupled receptor agonists and regulated by unique subsets of the cAMP degrading phosphodiesterases. The selective activation of individual PKA isoforms thus leads to phosphorylation of unique subsets of downstream targets.

AKAP complex regulates Ca2+ re-uptake into heart sarcoplasmic reticulum.

The beta-adrenergic receptor/cyclic AMP/protein kinase A (PKA) signalling pathway regulates heart rate and contractility. Here, we identified a supramolecular complex consisting of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2), its negative regulator phospholamban (PLN), the A-kinase anchoring protein AKAP18delta and PKA. We show that AKAP18delta acts as a scaffold that coordinates PKA phosphorylation of PLN and the adrenergic effect on Ca(2+) re-uptake. Inhibition of the compartmentalization of this cAMP signalling complex by specific molecular disruptors interferes with the phosphorylation of PLN. This prevents the subsequent release of PLN from SERCA2, thereby affecting the Ca(2+) re-uptake into the sarcoplasmic reticulum induced by adrenergic stimuli.

Transgenic fruit-flies expressing a FRET-based sensor for in vivo imaging of cAMP dynamics.

3'-5'-cyclic adenosine monophosphate (cAMP) is a ubiquitous intracellular second messenger that mediates the action of various hormones and neurotransmitters and influences a plethora of cellular functions. In particular, multiple neuronal processes such as synaptic plasticity underlying learning and memory are dependent on cAMP signalling cascades. It is now well recognized that the specificity and fidelity of cAMP downstream effects are achieved through a tight temporal as well as spatial control of the cAMP signals. Approaches relying on real-time imaging and Fluorescence Resonance Energy Transfer (FRET)-based biosensors for direct visualization of cAMP changes as they happen in intact living cells have recently started to uncover the fine details of cAMP spatio-temporal signalling patterns. Here we report the generation of transgenic fruit-flies expressing a FRET-based, GFP-PKA sensor and their use in real-time optical recordings of cAMP signalling both ex vivo and in vivo in adult and developing organisms. These transgenic animals represent a novel tool for understanding the physiology of the cAMP signalling pathway in the context of a functioning body.

Compartmentalized cAMP/PKA signalling regulates cardiac excitation-contraction coupling.

The sympathetic control over excitation-contraction coupling (ECC) is mediated by the cAMP/PKA signalling pathway. However, in the myocyte, the same signalling pathway is responsible for triggering a plethora of diverse intracellular functions the control of which must be independent of the regulation of ECC. Here we discuss what are the molecular mechanisms leading to selective modulation of ECC in cardiac myocytes with a particular focus on the role of spatial confinement of PKA subsets and the compartmentalization of cAMP.

Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway.

beta-Adrenergic signaling via cAMP generation and PKA activation mediates the positive inotropic effect of catecholamines on heart cells. Given the large diversity of protein kinase A targets within cardiac cells, a precisely regulated and confined activity of such signaling pathway is essential for specificity of response. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP]i in a stimulus-specific manner. By performing real-time imaging of cyclic nucleotides in living ventriculocytes we identify a prominent role of PDE2 in selectively shaping the cAMP response to catecholamines via a pathway involving beta3-adrenergic receptors, NO generation and cGMP production. In cardiac myocytes, PDE2, being tightly coupled to the pool of adenylyl cyclases activated by beta-adrenergic receptor stimulation, coordinates cGMP and cAMP signaling in a novel feedback control loop of the beta-adrenergic pathway. In this, activation of beta3-adrenergic receptors counteracts cAMP generation obtained via stimulation of beta1/beta2-adrenoceptors. Our study illustrates the key role of compartmentalized PDE2 in the control of catecholamine-generated cAMP and furthers our understanding of localized cAMP signaling.

Improvement of a FRET-based indicator for cAMP by linker design and stabilization of donor-acceptor interaction.

Förster resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for a variety of intracellular molecular events. Often, however, the poor dynamic range of such reporters prevents detection of subtle but physiologically relevant signals. Here we present a strategy for improving FRET efficiency between donor and acceptor fluorophores in a green fluorescent protein (GFP)-based protein indicator for cAMP. Such indicator is based on protein kinase A (PKA) and was generated by fusion of CFP and YFP to the regulatory and catalytic subunits of PKA, respectively. Our approach to improve FRET efficiency was to perform molecular dynamic simulations and modelling studies of the linker peptide (L11) joining the CFP moiety and the regulatory subunit in order to define its structure and use this information to design an improved linker. We found that L11 contains the X-Y-P-Y-D motif, which adopts a turn-like conformation that is stiffly conserved along the simulation time. Based on this finding, we designed a new linker, L22 in which the YPY motif was doubled in order to generate a stiffer peptide and reduce the mobility of the chromophore within the protein complex, thus favouring CFP/YFP dipole-dipole interaction and improving FRET efficiency. Molecular dynamic simulations of L22 showed, unexpectedly, that the conformational behaviour of L22 was very loose. Based on the analysis of the three principal conformational states visited by L22 during the simulation time, we modified its sequence in order to increase its rigidity. The resulting linker L20 displayed lower flexibility and higher helical content than L22. When inserted in the cAMP indicator, L20 yielded a probe showing almost doubled FRET efficiency and a substantially improved dynamic range.

Study of cyclic adenosine monophosphate microdomains in cells.

Cyclic adenosine monophosphate (cAMP) controls the physiological response to many diverse extracellular stimuli. To maintain signal specificity, cAMP-mediated signaling is finely tuned by means of a complex array of proteins that control the spatial and temporal dynamics of the second messenger within the cell. To unravel the way a cell encodes cAMP signals, new biosensors have recently been introduced that allow imaging of the second messenger in living cells with high spatial resolution. The more recent generation of such biosensors exploits the phenomenon of fluorescence resonance energy transfer between the green fluorescent protein- tagged subunits of a chimeric protein kinase A, as the way to visualize and measure the dynamic fluctuations of cAMP. This chapter describes the molecular basis on which such a genetically encoded cAMP sensor relies and the tools and methods required to perform cAMP measurements in living samples.

Measuring dynamic changes in cAMP using fluorescence resonance energy transfer.

cAMP is a ubiquitous second messenger that controls numerous cellular events including movement, growth, metabolism, contraction, and synaptic plasticity. With the emerging concept of compartmentalization of cAMP-dependent signaling, a detailed study of the spatio-temporal intracellular dynamics of cAMP is required. Here we describe a new methodology for monitoring fluctuations of cAMP in living cells, based on the use of a genetically encoded biosensor. The regulatory and catalytic subunits of the main cAMP effector, the protein kinase A (PKA), fused with two suitable green fluorescent protein (GFP) mutants is used for measuring changes in fluorescence resonance energy transfer (FRET) that correlate with changes in intracellular cAMP levels. This method allows the study of cAMP fluctuations in living cells with high resolution both in time and in space.

Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases.

Cardiac myocytes have provided a key paradigm for the concept of the compartmentalized cAMP generation sensed by AKAP-anchored PKA. Phosphodiesterases (PDEs) provide the sole route for degrading cAMP in cells and are thus poised to regulate intracellular cAMP gradients. PDE3 and PDE4 represent the major cAMP degrading activities in rat ventriculocytes. By performing real-time imaging of cAMP in situ, we establish the hierarchy of these PDEs in controlling cAMP levels in basal conditions and on stimulation with a beta-adrenergic receptor agonist. PDE4, rather than PDE3, appears to be responsible for modulating the amplitude and duration of the cAMP response to beta-agonists. PDE3 and PDE4 localize to distinct compartments and this may underpin their different functional roles. Our findings indicate the importance of distinctly localized PDE isoenzymes in determining compartmentalized cAMP signaling.